Abstracts

Keynote Session

Frontiers Lecture Series

5 HIGH THROUGHPUT FLOW CYTOMETRY AND THE NIH ROADMAP MOLECULAR LIBRARIES INITIATIVE

Larry A. Sklar¹, JeffreyArterburn², Bruce Edwards³, Tudor I Oprea⁴, Eric R Prossnitz⁵, Herbert G Tanner^{6 1}University of New Mexico, Cancer Researcn and Treatment Center and Pathology, Health Sciences Center, Albuquerque, New Mexico; ²New Mexico State University, Chemistry and Biochemistry, Las Cruces, New Mexico; ³University of New Mexico, Cancer Research and Treatment Center and Pathology, Albuquerque, New Mexico; ⁴University of New Mexico, Biochemistry and Molecular Biology, Albuquerque, New Mexico; ⁵University of New Mexico, Cell Biology and Physiology, Albuquerque, New Mexico; ⁶University of New Mexico, Mechanical Engineering, Albuquerque, New Mexico

The high throughput (HT) flow cytometry platform HyperCyt is adept at both cell and particle-based assays and is compatible with both high content and multiplex analysis. Our cell-based assays have initially been directed against G protein-coupled receptor (GPCR) targets where we have identified novel small molecule ligands for peptide and steroid receptors. We have developed general particle-based multiplexed approaches compatible with assemblies of soluble membrane receptors, receptor tails, proteases, kinases, nucleases, etc. We have probed the mechanism of partial agonism and the steps in signal transduction using flow cytometry-based kinetic analysis with soluble GPCR. We have also developed approaches probing cell-cell adhesion, nanoscale integrin conformational changes, and responses to nanoscale intercellular forces. The NIH Roadmap Molecular Libraries Initiative (MLI) has given us the opportunity, through the New Mexico Molecular Libraries Screening Center (NMMLSCN, http://nmmlsc/) to implement HT flow cytometry for the international research community. The MLSCN has expertise in: 1) target and assay development; 2) the integration of flow cytometric and virtual screening to enhance the discovery process; and 3) medicinal chemistry for the optimization of active molecules and the development of imaging agents. Through MLI and collaborations with investigators outside the MLSCN, we are currently developing cell-based assays for cytotoxicity where both cell viability and cell number can be recorded, bacterial virulence, multi-drug resistance, androgen response, protein expression, and generic cell responses, to name a few. We are also working on particle-based multiplexed protein-protein assays for interactions between Bcl-2 family members and generic protein-oligonucleotide interactions.

6 THE PHARMACODYNAMICS OF MOLECULAR CANCER THERAPEUTICS

David Hedley^{1 1}Princess Margaret Hospital, Toronto, Ontario, Canada

Our approach to cancer is being revolutionized through rapid progress understanding the molecular mechanisms, coupled to rational drug design programs producing highly selective agents to target these mechanisms. Despite the current excitement, there are formidable obstacles to making effective molecular cancer therapeutics a clinical reality. Unlike classical chemotherapy and radiotherapy, the drugs are highly selective in their actions, and effective only when their molecular target is playing a significant role driving the malignant process. Due to the complex, multigenetic basis of cancer development, it is unlikely that a single molecular targeted agent could achieve long term cancer control in the clinic. More likely, optimal treatment will consist of combinations of agents, rationally selected based on understanding the downstream interactions of drug targets, and individualized by analysis of the patient´s tumour tissue. Fluorescence-based techniques using flow or image cytometry have major advantages studying complex biology, because they address the problems of cellular heterogeneity, and are able to study molecular interactions through the use of multiple fluorescence labels. The development of phosphospecific antibodies has allowed the introduction of techniques to study signal transduction at the single cell level, including the analysis of complex signaling interactions. This is of particular importance to molecular oncology, since a large number of agents currently in clinical trial inhibit signaling pathways. As well as measuring baseline activity to identify if the drug target is being expressed in the cancer cells, cytometric methods can also be used to monitor pharmacodynamic effects during treatment. Pharmacodynamics is a branch of pharmacology that asks how drugs impact on the host tissues, whereas pharmacokinetics studies how drugs are metabolized and eliminated by the patient. During the early phases of drug development, it is important to show that the drug is interacting with its target in vivo, and to determine the relationships between drug dose and the extent of target inhibition. Along with others, our group has been able to develop pharmacodynamic markers based on flow cytometry and fluorescence image analysis, validate these using experimental systems, and then translate the methods to the actual clinical situation. Pharmacodynamic information is critical to understanding what is going on in a patient´s cancer cells during treatment, in order to explain why treatment works in some patients but not in others. Although still in its infancy, cytometrybased pharmacodynamics has the potential to play a major role bridging between basic science, pathology, pharmacology, and molecular oncology.

7 SEARCHING FOR AN HIV VACCINE: THE ROLE OF FLOW CYTOMETRY

Mario Roederer^{1 1}ImmunoTechnology Section, Laboratory of Immunology, Vaccine Research Center, NIAID, NIH, Bethesda, Maryland

Vaccine development to protect against HIV development is actively proceeding on two fronts: generation of a sterilizing (neutralizing) humoral response, and generation of an effective cellular response. To date, it has been impossible to generate an antibody response of sufficient potency that sterilizing vaccination is possible. Nonetheless, a vaccine can be considered successful on a global scale if the induced cellular response is sufficient to dampen viral loads (reducing transmission) as well as reducing morbidity and mortality after infection. We use a variety of adjuvants, delivery mechanisms, and immunogens to induce a variety of T cell responses. By challenging vaccinated nonhuman primates with live virus, we hope to identify the kinds of responses that are best correlated with protection as measured by a reduction in peak viral load, set point viral load, and amelioration of the dramatic destruction of memory CD4 T cells during acute infection. As we move forward through phase I and II clinical trials in humans, and prepare for phase III, we are keen to determine whether or not these types of responses are induced in humans as well. The primary tool for determining the quantity and quality of the T cell response is flow cytometry. Using sophisticated instrumentation, software, reagents, and techniques, we are able to measure five or more different functional responses simultaneously from each cell (e.g., cytokine profile, cytotoxic potential, proliferative capacity). These complex combinations of functions reveal that there are a number of distinct “flavors” of T cell responses present in either naturally infected or vaccinated subjects; the task now is to identify which of these flavors is most suited to protection from challenge. In addition, by studying rapid HIV progressors vs. long term nonprogressors, we are beginning to identify differences in T cell responses that are correlated with disease pathogenesis, perhaps pointing us towards desirable types of vaccine responses. Significant challenges remain, however. The automation of the sample analyses, the automation of the data analyses, compliance with GLP, and validation of these procedures is an enormous challenge. Integration and presentation of the highly complex datasets is still intensely laborious and challenging. In these terms, flow cytometry technology development is shifting from the instrumentation to the software and data interpretation aspects, which have yet to catch up to the rapid growth of the hardware capabilities.

8 FRONTIER LECTURE 4: HUMAN STEM CELL BIOLOGY AND POTENTIAL APPLICATIONS

Mickie Bhatia^{1 1}McMaster University, Hamilton, Ontario, Canada

Dr. Bhatia’s program has characterized signaling pathways that regulate human stem cells and initiated new approaches of changing human stem cell behaviour. Using human to mouse xenotransplant systems to define human stem cells in the whole body, he has shown that growth factors normally abundant during fetal development can be introduced to adult stem cells and change their growth properties and ability to form other cell types. In parallel, Dr. Bhatia has demonstrated that cord blood stem cells have the potential of regenerating a damaged pancreas and correcting diabetic-like symptoms. Where human embryonic stem cells (hESCs) have been explored, his work provides evidence of the ability to generate blood stem cells from hESC lines and novel ways of growing hESC lines in culture. These findings provide novel procedures that may be useful in cell transplantation based regenerative therapies and continue to compare the effectiveness of adult and embryonic stem cells alike.

Plenary Sessions

Speckles correspond to regions of the size of the point-spread-function with a higher fluorophore density and serve as fiduciary marks of the dynamics of the underlying structure. Although conceptually simple, the interpretation of FSM time-lapse sequences requires sophisticated computational tools for the tracking and statistical analysis of the intrinsically stochastic and highly convoluted signals. In this lecture I will cover the basic design of algorithms that simultaneously track thousands of speckles; and statistical models that allow the conversion of positional fluctuations and speckle intensity variations into spatiotemporally resolved maps of transport, turnover, and viscoelasticity of the probed structure. I will then report how quantitative FSM yielded major discoveries of the dynamic organization of the actin cytoskeleton in migrating cells and of the transient interaction of the cytoskeleton with other cellular assemblies such as focal adhesions. With a second set of examples I will indicate how we exploit the submicron resolution of this technique to infer molecular mechanisms of force generation during cell migration. Finally, I will illustrate the power of FSM as a general quantitative method for imaging the dynamics of any multiprotein structure in cells and in vitro.

13 FACILE GENERATION OF BIOSENSORS TO STUDY ENDOGENOUS PROTEIN ACTIVATION IN LIVING CELLS

Klaus Hahn^{1 1}University of North Carolina at Chapel Hill, Department of Pharmacology, Chapel Hill, North Carolina

We are developing new methods for studying the spatio-temporal dynamics of signaling with minimal perturbation. By conjugating novel dyes to affinity reagents, activities of endogenous proteins can be studied with high sensitivity. This approach opens the door to high throughput generation of biosensors via phage display and other screening for biosensor affinity elements. The approach also readily permits analysis of multiple signaling activities in the same cell.

14 MULTIPLEXED HYBRID CYTOMETRY BASED APPLICATION PLATFORM TECHNOLOGY: A TOOL TO FIGHT THE HIV PANDEMIC IN THE 21ST CENTURY

Francis Mandy^{1 1}Health Canada, Ottawa, Ontario, Canada

In sub-Saharan Africa about 12 million people perish every year from: HIV, malaria and tuberculosis. For the majority of the individuals who make up these gruesome annual statistics, the cause of death is uninvestigated. The introduction of affordable antiretroviral therapy (ART) in parts of Africa provides an opportunity to establish infrastructure to support laboratory medicine. This movement is a significant step in the direction of an effective health care system. Conventional laboratory medicine from resource rich regions is incompatible with African economical, cultural and political realities. In sub-Saharan countries 38% of the population exists on <$1 a day, with a gross national income per capita <$500. In the past, in most resource limited regions, founding agencies’ concentrated on disease prevention and provision of care. Until now, little effort has been exerted to build sustainable laboratory capacity. With the introduction of ART in Africa, health care policy makers and clinical scientist recognize the urgent need for affordable and sustainable laboratory infrastructure to support the diagnosis and treatment of HIV. For 25 years, flow cytometry has been the CD4 T-cell enumerating devic, to monitoring immune status of HIV infected individuals. Recently, numerous companies introduced affordable and robust cytometers both the flow and none-flow variety. Currently, the lower cost, dedicated instruments are the most popular choice for CD4 T-cell counting in Africa. Accumulative sales of these instruments are reaching significant numbers. When establishing affordable and sustainable comprehensive laboratory infrastructure is the overall long term objective; they in fact may turn out to be not the most cost effective option. In this presentation features of the hybrid flow based application platform (HyFAP) will be covered. The focus is on how to harness HyFAP and make economical sense in sub-Sahara Africa. They run both cell and microsphere based assays. Evidence will be presented to illustrate how a multi-functional and multiplexing capacity is a realistic option in resource poor countries. In the future, HyFAP will be connected to GSM wireless external quality monitoring services (WEMS). The integration with WEMS will assure minimum global standards for immunology laboratories. With HyFAP, it is possible to build laboratory capacity to provide rapid, accurate affordable and reliable diagnostic tests to battle infectious diseases in most resource poor regions of the globe.

15 REGULATION AND THERAPEUTIC TARGETING OF HUMAN LEUKEMIA STEM CELLS

Kristin Hope¹, Liqing Jin², Eric Lechman², John Edgar Dick^{3 1}University of Toronto, Toronto, Ontario, Canada; ²University Health Network, Cell and Molecular Biology, Toronto, Ontario, Canada; ³University of Toronto, Medical Genetics & Microbiology, Faculty of Medicine, Toronto, Ontario, Canada

In acute myeloid leukemia (AML) the leukemic clone is organized as a hierarchy originating from rare leukemic stem cells (LSC). Our interest has therefore been in identifying and therapeutically exploiting the molecular mechanisms that are uniquely required by these LSC. Although gene expression analysis is a common way of identifying the molecular players in stem cell function, few have explored the potential for microRNAs (miRNAs) in such a regulatory role. MiRNAs are 22 nucleotide (nt) non-coding RNAs processed from hairpin precursors that regulate translational repression of target genes. MiRNAs have been implicated in directing diverse biological processes including neoplasia. The identification of a set of embryonic stem cell specific miRNAs suggests that these may be involved in maintenance of the pluripotent state while a study linking miRNAs to fate determination showed ectopic expression of a specific miRNAs in hematopoietic progenitors could promote B or T cell differentiation. To address the role of miRNAs in the regulation of LSC we performed miRNA array analysis on 4 purified fractions based on CD34 and CD38 expression from 6 primary AMLs. We identified a unique miRNA signature that discriminates the CD34⁺CD38- fraction from more mature populations. One candidate, mir155, was also found to be differentially highly expressed in the stem cell fraction of normal cord blood by affymetrix array. RNAi-mediated knockdown of mir155 in a novel CD34⁺ leukemic cell line resulted in a loss of CD34 expression, an increase of differentiation antigen expression and significantly reduced proliferative potential. Our results are suggestive of a role for microRNAs in the regulation of LSC and leukemogenesis. In order to cure AML, LSC must be effectively targeted, however as existing therapeutic strategies target cycling cells and LSC are largely quiescent, new approaches must be found. We have shown previously that an activating monoclonal antibody (H90) directed against the adhesion molecule CD44 can release the AML differentiation block when administered in vitro. To address whether CD44 activation can act at the level of the LSC, H90 was administered to NOD/SCID mice transplanted with primary AML cells. We show that H90 greatly impairs leukemic repopulation by up to 95% compared to mice injected with control antibody. In addition, posttreatment grafts exhibited a much reduced level of primitive cells and an increase in immunophenotypically differentiated cells. The absence of leukemia in serially transplanted mice established direct targeting of LSC. The mechanism of H90 induced LSC loss involves both a promotion of LSC commitment and an impairment of their homing to supportive microenvironments.

16 DIAGNOSING PNH AND PREVIOUSLY UNDIAGNOSED ACUTE LEUKEMIAS WITH FLAER AND MULTIPARAMETER FLOW CYTOMETRY

D. Robert Sutherland¹, Nancy Kuek¹, Jeff Davidson¹, Sylvia Lynn Bamford², Michael Keeney^{3 1}University Health Network, Clinical Flow Cytometry, Toronto, Ontario, Canada; ²London Health Sciences Centre, Flow Cytometry, London, Ontario, Canada; ³London Health Sciences, Hematology/ Flow Cytometry, London, Ontario, Canada

Paroxysmal Nocturnal Hemoglobinurea (PNH) is an acquired Hematopoietic Stem Cell disorder caused by a somatic mutation in the X-linked pig-a gene. This results in a partial or absolute deficiency of all glycophosphatidyl-inositol (GPI)-linked proteins/glycoproteins. The classical approach to diagnosis of PNH by cytometry involves the loss of at least 2 GPI-linked antigens (typically CD55 and CD59) on two different cell lineages (RBCs and Neutrophils). The bacterial lysin Aerolysin binds to the GPI moiety of cell surface GPI-linked molecules and causes lysis of normal cells but not GPI-deficient PNH cells. FLAER is a fluorescently-labeled inactive variant of aerolysin that does not cause lysis of cells to which it binds and this reagent is becoming more widely used in the diagnosis of PNH by Flow. In a single tube assay, we have combined FLAER with CD45, CD33 and CD14 that allows the simultaneous analysis of FLAER and the GPI-linked CD14 structure on neutrophil and monocyte lineages. Comparison to standard CD55 and CD59 analysis shows excellent agreement. The assay can be performed up to 48 hours after sample draw and data interpretation is straightforward. Additionally, we were able to detect fewer than 5% PNH-like monocytes and neutrophils in several cases of aplastic anemia, which we were otherwise unable to detect using CD55 and CD59 on red blood cells. Due to the higher signal to noise ratio, the method shows increased sensitivity in our hands over single (CD55 or CD59) parameter analysis. Interestingly, we have also detected leukemic blasts which show aberrant FLAER staining in several samples sent to us for ‘PNH testing’ including 1 case each of AML-M1, erythroleukemia (M6), a case of acute leukemia developed in a patient with a history of CMML and two cases of acute myelomonocytic leukemia. In all of these cases, the neutrophils stained normally with FLAER, thereby ruling out PNH, while the gated CD33bright cells failed to express CD14 and bound lower levels of FLAER.

22 DO NOT MIND THE GAP: PROTEIN TRAFFICKING BETWEEN THE ENDOPLASMIC RETICULUM AND THE GOLGI APPARATUS IN PLANT CELLS

Federica Brandizzi^{1 1}University of Saskatchewan and Michigan State University, Saskatoon/East Lansing, Saskatchewan/Michigan, Canada

Secretory materials are synthesized on the surface of the endoplasmic reticulum (ER)1. They are then shipped from the ER to the Golgi apparatus to be sorted either back to the ER or to distal secretory compartments such as vacuoles and plasma membrane. It is vital for a cell to regulate protein transport between these organelles. The ER and Golgi are closely associated in plant cells2,3 (Fig. 1). How these two organelles communicate with each other in plant cells is an important question that remains largely unanswered. To provide further understanding of the regulation of protein export from the plant ER, we have explored the mechanisms of protein trafficking between the ER and the Golgi apparatus using live cell imaging techniques. It appears that plant cells contain multiple mobile Golgi stacks distributed over the entire cytoplasm. These stacks move with the ER by means of actin-myosin motors3,4. The domains of the ER dedicated to the export of proteins, the ER export sites (ERESs), form secretory units that move along the surface of the ER together with the Golgi stacks4,5. We also found that the integrity of Golgi and ERESs is regulated by the activity of specific GTPases, such as Sar1 and Arf14,5. Finally, we determined the existence of a stringent signal-regulated mechanisms for ER export of multispanning, type I and type II membrane proteins6. For example, we found that mutations of a specific di-acidic motif (DXE) in the cytosolic tail of proteins such as CASP, a Golgi matrix protein with a type II membrane topology7, cause a reduction of the export of this protein from the ER6. ER export of type I and multispanning membrane proteins appears to be similarly regulated6. Our results indicate that in plant cells the ER and Golgi form a dynamic membrane system whose components continuously cycle through the ER via a regulated membrane trafficking pathway. References: 1. Nicchitta CV. Curr Opin Cell Biol 2002;14(4):412-6. 2. Boevink et al. Plant J 1998;15(3):441-7. 3. Brandizzi et al. Plant Cell 2002;14(6):1293-309. 4. daSilva et al. Plant Cell 2004;16(7):1753-71. 5. Stefano et al. Plant J 2006; in press. 6.

Hanton et al. Plant Cell 2005;17(11):3081-93. 7. Renna et al. Plant Mol Biol 2005;58(1):109-22.

Confocal image of a tobacco leaf epidermal cell transformed with ERD2-YFP, a Golgi and ER marker3. Note that Golgi bodies (arrow) are in close association with the ER network. Scale bar = 5 microns

Parallel Sessions

23 COHERENT ANTI-STOKES RAMAN SCATTERING (CARS) MICROSCOPY FOR LABEL-FREE, CHEMICALLY-SELECTIVE BIOMEDICAL IMAGING

Conor L. Evans¹, Jeanette Kurian¹, Eric O. Potma², Mehron Pourgish’Haag³, Daniel Cote³, Charles P. Lin³, X. Sunney Xie^{1 1}Harvard University, Chemistry and Chemical Biology, Cambridge, Massachusetts; ²University of California, Irvine, Chemistry, Irvine, California; ³Massachusetts General Hospital, Wellman Center for Photomedicine, Boston, Massachusetts

Advances in biomedical imaging have revolutionized our ability to visualize living organisms at the cellular and sub-cellular levels. Despite these developments, in vivo imaging with chemical contrast remains a challenge as molecular selectivity typically requires the introduction of specific labels. Many applications in biology and medicine would significantly benefit from a noninvasive imaging technique that circumvents such exogenous probes. In vivo microscopy based on vibrational spectroscopic contrast offers a unique approach for visualizing tissue architecture with chemical specificity. Coherent-anti-Stokes Raman scattering (CARS) microscopy is a non-linear imaging technique capable of rapid vibrational imaging of thick biological specimens. Backscattering of the intense forward propagating CARS light in tissue gives rise to a surprisingly strong epi-CARS signal that makes in vivo imaging possible. This substantial signal allows for real-time monitoring of dynamic processes, such as the diffusion of chemical compounds, in tissues [Evans et al. PNAS 102, 16807 (2005)]. By specifically tuning into the CH2 stretching vibrational frequency, we demonstrate CARS imaging and spectroscopy of lipid rich tissue structures in mouse skin, including sebaceous glands, corneocytes and adipocytes, with unprecedented contrast at subcellular resolution.

27 HYPERCHROMATIC CYTOMETRY PRINCIPLES FOR CYTOMICS BY SLIDE BASED CYTOMETRY

Attila Tárnok¹, Mittag Anja¹, Wiebke Laffers², Dominik Lenz³, Andreas Gerstner^{2 1}Cardiac Center, University of Leipzig, Research Laboratory, Pediatric Cardiology, Leipzig, Saxony, Germany; ²University Bonn, Bonn, Nordrhein-Westfalen, Germany; ³Purdue University, Purdue University Cytometry Laboratories, West Lafayette, Indiana

Multicolor and polychromatic analysis of biological specimens has become increasingly important due to the emerging new fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics. Combining different technologies and staining methods polychromatic analysis can be pushed further forward to virtually measure anything stainable in a cell. We termed this approach hyperchromatic and present different components suitable to be combined for achieving this task. For cell analysis Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, it is a non-consumptive method, i.e. the analyzed sample is fixed on the slide and can following manipulation of the object be subsequently reanalyzed. In this overview we demonstrate on a SBC instrument, the Laser Scanning Cytometer, various approaches for hyperchromatic analysis. The different components demonstrated here are: 1) polychromatic cytometry (staining of the specimen with eight or more different fluorochromes simultaneously), 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). Based on the feature of relocating cells that are immobilized on a microscope slide the identical cells can be subsequently reanalyzed and the data collected on the single cell level after manipulation steps. With the intelligent combination of several different techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

28 A MULTIMODE ENDOSCOPE FOR SIMULTANEOUS MACROSCOPIC AND MICROSCOPIC IMAGING OF TISSUES IN VIVO

Silas J. Leavesley¹, Bartlomiej Rajwa², J. Paul Robinson^{3 1}Purdue University, Biomedical Engineering, Weldon School of Biomedical Engineering, West Lafayette, Indiana; ²Purdue University, Basic Medical Sciences,Veterinary Medicine, West Lafayette, Indiana; ³Purdue University, Basic Medical Sciences & Biomedical Engineering,West Lafayette, Indiana Diagnosis of pathologies often includes gathering information using various diagnostic medical imaging modalities, and as a result of this information, the possible biopsy of tissues for further analysis. In many cases, the region(s) in which to perform a biopsy are detected and delineated by diagnostic images. Pathologies of the alimentary, respiratory, and urinary systems often make use of endoscopic imaging modes (mainly traditional and fluorescence endoscopy) to detect potential lesions, and use biopsy as a method for confirming the nature of the detected lesion. Thus, increasing the sensitivity of endoscopic diagnosis will aid in correctly delineating the boundaries of detected lesions, and increasing the specificity of endoscopic techniques may help to decrease the total number of biopsies taken. This work outlines the development of a new type of endoscope that features a forward-facing macroscopic video imaging port, and a side-facing microscopic imaging port. Both of these modes of imaging can operate simultaneously, and both can be operated in reflected light and fluorescence modes, allowing a physician to “zoom-in” on potential lesions and inspect their cellular makeup. The ability to inspect these suspected areas should decrease the number of false positive diagnoses, which could serve to decrease the number of biopsies, in many cases. This is appealing for two reasons, first it will serve to reduce tissue damage and pain to patients; second, it will reduce some costs associated with unnecessary biopsies. In addition, with the advanced software used in this system, the physician has greater control over what is actually imaged. Thus, this endoscope advances the technology in this field by delivering quality, high-resolution microscopic images with simultaneous macroscopic location information.

Image Processing and Analysis 1

29 TOPOLOGY PRESERVING STACS SEGMENTATION OF PROTEIN SUBCELLULAR LOCATION IMAGES

Amina Chebira¹, Gowri Srinivasa¹, Lionel Coulot², Heather Kirschner³, Jose M. F. Moura³, Jelena Kovacevic¹, Elvira Garcia Osuna⁴, Robert F. Murphy⁵

¹Carnegie Mellon University, Biomedical Engineering, Carnegie Institute of Technology, Pittsburgh, Pennsylvania; ²EPFL, Lausanne, Switzerland; ³Carnegie Mellon University, Electrical and Computer Engineering, Pittsburgh, Pennsylvania; ⁴Carnegie Mellon University, Biomedical Engineering, Pittsburgh, Pennsylvania; ⁵Carnegie Mellon University, Biological Sciences and Biomedical Engineering, Pittsburgh, Pennsylvania We present an algorithm for the segmentation of multicell fluorescence microscopy images. Such images abound and a segmentation algorithm robust to different experimental conditions as well as cell types is becoming a necessity. In cellular imaging, among the most often used segmentation algorithms is seeded watershed. One of its features is that it tends to oversegment, splitting the cells, as well as create segmented regions much larger than a true cell. This can be an advantage (the entire cell is within the region) as well as a disadvantage (a large amount of background noise is included). We present an algorithm which segments with tight contours by building upon an active contour algorithm—STACS proposed by Pluempitiwiriyawej at al. We adapt the algorithm to suit the needs of our data and use another technique, topology preservation proposed by Han et al, to build our topology preserving STACS (TPSTACS). Our algorithm significantly outperforms the seeded watershed both visually (see Figure, seeded watershed on the left, TPSTACS on the right) as well as by standard measures of segmentation quality (see Table).

33 IMAGE ANALYSIS AND METADATA PROCESSING FOR CELL STRUCTURE AND FUNCTION DESCRIPTION. EXAMPLES OF APPLICATION AS A NEW DIAGNOSTIC APPROACH IN THREE DIFFERENT FIELDS: HAEMATOLOGY, TOXICOLOGY AND MICROBIOLOGY

Maria Cristina Albertini¹, Marco Rocchi², Augusto Accorsi¹, Laura Teodori^{3 1}University of Urbino, Institute of Biochemistry C.Fornaini, Urbino, Italy; ²University of Urbino, Institute of Biomedical Engineering, Urbino, Italy; ³ENEA CR CASACCIA, BIOTEC-MED, Rome, Italy

In order to describe biological states and processes, the collection and the evaluation of a large amount of image data are becoming more and more important. Consequently, the search for suitable complex statistics and the development of algorithms, allowing us to model the data and compile the knowledge gained from bio-imaging is the next challenge. We used a platform of statistical methods such as: multivariate statistics (PCA, discriminant analysis and multidimensional scaling) circular statistics (analysis of directional data) and shape analysis, to express analytical results from captured meaningful cell image information acquired from optical, electron and atomic force microscopy. All these microscopies have added high-resolution spatial dimensions information that enabled us to quantitatively detect cell orientation, surface properties and metabolic features. We reported examples of application to three different research areas: haematology, microbiology and toxicology. By this approach we were able to: i) diagnose some shape related RBC pathologies based on morphological automated analysis (essential hypertension prediction); ii) evaluate water Vibrio alginolyticus contamination, distinguishing between viable-but-not-culturable-cells to culturable-cells; iii) quantitatively analyse physiological and morphological alterations from static magnetic field exposure, demonstrating altered cell shape, diverse orientation and abnormal membrane structure; iv) increase the power of the comet assay DNA damage evaluation, distinguishing different DNA damages from chemical/physical noxia. In conclusion, the results demonstrated that through combining the imaging of cells with powerful image analysis algorithms, one can acquire a deeper knowledge on multiple biochemical and morphological pathways at the single-cell level to be used as a diagnostic tool.

34 NONINVASIVE FORWARD-SCATTERING SYSTEM FOR RAPID DETECTION, CHARACTERIZATION, AND IDENTIFICATION OF LISTERIA COLONIES. IMAGEPROCESSING AND ANALYSIS

Bulent Bayraktar¹, Padmapriya P Banada², J. Paul Robinson³, Arun K. Bhunia², E. Daniel Hirleman⁴, Bartlomiej Rajwa^{3 1}Purdue University, Electrical and Computer Engineering, Schools of Engineering, West Lafayette, Indiana; ²Purdue University, Molecular Food Microbiology Laboratory, Department of Food Science, West Lafayette, Indiana; ³Purdue University, Basic Medical Sciences, Veterinary Medicine, West Lafayette, Indiana; ⁴Purdue University, School of Mechanical Engineering, Engineering, West Lafayette, Indiana

Bacterial contamination by Listeria monocytogenes not only puts the public at risk but also is costly for the food-processing industry. Traditional biological and chemical methods for pathogen identification require complicated sample preparation for reliable results. Optical scattering technology has been used for identification of bacterial cells in suspension, but with only limited success. Extracellular materials produced by a culture in a colony along with cellular arrangements may provide unique signatures that are necessary for differentiation of colonies by light scattering. We have developed a noninvasive optical forward-scattering system for rapid detection, characterization, and identification of Listeria colonies grown on solid surfaces. The presented work includes application of computer vision and pattern recognition techniques. Bacterial colonies with a diameter of approximately 1.8 to

1.9 mm and a thickness of around 0.3 to 0.4 mm (measured along the optical axis) were analyzed with a laser scatterometer. Circular scatter patterns formed by bacterial colonies illuminated by red laser light were analyzed using Zernike (continous) or Tchebichef (discrete) moment invariants. Discrete moments do not possess the discretization errors inherent in continuous moments. This important advantage allows better fulfillment of orthogonality and invariance properties. Use of discrete moments improves the speed of our image processing algorithms. This is attained by calculating discrete polynomial coefficients directly from their definitions employing arbitrary precision arithmetic, rather than using recurrence relationships. The constructed coefficients are stored in look-up tables, and retrieved during the process of image analysis. This approach also eliminates a potential of numerical instability due to inadequate numeric precision. Principal component analysis and hierarchical clustering were performed on the results of feature extraction for colony differentiation. Classifications using linear discriminant analysis, partial least squares, and neural networks were used to test the feasibility of automated determination of bacteria pathogenicity on the basis of colony scatter patterns. The overall system is robust and can be extended into an automated user-friendly device for detection and differentiation of various pathogenic bacteria.

Flow Instrumentation 1

35 ENHANCED DIFFERENTIATION MODULE (EDM) FOR CLINICAL FLOW CYTOMETRY ANALYSIS

J. Paul Robinson¹, Kathy Ragheb², Cheryl Holdman³, Valeri Patsekin⁴, Christakis Christodoulou⁵, Bill Kiroviac⁶, Paul Church⁷, Todd Lary^{7 1}Purdue University, Basic Medical Sciences & Biomedical Engineering, West Lafayette, Indiana; ²Purdue Unviersity, Basic Medical Sciences, West Lafayette, Indiana; ³W. Lafayette, Indiana; ⁴Purdue University, W. Lafayette, Indiana; ⁵Beckman-Coulter, Engineering, Physical Sciences and Engineering, Miami, Florida; ⁶Beckman-Coulter, Miami, Florida; ⁷Miami, Florida

We report on a new technology for flow cytometry to improve the light scatter collection of a routine instrument. The Enhanced Differentiation Module (EDM) developed for the FC500 and Altra flow cytometers provides several new scatter options for routine flow cytometry. Forward Angle Light Scatter (FALS) has been a standard feature of almost all flow cytometers for over 30 years. While inital developments in the field included studies of many angles of scatter, almost all instruments adopt a standard 2-4 degree scatter detection angle. The EDM module developed by Beckman-Coulter uses a fiber optic array with multiple rings at preset angles to collect significantly enhanced scatter signatures. Inital tests of the EDM were performed on a standard 2 laser FC500 benchtop cytometer. In the study, two identical instruments were run side-by-side with one instrument modified to accept the EDM. We present data showing the imact of using the different angles of scatter on very small beads, blood cells and regular cell cultured in flasks with a variety of treatements. The EDM as installed allows the use of individual or combined collection rings in addition to the implementation of neutral density filters to reduce the signal intensity on the detector if necessary. A very useful feature of the device was the ability to modify the gains on each detector to achieve optimal differentiation for any particular sample type. Current flow cytometry essentially assumes that a single forward scatter signal is adequate for most use. We have shown that this is seriously mistaken perception. Multiple scatter angles undoubtedly allow for differentiation of morphologically distinct populations otherwise inseparable by a standard scatter detector. We believe that this modification of a routine instrument will open new opportunities for cellular differentiation for malignant cells, cells which have phagocytosed particles, minor size changes and other refractive index alterations that differentiate cells or particles.

40 BIOINFORMATICS DATA STANDARDS FOR FLOW CYTOMETRY

Ryan R. Brinkman¹, Josef Spidlen¹, Adam S. Treister², Clayton Smith¹, Michael B. Ochs³, Robert Gentleman⁴, Charles Schmitt⁵, Perry Haaland^{5 1}British Columbia Cancer Research Centre, Terry Fox Laboratory, Vancouver, British Columbia, Canada; ²Tree Star, Inc., Ashland, Oregon; ³Fox Chase Cancer Center, Philadelphia, Pennsylvania; ⁴Fred Hutchinson Cancer Reserch Center, Seattle, Washington; ⁵BD Technologies, Research Triangle Park, North Carolina

We are developing free, open source and commercial software compatible bioinformatics standards and software tools for flow cytometry. The scope of this project includes development of an ontology, a data base schema, statistical and visual analysis tools, and a gating standard in the Extensible Markup Language (XML). The gating standard is presently our most well-developed component. Gating in flow cytometry is a process for defining a boundary that delineates the characteristics of particles for further analysis or sorting. During gating users define regions around a group of events of interest and then choose to include or exclude these events using some form of Boolean logic. Although flow cytometry has a successful data format standard, there is no shared representation of gates. This prevents a variety of collaborative opportunities to recreate experimental methods and results. We have developed a proposal on how to form gate definitions that can facilitate the interchange and validation of data between different software packages, and provide the means to better integrate methods with results in reporting. Specifically, we have developed a detailed description of the gating specification, a W3C Schema usable to validate XML documents, user documentation, and a set of examples of gating XML files. A software tool that implements the specification includes functionality to read a Flow Cytometry Standard data file, an accompanying XML file which describes the gates of interest, and the ability to process this information to provide descriptive statistics. Robustness problems are especially serious in geometric computation, since numerical errors can propagate into the combinatorial computations and result in complete failure of the algorithm. Our example implementation uses algorithms which are based on a user-definable precision model and contains code for robust geometric computation. As a result, the software tool will be useful to validate compliance of alternative software tools that implement the gating standard. The gating specification currently supports gating in n dimensions, including rectangular gates (e.g., range gates, 3D box regions, and n-dimensional hyper-rectangular regions), polygon gates, ellipsoid gates, decision tree structures and Boolean collections of the any of the types of gates. Support for platform independent validation of 2D gates is available from the JTS Topology Suite, and support for higher dimension polytopes through platform specific implementations based on Qhull.

Cell Physiology 1

41 PLASTICITY OF THE TUMOUR CELL GLYCOCALYX

Paul J Smith¹, Emeline Furon¹, Sally Chappell¹, Marie Wiltshire¹, Robert A Falconer², Laurence Patterson², Andrew Goater³, David Morris³, Julian PH Burt³, Rachel J. Errington^{4 1}Cardiff University, Cardiff, Wales, United Kingdom; ²Bradford University, Institute of Cancer Therapeutics, Bradford, West Yorkshire, United Kingdom; ³University of Wales, Bangor, Institute of Bioelectronic and Molecular Microsystems, Bangor, Gwynedd, United Kingdom; ⁴Cardiff University, Medical Biochemistry, Medicine, Cardiff, Wales, United Kingdom

Phenotypic plasticity (PP) in tumour cell populations has the undesirable potential to maximise Darwinian fitness for the selection of metastatic and drug-resistant variants. Changes in surface carbohydrate expression have been implicated in this process - the surface glycocalyx altering cell behaviour within a multicellular environment and assisting metastatic spread through enhanced cell detachment. Polysialic acid (PSA), providing polyanionic cell-surface decoration of the neural cell adhesion molecule (NCAM), is thought to be associated with the property of the early dissemination of cells from the primary tumour mass of small cell lung cancer (SCLC) - a common metastatic cancer expressing chemoresistance. Our aim was to explore the plasticity of the glycocalyx, and NCAM decoration in particular, together with the impact on gene expression and cellular dielectric properties. We have used a SCLC model (NCI-H69) and substrate adhesion as a selective principle to provide extremes of reversible phenotypic behaviour reflecting adherent versus non-adherent growth. We found reduced PSA-decoration of NCAM in adherent cells using flow cytometry and confocal imaging. PSA decoration could be re-gained by the growth of adherent cells on non-attaching hydrophobic surfaces without any induction of cell death. PSA reduction was accompanied by a downregulation of the critical glycolytic pathway genes GNE and FUT8, rather than modulation of specific polysialyltransferase genes. The reductions in NCAM decoration observed in vitro were re-iterated when cells were grown as mouse xenografts. The in vitro changes were accompanied by an increase in cytoplasmic membrane permittivity, but no change in zeta potential, for adherent cells as determined by electrokinetic analysis and multishell modelling. We outline a new probeless approach analysing the metastatic phenotype for diagnostics and drug screening purposes. There was no evidence that PP acted to modify pathways for cell cycle control, multi-drug resistance or tumour growth potential per se. However, cDNA microarray analysis revealed that genes modulated during acquisition of the adherence phenotype were linked through VEGF, with significant down-regulation of pro-metastasis, angiogenesis and ECM-related genes. Furthermore, the most prominent proteoglycan expression gained upon acquisition of adherence was glypican-3 - the encoding gene GPC3 being a potential tumour suppressor in SCLC. We conclude that PP can reversibly generate cellular phenotypes with elevated metastatic potential without recruiting de novo drug resistance - providing new targets for therapeutic control.

42 LIPID RAFTS REGULATE PDGFR CLUSTERING, ACTIVATION, INACTIVATION AND DOWNSTREAM SIGNALING IN A CELL CONFLUENCE-DEPENDENT MANNER

György Vereb¹, László Ujlaky-Nagy¹, János Szöllõsi^{1 1}University of Debrecen Medical and Health Science Center, Debrecen, Hungary

PDGF receptors are transmembrane tyrosine kinases that play an important role in the development and proliferation of glial tumors. Key events in their activation are di- (oligo)merization followed by trans-phosphorylation and downstream signaling cascades. Our aim was to reveal how cell confluence-dependent PDGFR activation is regulated by the molecular environment of receptors in the cell membrane. PDGF receptors were labeled with immunfluorescence. Their spatial arrangement and relationship to lipid rafts decorated with fluorescent choleratoxin B subunits (CTX-B) were determined by confocal laser scanning microscopy. Intracellular calcium levels as a measure of receptor activation in response to PDGF were measured with ratio videomicroscopy. The phosphorylation of receptors was assessed with anti-phosphotyrosine antibody in Western-blot and in situ immunofluorescence experiments. The glioblastoma cell lines A172 and T98G express primarily PDGFR beta. The number of receptors in the cell membrane increased as cell cultures reached confluence. Parallel to this, calcium responses evoked by PDGF were 2-phased and prolonged in an increasing portion of cells. In contrast, PDGFR mRNA levels decreased with confluence, while the rate of spontaneous internalization increased. Receptors showed a non-random, clustered distribution in the cell membrane. The overlap of receptor clusters with CTX-B-labeled lipid rafts was substantial. The cross-correlation coefficient characterizing the overlap increased with cell confluence. Furthermore, receptors showed higher relative phosphorylation in rafts than outside rafts. Interestingly, PDGF stimulation increased PDGFR phosphorylation much more in the non-confluent cells. However, inhibition of tyrosine phosphatases reverted this situation and the larger extent of spontaneous, as well as ligand-induced phosphorylation in confluent cells became apparent. Crosslinking of the lipid rafts by CTX-B at 37 C led to the aggregation of lipid rafts and sequestration of PDGFR clusters from them. Reducing the cholesterol content of the cell membrane by methylbeta-cyclodextrin dispersed lipid rafts and PDGFR clusters, decreased their overlap, and almost completely abolished phosphorylation and calcium response to PDGF stimulus. We conclude that raft localization of PDGFR has a functional consequence and is linked to regulation of proliferation as cells reach confluence inasmuch as both the receptor and tyrosine phosphatases are increasingly clustered in lipid rafts upon reaching confluence, whereby a greater turnover of the receptors´ activation cycle allows a more efficient downstream signaling.

46 EVALUATION OF ANTI-CANCER TARGETS ON CIRCULATING TUMOR CELLS TO PREDICT THERAPEUTIC SUCCESS

Arjan Tibbe¹, Joost Swennenhuis¹, Gerald Doyle², Dave Chianese², Jan Keij², Chandra Rao², Mark Connelly², John Verrant², Leon WMM Terstappen^{2 1}Immunicon Europe Inc., Enschede, Netherlands; ²Immunicon Corporation, Huntingdon Valley, Pennsylvania

The CellSearch^TM System has been used in multi-center prospective studies to demonstrate that presence of tumor cells in blood of patients with metastatic carcinomas is associated with poor survival prospects. Failure to eliminate Circulating Tumor Cells (CTCs) after one cycle of therapy in these studies strongly suggests that these patients are on a futile therapy. Assessment of the presence of therapeutic targets on the tumor should enable the appropriate choice of therapy. Here we demonstrate that anti-cancer targets can be identified on CTCs before initiation of therapy. In the CellSearch^TM System CTCs from 7.5 mL of blood are identified as Cytokeratin(CK)⁺, CD45- nucleated cells after EpCAM immunomagnetic selection. CTCs are identified by the CellTracks^® Analyzer II at the upper surface of a cartridge where they are held by a magnetic field. Three of the six colors currently available in the analyzer are used for the identification of the CTCs and the others are available for further interrogation of CTCs. Fluorescently labeled antibodies that recognize treatment targets associated with known therapies such as HER2, IGF-1, Bcl-2 and EGFR can be assessed on the CTCs. For expression of therapeutic targets at the molecular level we demonstrated that CTCs can be preserved for cytogenetic analysis. After the fluid in the cartridge is removed the cells are fixed and maintain their original position. Hybridization conditions remove the fluorescent probes used for the original identification. Since the system knows their original position, the cells can be reexamined for the presence of probes of interest. The example shows a CTC and a leukocyte before and after hybridization (left and right from the dotted line) for chromosome 1, 7, 8 and 17. Grey color in the overlay shows the DAPI stained nucleus after hybridization. Expression level of specific drug related molecular targets can be detected on CTCs and can be assessed at the protein or gene level. In addition, effect of therapy can be assessed after administration of therapy by a change in the number of the CTC after days or weeks of therapy rather than months.

Calibration and Standardization

47 COMPREHENSIVE CHARACTERIZATION OF FLOW CYTOMETER FLUORESCENCE MEASUREMENT WITH A MINIMAL BEAD SET

Robert A. Hoffman¹, Joseph Trotter², Alan Stall^{3 1}BD Biosciences, San Jose, California; ²BD Biosciences, San Diego, California; ³BD BioSciences Pharmingen, San Diego, California

Many instrument performance factors contribute to the accuracy and precision of flow cytometer measurements of fluorescence. The major factors are linearity, illumination uniformity, illumination noise, detection efficiency (Q), optical background (B), and electronic noise. Deviation from linearity (direct proportionality) is the measure of accuracy. The combined effect of illumination uniformity and illumination noise contributes a constant variance to the overall precision (CV) of fluorescence measurements independent of how bright the fluorescence signal is. Detection efficiency (the number of photoelectrons generated per fluorochrome molecule passing through the illumination beam), optical background and electronic noise are the primary factors that determine the CV´s of low-level signals. To simply measure all these fundamental instrument characteristics, we have developed a method of data acquisition and data analysis using as sample a mixture of beads with 3 different intensity levels (dim, midlevel and bright). Linearity is measured using the dual pulse method (ref. 1) by measuring the ratio of median channel of two bead intensities at varying photomultiplier (PMT) voltages. Electronic noise is determined from the broadening of a population CV as the PMT voltage is decreased. The contribution of illumination variability to measurement variance is estimated from the bright bead CV. Q and B (ref. 2) are determined from the CV´s of the midlevel and dim beads respectively after correcting for electronic noise and illumination variance. Knowledge of the intrinsic bead (sample) CV is necessary in order to perform an accurate instrument characterization. Methods to determine the intrinsic bead CV will be presented. We will also show examples of instrument characterization using the 3-bead method and show how the measured characteristics can be used to predict performance of a wide variety of applications. REFERENCES 1. Bagwell, C. B., Baker, D., Whetstone, S., Munson, M., Hitchcox, S., Ault, K. A. and Lovett, E. J. (1989). A simple and rapid method for determining the linearity of a flow cytometer amplification system. Cytometry 10, 689-94. 2. Chase, E. S. and Hoffman, R. A. (1998). Resolution of dimly fluorescent particles: a practical measure of fluorescence sensitivity. Cytometry 33, 267-79.

48 INITIAL RESULTS FROM NATIONAL SURVEY OF Q AND B VALUES

Eric Chase¹, Raymond Lannigan^{1 1}Cytek Development, Fremont, California

Initial measurements of Q and b values on several flow cytometers in the US are presented. The rapid method used to determine Q and b was discussed in Cytometry (Resolution of Dimly Fluorescent Particles: A Practical Measure of Fluorescence Sensitivity; E.S. Chase and Robert Hoffman; Cytometry, 33:267). Duke FC3M beads were used for the data. Sources of error in the Q and b measurements are presented: log amp nonconformity, spectral mismatch between the hard dyed beads and target dyes, filter variation from instrument to instrument, and MESF assignment of the beads themselves. Measured Q values are compared to estimates of Q values calculated from the design of flow cytometers and the dye characteristics. Estimated Q values are within a factor of 2 of observed values. Ratios of calculated Q values between FL1 and other channels were used to estimate the accuracy of PE MESF assignments by various bead manufacturers. Calculated ratios of Q are expected to be more accurate than the calculated values. Sources of the large variance of Q from instrument to instrument are discussed.

51 QUANTITATIVE CHARACTERIZATION OF CONFOCAL MICROSCOPE PERFORMANCE

Edward H. Cho¹, Stephen J. Lockett^{1 1}SAIC-Frederick, Frederick, Maryland

The confocal microscope is now in wide-spread use for quantitatively analyzing molecular pathways inside living cells, and therefore convenient characterization of its performance is essential. However, methods developed to date for evaluation confocal microscopes are generally rather time consuming to implement and it is hard to compare different instruments because they do not yield measurements in absolute terms. Thus, we built a highly stable, uniform and isotropically-emitting light source with an intensity equivalent to a dim, fluorescence-labeled cell sample for calibrating the emission light path of optical microscopes. The source consisted of a battery-driven light emitting diode placed behind a near-Lambertian diffuser and housed inside a coverslip bottomed 35 mm cell culture dish. Since the emission from the source was both uniform and isotropic, the image intensity did not vary significantly as a function of the distance from the source to the objective lens. This had the major practical advantage that the same intensity was obtained without any precise adjustment when the source was removed from and replaced on the microscope. We mathematically modeled the emission light path of laser-scanning confocal microscopes using the Poission and Gaussian distributions to represent photon loss and amplification noise respectively. The model enabled us to derive procedures for measuring the absolute photon detection efficiency, dynamic range, linearity, uniformity over the detection area, amplification noise and background noise. Analysis of light source images acquired using a very common confocal microscope model showed that 0.3% of photons emitted from a sample are recorded in the image. This was as high as can be expected given the inherent limitations of the optical components and photomultiplier tubes (PMT). As expected, image intensity was proportional to intensity from the light source and the efficiency of the confocal microscope was independent of PMT gain. However, interestingly amplification noise was proportional to gain, leading us to demonstrate that the highest dynamic range is achieved with relatively low gain and 12-bit digitization. Practical applications of the light source for checking the transmission of optical components in the emission light path were tested by exchanging different components (e.g. objective lens, emission filters, dichroic mirrors) in the emission light path. Funded by NCI Contract NO1-CO-12400.

52 MEASUREMENT OF STABLITY AND PRECISION OF LIGHT DETECTION IN OPTICAL MICROSCOPY

Tytus Bernas¹, Elikplimi KWAKU Asem², J. Paul Robinson³, Bartlomiej Rajwa^{4 1}Purdue University, Bindley Bioscience Center, West Lafayette, Indiana; ²Indiana University, Pharmacology, School of Medicine, West Lafayette, Indiana; ³Purdue University, Basic Medical Sciences & Biomedical Engineering, West Lafayette, Indiana; ⁴Purdue University, Basic Medical Sciences,Veterinary Medicine, West Lafayette, Indiana

Currently available techniques of calibration and standardization of optical microscopes provide estimation of stability and measurement precision (noise) of an imaging system at single level of signal. In addition only the total noise level, but not its characteristics (spectrum), is measured. We propose a novel technique for estimation of time variability of signal and noise in microscopic imaging. The method requires registration of a time series of images of any stationary microscope specimen. The analysis is multi-step process, which separates monotonic, periodic and random components of pixel intensity change in time. The technique allows simultaneous determination of dark, photonic and multiplicative components of noise. Consequently, confidence interval (noise) is obtained for each level of signal along with the respective confidence interval (noise). The proposed algorithm can be applied to detect mechanical instability of a microscope and instability of illumination source. In addition, photobleaching kinetics may be characterized at each level of fluorescence intensity. The technique is validated using datasets of biological images with known signal and noise characteristics. The method is then applied to assess performance of photomultipliers in a confocal microscope and CCD cameras in a wide-field microscope.

Advanced Microscopy and Image Acquisition 2

53 IMPROVING SINGLE MOLECULE FÖRSTER RESONANT ENERGY TRANSFER MEASUREMENTS BY PULSED INTERLEAVED EXCITATION AND FLUORESCENCE CORRELATION SPECTROSCOPY

Steffen Rüttinger¹, Benedikt Kraemer², Martin Roos³, Eberhard Hildt³, Felix Koberling², Rainer Macdonald^{1 1}Physikalisch-Technische Bundesanstalt, Berlin, Germany; ²PicoQuant, Berlin, Germany; ³Robert Koch-Institut, Berlin, Germany

Förster Resonant Energy Transfer (FRET) is a well-known effect with first applications as nanometric spectroscopic ruler dating back to 1978 [1]. With the recent advances in sensitive fluorescence detection techniques single pair FRET (spFRET) is used to detect e.g. co-localization of molecules or to measure conformational changes on a single molecules. However, in spite of the strong distance dependency of the energy transfer efficiency quantitative results are difficult to obtain and FRET experiments in solutions are generally interpreted qualitatively. One problem is the so called zero efficiency peak [2] caused by FRET pairs with missing or non fluorescent acceptor [3]. Furthermore, the analysis is hampered by the presence of crosstalk due to imperfect spectral filtering, direct excitation of the acceptor as well as not directly measurable excitation and quantum efficiencies of the fluorophores and sensitivities of both detection channels To identify molecules diffusing across the interaction volume and exhibiting non-fluorescent or missing absorbing dye, we applied (dual color) pulsed interleaved excitation for FRET measurements [4] (PIE-FRET). Combined with time correlated single photon counting (TCSPC), the presence of a fluorescing acceptor is detected without relying on the occurrence of energy transfer. Contributions originating from the zero efficiency peak can be identified unambiguously and subsequently eliminated to obtain meaningful FRET-histograms. Since direct acceptor excitation, molecular brightness of donor and acceptor fluorophores and crosstalk or leakage is determined by analyzing the same data-set with FCS, all quantities required to interpret FRET-measurements correctly are available. To demonstrate the advantages of our approach, we investigated a poly-proline assay labeled with Alexa 555 and Alexa 647 as donor and acceptor, respectively. [1] L. Stryer., Ann. Rev. Biochem, 47:819–846, 1978. [2] A. Deniz et al., Proc. Natl. Acad. Sci. USA Biophysics, 96:3670–3675, 1999. [3] B. Schuler et al., Nature, 419:743–747, 2002;

A. Dietrich et al., Reviews in Molecular Biotechnology, 82:211–231, 2002; S. Weiss, Science, 283:1676-1683, 1999. [4] D. Lamb, Picoquant, 10th International Workshop 2004.

54 EXTENDED DEPTH OF FIELD IMAGING WITH THE IMAGESTREAM EDF IMAGING FLOW CYTOMETER SYSTEM

William Ortyn¹, David Basiji¹, Keith Frost¹, Richard Bauer¹, Richard Esposito¹, Cathleen Zimmerman¹, David Perry¹, Philip Morrissey¹, Thaddeus George¹, Brian Hall^{1 1}Amnis Corp., Seattle, Washington

Confocal microscopy provides the ability to synthesize an image of a cell from multiple focal planes bringing all features simultaneously into focus. This capability is desirable for a wide range of cell analysis applications including co-localization studies, quantifying the translocation of molecules between cellular compartments and the enumeration of FISH probes randomly located in a nucleus. In this presentation we disclose a technique to perform high speed, extended depth of field (EDF) fluorescence imaging in flow, whereby, imagery from thousands of cells is collected in less than a minute with the entire cell simultaneously in focus. We provide a theoretical treatment of the underlying mechanism and show the discrete steps in the process of generating EDF imagery. We demonstrate the effectiveness of the methodology by comparing large focus pans of several thousand beads collected with the standard ImageStream and the ImageStream EDF systems. Visual observation of both standard and EDF collection modes shows that the EDF method maintains good focus while the standard collection mode exhibits substantial blurring. We then analyze photometric and morphological bead parameters to quantitatively assess the benefits of the method. Finally, we apply the method to the enumeration of FISH probes in a comparative study. We show EDF and non-EDF cell imagery to visually demonstrate the benefits of the method and then apply an automated classifier to both image sets demonstrating significant increase in the efficacy of chromosome enumeration using the EDF imagery.

55 ABOUT A NOVEL LIGHT MICROSCOPE ARCHITECTURE

Rainer Uhl^{1 1}Ludwig-Maximilians-Universitat Munchen, BioImaging Center, Martinsried, Germany

Human Vision comprises a sequence of complex interactions between eye and brain. The microscope extends this domain into the microcosm. While conventional light microscopes are a mere extension of the optical apparatus of the eye, modern imaging microscopes can assume many more functions usually associated with the retinal part of the eye and the brain itself. They thus comprise the front-end of a computer-based intelligence and turn the microscope into a quantitative measuring device. We will present a novel light microscope architecture (iMIC), which aims at the highest possible degree of automation and high throughput. In sharing this goal with other approaches, the iMIC isn´t restricted to the most fundamental imaging techniques, instead it constitutes a seamless integration of all conceivable high end imaging and sample manipulation techniques into a single, highly versatile instrument. These include: • Time-lapse studies with genuine real-time performance; • FRET measurements at two freely selectable emission wavelengths; • TIRF measurements with dynamically variable TIRF-

angle; • FRAP or other techniques (e.g. laser microdissection, optical tweezers) requiring the positioning of a laser beam in the object field; • Widefield, structured illumination or slit-scan confocal measurements with incoherent illumination at any freely selectable wavelength between 340 and 680 nm. • Multispectral confocal laser-scanning with fully digital scan-control and close to theoretical sensitivity, and • Multiphoton excitation experiments with maximal photon collection efficiency.

56 CELL AND TISSUE RELATED SCANNING FLUORESCENT VIRTUAL MICROSCOPY USING A DEDICATED DESK TOP SCANNER SYSTEM

Bela Molnar¹, Viktor SEBESTYEN Varga², Attila Tagscherer³, Tibor Virag⁴, Viktor Kamaras⁴, Zsolt Tulassay^{2 1}Semmelweis University II, Budapest, Hungary; ²Semmelweis University, II.Dept. of Medicine, Cell Analysis Lab, Budapest, Hungary; ³Semmelweis University of Medicine, Budapest, Hungary; ⁴3DHISTECH LTD, Budapest, Hungary

Background and aims : Scanning fluorescent microscopy on traditional microscopes are slow, inconvenient and hardly limited in the automation opportunities. We aimed to develop a desk top size fluorescent slide scanner for slide loading and identification (upto 10 slides), including epifluorescent illumination with enhanced filter numbers (8), with increased fluorescent light source life time and and a modular construction for application specific camera selection. Methods:In a public.private partnership (Semmelweis Uni.- 3DHISTECH LTD., Budapest, Hungary) a desk top, slide loading and scanner mechanics was developed incorporating traditional optical elements from a high quality microscope ( Axioplan 2 Imaging, Carl Zeiss, Germany) without any ocular. The system performance was tested from the following point of views: automated slide loading, slide identification, region of interest determinations, focusing, multichannel slide scanning. The system´ fluorescent quantitative calibration was done with the X-cyte fluorescent light source without and with a special bead based algorythm or using fluorescent slides( FITC, Rhodaminm Hoechst). The linearity and quantitative accuracy was tested using fluorescent beads. Routine software applications in cytology and histology were developed towards pseudocoloured visualisation, counting, TMA in a virtual microscopy environment. Results: Systems mechanics was robust to load and identify the barcode labelled slide set (1-10). The systems linearity was proven in the range of 1 to the 106. The CV of the measured PI labelled lymphocyte slide was 4.3%, CV of the bead measurements was below the given exclusion criterias using the fluorescent slide based white field compensation, only. Multichannel , pseudocolorised cytology and histology virtual microscopy was easily applied for visual analysis. Transmitted and fluorescent TMA evaluation was successfully implemented. Conclusions: Developing a desktop fluorescent slide scanner and sophisticated virtual microscopy programms the traditional microscopy based scanning fluorescent microscopy could be enhanced for everday use in fluorescent cellular and histology research.

Image Processing and Analysis 2

61 IMAGE CYTOMETRY PROFILING OF CELL CYCLE PHENOTYPES IN GENOME-WIDE SIRNA KNOCKDOWN CELLS

Yan Feng¹, Jonathan Hoyt², Yong-Chuan Tao², Timothy Mitchison^{3 1}Novartis Institute for Biomedical Research, Genome and Proteome Sciences, Cambridge, Massachusetts; ²Cambridge, Massachusetts; ³Harvard Medical School, Systems Biology, Boston, Massachusetts

We used a multi-parameter imaging cytometry based assay and genome-wide siRNA knockdown to characterize genes and pathways that regulate cell cycle progression. A series of statistical methods, including supervised and unsupervised classification, and genome-wide screen normalization, were used to identify cell cycle stage of each individual cell and generate a profile for each gene knockdown. A series of tests were used to identify potential false-negative or off-target effects. System level analysis was then performed by putting the cell cycle profile of each gene knockdown into functional genomics context. Implication of new cancer drug targets will also be discussed.

62 IMAGE ANALYSIS FOR STRUCTURAL GENOMICS REVEALS NOVEL PROCESSES IN TUMOR PROGRESSION AND APOPTOSIS

Bart J. Vermolen¹, Ian Theodore Young¹, Sabine Mai², Sherif Louis², Vered Raz³, Yuval Garini^{1 1}Delft University of Technology, Delft, Netherlands; ²University of Manitoba, Winnipeg, Manitoba, Canada; ³Leiden, Zuid-Holland, Netherlands

Major success has been achieved in recent years in understanding the function of the genome. It also became evident that the structure and organization of the genome in the nucleus is important, it changes along the cell cycle and during cancer progression. For structural and organizational studies to take place, it is required to apply markers, probe regions of interest (wetware), and acquire images (hardware), tasks that have reached a mature state. For successful interpretation, a last step has to be made: analysis of the images (software). To obtain quantitative results, the first two components (wetware and hardware) have to be taken into account in the software and algorithms. We studied the spatial organization of telomeres and centromeres. Telomeres were studied during the cell cycle of normal mammalian cells and after c-Myc deregulation and centromeres were studied in normal, senescent and apoptotic human mesenchymal stem cells. We will present the algorithms that were developed and some of the results. Novel processes have been discovered, including the cell-cycle dependence of the telomeres, telomere aggregates during tumor progression and the redistribution of centromeres during apoptosis.

63 MICRONUCLEI FORMATION, MORE THAN MEETS THE EYE? A MULTIPARAMETRIC HIGH CONTENT ANALYSIS STUDY USING LASER SCANNING CYTOMETRY

Raffi Manoukian¹, Satin Sawant¹, Gloria Juan^{1 1}Amgen, Inc., Thousand Oaks, California

Genotoxicity leading to chromosomal damage during cell division is an important consideration when screening for novel small molecule compounds in vitro or for monitoring their effects in vivo. Chromosomal mutation not only leads to a genotoxic and cytotoxic state but also may play an important role in carcinogenesis. Under such conditions, chromosome fragments or lagging whole chromosomes accumulate in the cytoplasm in anaphase, as they are unable to reach the spindle poles during mitosis. These residual minute chromatin masses, which are adjacent to the larger main nucleus, are called micronuclei (MN). The presence of micronuclei in cells can thus be used as a means to quantitate chromosome damage. Recent work has shown that High Content Analysis (HCA) MN assays allow for a more automated, objective, and faster approach, and yet these and other studies were limited to a single cell line. Other groups have shown that using one cell line as a standard for MN could be misleading, as it seems that onset and severity of micronucleation vary greatly between lineages. In this study we examine multiple and more clinically relevant cell lines as well as white blood cells from the peripheral blood in an attempt to not only enumerate MN formation but to correlate it to multiple endpoints such as cell cycle modulation, DNA synthesis inhibition and apoptosis. This multiparametric approach can be achieved by performing high content analysis using laser scanning cytometry and quantitative imaging as the investigative platform. We demonstrate that HCA can extract more indepht information, resulting in a correlative matrix of MN formation, cell lineages and functional endpoints.

64 HISTONE HYPERACETYLATION CAUSES REPOSITIONING OF CENTROMERES AND TOPOLOGICAL REORGANISATION OF CHROMATIN IN HUMAN PROSTATE CANCER

Vicky Kyle², Perry Maxwell³, Peter Hamilton^{3 1}Queen’s University Belfast, Centre for Cancer Research and Cell Biology, Biomedical Imaging and Informatics, Belfast, Northern Ireland, United Kingdom; ²Napier University, Edinburgh, Biomedical Science, Edinburgh, Scotland, United Kingdom; ³Queen’s University Belfast, Pathology, Belfast, Northern Ireland, United Kingdom

Quantitative changes in nuclear chromatin and epigenetic status occur in prostatic neoplastic progression, suggesting a functional role for nuclear topology in tumour development. In vitro studies on normal and malignant prostate cell lines highlighted quantitative differences in the chromatin organisation of G0 phase nuclei using high resolution texture analysis. The basis for changes in chromatin phenotype in prostate cancer are unknown, however, we expect that histone acetylation may play a central role. The aim of this study was to explore the impact of Trichostatin A (TSA) induced histone hyperacetlyation on centromeric topology and chromatin organisation in prostate cancer cells. Prostate cancer cell lines LNCaP, DU145 and PNT1A were grown onto glass slides and treated with 0, 12 and 100 ng/ml TSA for 24 hours. High resolution computerised texture analysis was used to measure changes in chromatin phenotype. Centromeric FISH was carried out for chromosome 11 using a biotin labelled probe, labelled with an FITC anti biotin secondary antibody and the nuclei counterstained with propidium iodide. 2D measurements of intranuclear centromere position were made using Leica QWin. 3D Confocal microscopy was used to obtain serial optical sections of nuclei approximately 0.12µm apart. Serial sections were reconstructed using Analyze software (AnalyzeDirect) to reconstruct and render 3D composite images and a variety of measurements were made. It was shown that induced histone hyperacetylation by low dose TSA resulted in increased chromatin density and chromatin reorganisation. Untreated LNCaP nuclei show distinct centromeric topologies using 3-D FISH. The PNT1A and DU145 cell lines were most similar where the volume for centromeres was similar within and between nuclei. In trisomic nuclei, two of the centromeres were the same volume and one was approximately twice that size. The largest centromere was positioned equidistant from the two smaller sized centromeres and was closer to the centre of the nucleus. 2-D image analysis showed that increased chromatin density, was associated with concomitant rearrangement of chromosome 11 centromeres. In general, the centromeres separated, one moving to a central position while the other moved to a more peripheral location. Interestingly, when a third centromere was present, this did not impact the distance between the smaller centromeres, nor did its position change. The study of nuclear topology provides important insights into tumour development and the impact of epigenetic modifiers such as TSA on nuclear phenotype. In prostate cancer, cells show a unique centromeric arrangement for chromosome 11 which is modified following low doses of TSA which alter the global histone acetylation status of the genome.

Flow Instrumentation 2

Consequently, we have demonstrated single microfluidic switch sorting rates at 2000 sorts per second. Overall sample throughput and sort rates can be further increased through parallel implementation of this technology in which 10 to 100 microfluidic sorters are fabricated on a single chip. This paper discusses reliable sorting of human rare cell populations using a scalable, closed, high-throughput optical sorting system.

69 DESIGN OF A HIGH-THROUGHPUT BIOMEMS MICROFLUIDIC CYTOMETER/SORTER

James F. Leary¹, Rashid Bashir^{2 1}Purdue University, Basic Medical Sciences & Biomedical Engineering, West Lafayette, Indiana; ²Purdue University, Electrical/ Computer Engineering & Biomedical Engineering, Engineering, West Lafayette, Indiana

BioMEMS (Bio MicroElectroMechanical Systems) microfabrication technology is being designed, constructed, and tested for applications in genomics, proteomics, and drug delivery. A current limitation of BioMEMS microfluidic technology is overall cell throughput rates. A multidisciplinary team from our two laboratories is designing a novel, small, portable, high-throughput (> 100,000 cells/sec) exponentially staging system that can be used for these and other applications. The overall preliminary design (US patent filed and pending) consists of a disposable microfluidic chip of branching tree architecture for parallel and multi-stage processing. The system does not need to handle single cells in its initial fluidic stages. However, by the third or fourth fluidic stage, single cells can be sorted depending on the overall throughput rates and initial cell concentration. It is a closed system, thus eliminating biohazardous aerosols, and also facilitating sterility. The fluidic portion of the system (PDA-sized) can be placed, if desired, in a biohazard hood. The microfluidic chip is made of optically semi-transparent PDMS ((poly)dimethylsiloxane)), with built-in microfabricated, elastomeric valves. The excitation sources are small, inexpensive, super-luminescent LEDs (light emitting diodes) requiring only battery-level power. Photodetectors consist of avalanche photodiodes (APDs). The LEDs and APDs are arranged in patterns corresponding to the physical structure of the removable PDMS microfluidic chip which is placed between them. The present data acquisition system uses a National Instruments PXI data acquisition system with embedded controller running Real-Time LabView version 7.1 and 16-bit multifunction DAQ modules for digital signal processing and sort logic control. Preliminary data from this BioMEMS system will be presented as well as a discussion of the problems and promises of these new microfabricated approaches to flow cytometry and cell sorting.

70 A HIGHLY AUTOMATED PARALLEL SORTING (HAPS) SYSTEM THAT PROCESSES 2.5 X 109 CELLS PER HOUR UNDER THE CONTROL OF A SINGLE OPERATOR

Gary Durack¹, Paul Weiss^{1 1}iCyt Visionary Bioscience, Champaign, Illinois

Highly Automated Parallel Sorting (HAPS) can now be accomplished using a new type of cell sorting system that can process many times what is possible with a traditional FACS device. We demonstrated a HAPS configuration that can process 2.5 x 109 cells per hour. The system consisted of 16 HAPS channels, each operated with a throughput of approximately 50,000 cells per second. Each HAPS channel generated droplets at 80 KHz yielding a total system droplet output of 1.26 MHz. The HAPS system was controlled by a single operator through a graphical user interface (GUI). The HAPS system was used to measure, classify and sort 1.2 x 1010 cells over a four hour period. Recovery, purity and efficiency of HAPS were compared to traditional FACS which could classify and sort 4 x 108 cells over the same period. The HAPS system is scalable and can be expanded to achieve throughputs of several millions of cells per second.

Cell Physiology 2

71 FLOW CYTOMETRIC MEASUREMENTS OF OXIDATIVE STRESS IN HEMOGLOBINOPATHIES

Fibach Eitan¹, Johnny Amer^{2 1}Hadassah University Hospital, Jerusalem, , Israel; ²Hadassah University Hospital Jerusalem, Hematology, Jerusalem, , Israel

Oxidative stress represents an imbalance between oxidants and antioxidants. Oxidants include Reactive Oxygen Species (ROS), unstable reactive free-radicals possessing an unpaired electron, which can oxidize various molecules leading to cell damage. They are produced continuously in cells as by-product of metabolism and can be increased by environmental factors and pathological conditions. The antioxidants include reduced glutathione (GSH) - the most potent cellular ROS scavenger. Hemoglobinopathies are inherited disorders which result from a decreased synthesis of hemoglobin in thalassemia, or synthesis of an abnormal hemoglobin in sickle cell anemia (SCA). Although the primary lesion is in the globin genes, the clinical symptoms of these diseases could be mediated by oxidative stress. We developed flow cytometric techniques to measure multiple aspects of oxidative stress in various blood cells. ROS generation was measured by staining the cells with 2´-7-dichlorofluorescein; GSH – by staining with mercury orange; membrane lipid peroxidation – with fluor-DHPE and externalization of phosphatidyl serine (PS) moieties, a marker of membrane damage, by fluorochrome-conjugated Annexin-V. Specific sub-populations were gated based on their forward and side light scatter as well as by staining with antibodies to lineage-specific surface antigens, glycophorin A for RBC, CD61 for platelets and CD15 for neutrophils. Thus, various oxidative stress parameters could be assigned to each cell type. Our data indicate that cells derived from patients with beta-thalassemia or SCA have higher levels of ROS, PS and lipid peroxidation and lower levels of GSH compared with their normal counterparts. The results suggest that hemolysis, the major clinical feature, as well as thromboembolic complications and recurrent infections, which frequently occur in these diseases, could be the result of oxidative stress in the RBC, platelets and neutrophils, respectively, of these patients. Antioxidants, such as N-acetyl cysteine, vitamin C and vitamin E, reduced the oxidative stress of these cells and protect them from its deleterious consequences. This was observed by treating blood cells in vitro as well as in vivo – in a mouse model of beta-thalassemia and in patients treated with antioxidants. The results suggest that flow cytometry, a standard technology in most hematological labs, can be useful for measuring the oxidative status of various blood cells and for studying the effects of antioxidants or iron-chelators in patients with thalassemia and SCA as well as in other diseases where oxidative stress is involved in their clinical symptoms.

72 ESSENTIAL REQUIREMENT OF REDUCED GLUTATHIONE FOR THE ANTI-OXIDANT EFFECT OF THE FLAVONOID QUERCETIN

Roberta Ferraresi¹, Erika Roat¹, Leonarda Troiano¹, Enrico Lugli¹, Chiara Giovenzana¹, Maria Garcia Fernandez², Elisa Nemes¹, Milena Nasi¹, Marcello Pinti¹, Andrea Cossarizza^{1 1}University of Modena and Reggio Emilia, Dept. of Biomedical Sciences, Modena, Italy; ²University of Malaga, Dept. of Physiology, Malaga, Spain

Quercetin (3,3´,4´,5,7-pentahydroxyflavone, QU) is one of the most abundant dietary and frequently studied flavonoids, molecules with a variety of effects, including those patho-preventive, anti-oxidant, antiallergenic, anti-inflammatory, anti-proliferative and anti-viral. Qu shows an apparent opposite double action: in different models, it acts at the same time as a pro- and anti-oxidant. The mechanisms of these effects have not yet been completely clarified. We have studied by polychromatic functional flow cytometry the anti- or pro-oxidant effects of Qu. The U937 human cell line was treated with Qu 10, 50 and 100 uM for different periods of time, up to 24 hours. In living cells, we evaluated simultaneously several parameters, including hydrogen peroxide content by 2,7-dicholorodihydrofluorescein diacetate, superoxide anion content by hydroethidine, reduced glutathione (GSH) content by monobromobimane, mitochondrial membrane potential (MMP) by JC1, DNA content by Hoechst 33342, early/late apoptosis by phosphatidylserine exposure on the outer face of the plasma membrane by Annexin-V Alexa Fluor 647 and cell viability by Propidium Iodide. A 16-parameters CyFlow ML (Partec GmbH, Muenster, Germany), equipped with a blue solid state laser (488 nm, 200 mW), a UV Mercury lamp HBO (100 long life, 100 W), a red diode laser (635 nm, 25 mW), a green solid state laser (532 nm, 50 mW) and a CCD camera was used. For short periods of treatment, Qu exerted an anti-oxidant effect (decrease in hydrogen peroxide levels), whereas for long periods it showed a pro-oxidant activity (increase in superoxide anion), simultaneous loss in GSH content, depolarization of MMP and apoptosis. Thus, it appears that prolonged/strong treatments with Qu induce depletion of intracellular GSH stores, so that oxygen free radicals are no longer scavenged and the pro-oxidant effect prevails on the anti-oxidant effect. In conclusion, Qu exhibits a double action, anti- and pro-oxidant, which seems to depend on the cell oxidative balance and GSH content. Probably, when anti-oxidant system becomes inefficient, the consequent overproduction of oxygen radicals alters the cell redox state, activating the apoptotic program. The polychromatic flow cytometric approach, which has been used to obtain these data, is likely the best technology for understanding and better clarifying at the single cell level the complex relationship among anti-proliferative, pro-apoptotic, anti- and pro-oxidant effect of Qu as well as of other molecules.

SIMULTANEOUS ANALYSIS OF MULTIPLE CASPASE ACTIVITIES IN MOUSE T LYMPHOCYTES BY FLOW CYTOMETRY

William G. Telford¹, Beverly Z. Packard², Akira Komoriya^{2 1}National Institutes of Health (NIH), Experimental Transplantation and Immunology Branch, National Cancer Institute (NCI), Bethesda, Maryland; ²Oncoimmunin, Inc., Gaithersburg, Maryland

Caspases play a critical role as both signaling molecules and effectors in apoptosis. Cell-permeable fluorogenic caspase substrates have played an important role in identifying the activation of these proteases in apoptotic cells; however, fluorochrome and instrument limitations have generally limited their use to the detection of a single caspase. In this study, we have employed a series of PhiPhiLux fluorogenic caspase substrates (developed by Oncoimmunin, Inc.) conjugated to fluorescein, rhodamine- and Cy5-like fluorochromes for simultaneous analysis of multiple caspase activities. Mouse T lymphoma cell lines were simultaneously labeled with fluorescein-, rhodamine- and Cy5-like substrates specific for caspase 1, 3 and 8, and were analyzed on a flow cytometer equipped with a DPSS 561 nm laser capable of simultaneous rhodamine and Cy5 excitation and detection. This laser gave excellent excitation of rhodamine and adequate excitation of Cy5 while not causing laser light contamination in the fluorescein detector range of conventional flow cytometers, allowing simultaneous analysis of all three fluorochrome-linked substrates. Activation of caspase 1, 3 and 8 could therefore be simultaneously detected, allowing the activation order of multiple protease activities in early apoptotic cells to be assessed. In murine T lymphoma cells, caspase 8 activation was found to precede caspase 3 (as expected from previous biochemical data) and caspase 1 activated simultaneously with caspase 8. This technique allowed simultaneous temporal mapping of multiple caspase activities in live cells, information hitherto available only from in vitro biochemical studies.

Rare Event Detection and Stem Cell Technologies

77 DETECTION OF MIGRATING FIBROBLASTS IN THE PERIPHERAL BLOOD BY SLIDE BASED CYTOMETRY

Ulrich Sack¹, Christian Eimermacher¹, Anja Mittag², Attila TáRnok^{2 1}University of Leipzig, Institute for Clinical Immunology and Transfusdion Medicine, Leipzig, Saxony, Germany; ²Cardiac Center, University of Leipzig, Research Laboratory, Pediatric Cardiology, Leipzig, Saxony, Germany

Rheumatoid arthritis is an inflammatory, chronically proceeding event, in which aberrant fibroblasts play a crucial role in the pathogenesis and development of the disease. Our group has recently shown that fibroblasts isolated from synovial tissue are potential inductors of rheumatoid arthritis in SCID-Mice. Furthermore, fibroblasts transfected with interleukin (IL-11, IL-15) have been recovered in joints other than the one they were injected into, but notably not in other organs. This and the fact, that rheumatoid arthritis adversely affects several joints consecutively, leads to the hypothesis that a migration of fibroblasts in the peripheral blood occurs. We therefore developed a method to isolate fibroblasts from peripheral blood through means of magnetic beads (MACS) labelled with a fibroblast-specific antibody (Mab FibAS02), using an immunomagnetic cell seperation unit (autoMACS). Because of the rarity of fibroblasts in peripheral blood samples, the cytometric detection of these cells should be confirmed by cytological methods, in particular, through use of a Laser Scanning Cytometer (LSC). In contrast to a conventional Flow Cytometer the technology of the LSC allows a multi-parametric measurement of adherent cells, including a relocalization and optical evaluation, and therefore a morphological assessment of the cells of interest. Thus, it is possible to detect even very rare cells and, if necessary, to separate them from related populations.

78 PRACTICAL STRATEGIES FOR SUCCESSFUL RARE EVENT DETECTION AND ISOLATION OF HUMAN HEMATOPOIETIC STEM CELL POPULATIONS WITH FLOW CYTOMETRIC INSTRUMENTATION

Lora W Barsky¹, Ewa Zielinska¹, Mary A Price², Kimberly J Payne¹, Yanjia Zhang¹, Qian-Lin Hao¹, Yuhua Zhu¹, Yasmin K Parrish¹, Kenneth I Weinberg¹, Gay M Crooks^{1 1}Children’s Hospital of Los Angeles, Research Immunology, BMT and GISCT Program, University Southern California, Los Angeles, California; ²Barrow Neurological Institute, Neuroimmunology Research Laboratory, Phoenix,Arizona

Flow Cytometry (FCM) has been used to detect, characterize, and isolate rare cell populations for twenty years. The availability of sophisticated multi-parameter FCM has been pivotal for Stem Cell Biology, allowing the identification and isolation of rare cell types for further analysis. Technological improvements to flow cytometer instrumentation, such as high speed and digital electronics, have further facilitated rapid progress in the field. Nevertheless, isolation of rare hematopoietic stem cells (HSC) and committed progenitors remains a daunting task for many laboratories. The cell surface marker CD34 is present on 1% of human bone marrow and cord blood mononuclear cells and its use in FCM and magnetic bead separation has proved very useful as an initial enrichment step for primitive hematopoietic cells. However, the CD34⁺ population is functionally heterogeneous as it contains HSC and different types of lineage committed progenitors such as “Common Lymphoid Progenitors” (CLP). The numerous mature progenitors within the CD34⁺ population express the CD38 antigen. However, HSC are found in the 1-5% of the CD34⁺ cells that lack the CD38 antigen (i.e. CD34⁺CD38-). This CD34⁺CD38- population can be further defined based on CD7 expression. HSC (defined as CD34⁺CD38-lin-CD7-) are the majority of the CD34⁺CD38- population (and therefore 1 to 5 cells in 10,000 mononuclear cells) whereas, CLP (defined as CD34⁺CD38-lin-CD7⁺) are 5-10% of CD34⁺CD38- cells in cord blood (and therefore <1 in 100,000 mononuclear cells). Meaningful results from functional assays on HSC and CLP subsets require high cell purity (>99%), which is a particular challenge considering the initial frequencies of these cells range from 0.01% to as low as 0.0005% respectively. To achieve the high purity needed for sensitive functional and PCR assays, our laboratory protocol requires a series of enrichment steps. Our laboratory pre-enriches for CD34⁺ progenitors by magnetic bead separation prior to FCM sorting. For FCM sorting, we stain to utilize one or more “dump channels” to remove committed, lineage positive events from the negative non-committed progenitors, which are further characterized by specific HSC or CLP markers with remaining fluorescent parameters. Ultimately, sequential FCM sorting steps are performed to ensure pure populations. Here we present a discussion of the practical techniques used for successful detection and isolation of rare and ultra rare populations from the CD34⁺ compartment with state of the art instrumentation and software.

79 INHERENT LIMITATIONS IN RARE CELL DETECTION

Arjan Tibbe¹, Craig Miller², Leon Terstappen^{2 1}Immunicon Europe Inc., Enschede, Netherlands; ²Immunicon Corporation, Huntingdon Valley, Pennsylvania

Background. Circulating tumor cells (CTCs) in patients with carcinomas are extremely rare. In metastatic breast cancer, the presence of >5 CTCs in 7.5mL of blood has been associated with short survival. As this threshold has clinical implications, it is important to recognize the limitations associated with the detection and enumeration of CTCs. Methods. Poisson statistics was used to determine the probability of collecting CTCs from a patient in a certain blood volume as a function of the number of cells in its body. The statistical parameters for sample processing and final image analysis were empirically determined. All statistical parameters associated which each step of the process, from blood collection to final image analysis and CTC enumeration, were implemented into a model. Using the model a statistical analysis was performed on data generated from a multi-center clinical trial that utilized the CellSearch^TM System to isolate and enumerate CTCs in 7.5mL blood samples. Results. The theoretical limit of detection is determined by the probability of collection a CTC in a 7.5 ml blood sample acquired from the patient. The blood volume that needs to be acquired to collect at least one CTC increases very rapidly to volumes that are too large to acquire without patient suffering. Using the CellSearch^TM System we were able to collect and identify at least one CTC in a single 7.5 ml blood sample in 35% of the samples, if the total number of CTCs in the patient´s circulation system was 1000 CTCs and assuming a total blood volume of 5 liters. Using the model the reader was identified as the most critical step in the detection of CTCs. The reader will on average detect 1 CTC in 11% and 2 CTCs in 1% of the cases if no CTCs are present in the blood sample. In case the reader is error free the threshold level can be lowered to 1 CTC. This would mean that the presence of any CTC in the blood would be an identifier for a bad prognosis. Conclusions. Ultra low detection limits are only possible with a highly standardized and automated protocol as is used in the CellSearch^TM System. With this system it is possible to detect a single CTC in 7.5 ml of blood. The amount of blood that can be acquired from the patient is the limiting factor in reaching lower limits of detection than currently achieved by the CellSearch^TM System.

Image processing and Analysis 3

83 DETECTION AND ASSESSMENT OF CERVICAL INTRAEPITHELIAL NEOPLASIA (CIN) LESIONS BY DNA IMAGE CYTOMETRY

Sun Xiaorong^{1 1}Canada BC Cancer Agency;Wuhan Landing Early Cancer Detecting Center, Wuhan, Hubei, China

Objectives: To compare detection and prognostic assessment of CIN lesions between conventional cytology and DNA image cytometry (DNAICM) assisted cytology. Methods: The study enrolled 87 women. Cervical samples were collected employing cervix brushes which were then washed in SedFix. Two slides were prepared from each case: one slide was stained by Papanicolaou stain for conventional cytology, while the other slide was stained by Feulgen-Thionin method for measurements of the amount of DNA in the cell nuclei using an automated DNA imaging cytometer. Results: Of 87 cases 30 were called normal by conventional cytology. Of the total of 20 ASCUS cases called by conventional cytology, no CIN2 or greater lesions were found in the 7 cases that did not contain any cells with DNA amount greater than 5c, while CIN2 lesions were found in 11 out of 13 cases that had some aneuploid cells with DNA amount greater than 5c. Of 30 LSIL cases called by conventional cytology, CIN2 lesions were detected in 3 out of 7 cases that did not contain any aneuploid cells with DNA greater than 5c and in 22 out of 23 cases that contained aneuploid cells with DNA amount greater than >5c. Of the residual 7 cases that conventional cytology called HSIL, all case contained aneuploid cells containing DNA greater than 5c. If cytology were to be used to refer all cases of LSIL and HSIL to colposcopy and biopsy procedure to detect potential CIN2 or greater lesions then sensitivity, specificity, positive predictive value and negative predictive value would have been 58%, 84%, 87% and 54%, respectively. If DNA-ICM were to be used instead, and all cases having 3 or more cells with a DNA amount greater than 5c were to be referred to colposcopy and biopsy to detect potential CIN2 or greater lesions then sensitivity, specificity, positive predictive value and negative predictive value would have been 73%, 88%, 90% and 65%, respectively. We also compared Ki67 positivity in these samples and found that DNA-ICM results are comparable to this biomarker method. Conclusions This study demonstrates that DNA-ICM can be successfully used to detect significant (i.e. CIN2 or greater) lesions. It has been shown before that the presence of aneuploid cells containing DNA amount greater than 5c strongly predicts a progressing lesion that would lead to an invasive cancer unless treated. Therefore, the DNA-ICM approach provides also a prognostic assessment of CIN lesions.

84 CELL SIZE, SHAPE AND MEMBRANE ARE TARGETS OF STATIC MAGNETIC FIELDS AS DEMONSTRATED BY ELECTRON, OPTIC AND ATOMIC FORCE MICROSCOPY IN HUMAN GLIOBLASTOMA CELLS

Laura Teodori¹, Maria Cristina Albertini², Francesco Uguccioni², Elisabetta Falcieri³, Marco Rocchi⁴, Antonio Bergamaschi⁵, Andrea Magrini⁵, Raffaele Mucciato⁶, Augusto Accorsi^{2 1}ENEA - Casaccia, BIOTEC-MED, Rome, Italy; ²University of Urbino, Institute of Biochemistry G. Fornaini, Urbino, Italy; ³University of Urbino, Institute of Morphological Sciences, Urbino, Italy; ⁴University of Urbino, Istitute of Biomedical Engineering, Urbino, Italy; ⁵University of Rome ‘Tor Vergata’, Occupational Health, Rome, Italy; ⁶2M strumenti, Rome, Italy

Background. There are very few theoretical reasons which support that static magnetic fields (SMF) might cause or contribute to cancer or any other human health problems and there is very little laboratory or epidemiological evidence that connects SMF exposure and human health hazards. However, some experimental results demonstrated that SMF affect cellular structures and functions. In order to investigate the effect of exposure to increasing doses of SMF on cell morphology, human glioblastoma cells were exposed to SMF ranging between 80 and 3,000

G. Cell morphology was studied by electron, optic and atomic force microscopy. Results. Optical microscope images showed altered orientation and alignment of exposed cells as demonstrated by circular statistic analysis. TEM images of the 3,000 G treated cells revealed the presence of micronuclei and dense vacuolized cytoplasm. The modifications at 80 and 300 G were not remarkable. Scanning electron microscopy demonstrated a dose dependent cell shape and size modification as demonstrated by the roundness index. Loss of the long villi, and appearance of membrane roughness and blebs were also evident. The cellular actin distribution detected by FITC-phalloidin staining showed that SMF exposure produced actin filament contraction. The atomic force microscopy of the exposed cells demonstrated membrane reorganization and several significant dose dependent modifications such as disappearance of the ordered surface ripples and furrows typical of the unexposed cells and occurrence of surface membrane corrugation. The roughness index was also affected (p = 0.009) Conclusions. Our experimental procedures demonstrated that exposure to SMF affects size, shape orientation and membrane surface of glioblastoma cells.

85 IMAGING THE CELLULAR RESPONSE TO FLUID SHEAR BY APPLYING ULTRA-LOCALIZED FLOW FIELDS GENERATED BY ROTATING LASER-TRAPPED MICROSPHERES

Elliot Botvinick¹, Gregor Knoener², Michael W. Berns³, Halina Rubinsztein-Dunlop^{2 1}Beckman Laser Institute, Irvine, California; ²University of Queensland, Physics, Brisbane, Queensland, Australia; ³University of California, Irvine, Surgery, College of Medicine, Irvine, California

An advanced microscope system has been developed to measure spatial and temporal responses to fluid shear applied to local regions of the cell surface. The microscope is built on the RoboLase platform and incorporates fluorescent and wide filed imaging as well as Florescent Resonance Energy Transfer (FRET) measurements. The microscope also incorporates the laser-mediated tools of Fluorescence Recovery after Photobleaching (FRAP), laser ablation and laser tweezers. As a new ‘spin´ on applying fluid shear to the cell surface, spherical vaterite crystals are rotated near the cell surface to apply flow rate gradients generally confined to the length scale of the particle diameter. The crystals are held in a 3-D laser trap of circularly polarized light that applies optical torque to the crystal thus rotating it with respect to the surrounding liquid media. The optical torque can be quantified optically by measuring the polarization change of light passing through the crystal to provide a direct measure of the drag torque. Crystals a few microns in diameter can be rotated at hundreds of Hertz thus applying physiological shear stresses to confined regions of the cell surface. This mechanism of interaction allows the study of heterospatial force transduction and force-mediated cell properties. Furthermore this method provides an uncoupled control for complementary ligand coupled bead-pulling experiments, as the fluid-coupled stresses do not directly tie into the cytoskeleton, surface molecules or the cortical actin. By optically monitoring the laser power, rotation rate, and polarization change, the environment surrounding the crystal can be monitored for changes which may provide new insight as to the extent and role of surface molecules as they pertain to flow-induced function.

BioPharma Applications

89 DEVELOPMENT OF MULTILAYERED NANOPARTICLE SYSTEMS FOR NANOMEDICINE

James F. Leary¹, Tarl W Prow², Donald EUGENE Bergstrom^{3 1}Purdue University, Basic Medical Sciences, West Lafayette, Indiana; ²Johns Hopkins University, Baltimore, Maryland; ³Purdue University, Medicinal Chem & Molecular Pharmacology, Pharmacy & Pharmacal Sciences, West Lafayette, Indiana

Nanomedicine provides for a revolutionary new approach to regenerative medicine by a “bottom up” approach whereby tissues and organs are treated in parallel processing fashion at the single cell level using billions of smart, multilayered nanoparticles. Such nanoparticle drug delivery systems have immense promise for more accurate delivery of drugs with a lower rate of bystander cell mis-targeting. Such enclosed nanosystems also protect drugs from being broken down in the bloodstream, a common problem in drug delivery. By employing a multi-step process, the nanosystem can contain built-in error-checking to minimize bystander effects. Targeting can also be intracellular in order to bring a drug into close proximity of its molecular target inside single living cells. Since these nanosystems contain both diagnostic and therapeutic features, the conventional distinction between diagnostics and therapeutics is blurred. A new term “theragnostics” describes this new concept of simultaneous diagnostics and initial therapeutics. Therapeutic drugs or genes can be delivered inside single cells whereby the dose is controlled by biomolecular sensors on a cell-by-cell level. Multilayered nanoparticle systems are being constructed on a variety of core particle types (nanogold, quantum dots, paramagnetic). The nanosystems are built in a layer-by-layer assembly and are designed to disassemble layer-by-layer in reverse order in-vivo effectively creating a programmable nanoparticle system (Leary and Prow, patent pending). Nanosystems from 60 – 160 nm diameter are probably optimal for in-vivo therapeutics. Nanoparticle targeting to cells can be assessed using a combination of MACS magnetic sorting, fluorescence membrane tracking probes, fluorescence microscopy, flow cytometry and LEAP scanning cytometry/laser optoinjection. By using PCR amplifiable sequences, biodistribution of these nanoparticle systems can be studied in animals even when the nanoparticles in tissues cannot easily be located for conventional imaging techniques. The applications of this platform nanotechnology are numerous. Specific examples for cancer and antiviral treatments will be discussed.

90 CYTOMETRY AS AN AID IN DEVELOPMENT OF ANTIMICROBIAL AGENTS

Howard Shapiro¹, Nancy Perlmutter^{2 1}Howard M. Shapiro, M.D., P.C., West Newton, Massachusetts; ²Howard M. Shapiro, M.D., P.C., Allston, Massachusetts

The effects of antimicrobial agents on microorganisms are almost always assessed at the population or colony level. Modern instruments for determination of drug susceptibility typically measure increases in bacterial mass, as indicated by increasing turbidity of liquid media, or increases in products of bacterial metabolism, as indicated by reduction of tetrazolium dyes, changes in the pH or impedance of the medium, etc. Even when response to antibiotics is assessed by colony counts of treated and untreated cultures, essentially no information is obtained about changes in the physiology of individual organisms. Although modern multiparameter flow cytometry allows cell-by-cell analysis of the kinetics of changes in several physiological characteristics associated with the transition from a viable to a non-viable state, most studies of the interactions of antimicrobials and bacteria or fungi have measured only a single characteristic, typically membrane permeability or membrane potential. More recent studies, in which refined ratiometric methods were used for simultaneous measurement of both parameters, show that cell dynamics following exposure to antimicrobials may be more complex than initially expected, and that different species and strains may respond differently to different antibiotics. Although this complexity may preclude the development of simple, rapid cytometric clinical assays for drug susceptibility, it allows multiparameter cytometry to provide some otherwise unobtainable insights into mechanisms of antimicrobial action at the single cell level, usually within a few generation times after drug exposure, which facilitates work with slower growing organisms such as Helicobacter pylori and Mycobacterium tuberculosis. Cytometry also permits detection of small numbers of resistant organisms, and has pointed the way toward development of new combination therapies based on selectively increasing permeability of bacteria to otherwise nontoxic agents. Portions of this work were supported by NIH Grant AI063833.

91 DEVELOPMENT OF A PHOSPHATIDYLINOSITOL 3KINASE DRUG ACTIVITY BIOMARKER ASSAY IN PLATELETS

Rita Bowers¹, Philip Marder¹, Lisa Green¹, Andrew Faber¹, Phillip Schwier¹, Candice Horn¹, James Thomas^{1 1}Indianapolis, Indiana

The phosphatidylinositol 3-kinase (PI3K) family of enzymes plays a pivotal role in controlling proliferation, motility, and survival of cells. Recent reports have indicated that >30% of human colorectal, breast, and hepatocellular cancers display somatic mutations in the alpha isoform of PI3K. This (and other evidence) has provided a rationale for clinical development of new drugs that might block such aberrant PI3K activity. In order to bring targeted agents to market more quickly, development of selective drug activity biomarker assays has become essential for preclinical and early phase human studies. An ideal drug activity biomarker should be robust, reproducible, easily attainable, and closely linked to the molecular target of interest. Platelets contain a rich source of PI3K (including the alpha isoform) that can be activated through stimulation of protease-activated receptors (PARs) using thrombin or its derived peptide ligands. Once stimulated, this upstream PI3K activity activates platelets into a cascade of signaling events including conformational changes in the fibrinogen receptor. In this study, we used flow cytometry to measure downstream PAR-induced platelet activation as a surrogate biomarker for modulation of PI3K activity in tumors. Results described in this presentation reveal a robust (10 – 30 fold fluorescence increase) signal that was blocked by the selective PI3K inhibitors, Wortmannin (WORT) and LY294002 (LY). The assay is functional in both human and mouse systems. For human sample analysis, platelets are isolated using size exclusion chromatography and assayed in citrated plasma by activation through PAR-1 followed by reaction with PAC-1-FITC monoclonal antibody. For the mouse, platelets are activated in whole blood through PAR-4 and reacted with alexafluor-488-fibrinogen. Both human and murine systems were blocked (in vitro) by WORT and LY in a concentration dependent manner. Furthermore, mice were dosed intraperitoneally with WORT (4 mg/kg) or vehicle and citrated blood was collected after 1 hr. and tested (ex vivo) for PAR-4-induced activation. Significant inhibition of PAR-4induced platelet activation was observed in WORT treated normal and tumor-bearing mice. Results from these studies indicate that flow cytometric measurement of PAR-induced platelet activation could be a suitable drug activity biomarker assay for evaluation of putative PI3K inhibitors in the clinic.

92 QUANTITATIVE ANALYSIS OF THERAPEUTIC MONOCLONAL ANTIBODY LOCALIZATION TO ENDOSOMES AND LYSOSOMES USING THE IMAGESTREAM IMAGING FLOW CYTOMETER

Brian Hall¹, Philip Morrissey¹, Che-Leung Law², Kristine Gordon², David Lynch¹, Keith Frost¹, Thaddeus George¹, David Basiji¹, Cathleen Zimmerman¹, William Ortyn¹,

Richard Bauer¹, David J Perry¹, Richard Esposito^{1 1}Amnis Corp., Seattle, Washington; ²Seattle Genetics, Bothell, Washington

Monoclonal antibodies (mAb) have recently become clinically effective drugs for the treatment of a variety of human diseases. A number of these bind to cell surface determinants and thus are subject to cellular mechanisms of membrane clearing which include internalization into the endosomal/lysosomal degradation pathway. However, this pathway is complex and sorting to different compartments and recycling are known to exist. The efficacy of therapeutic mAb could be influenced by the rate of membrane clearing and progression to the degradative lysosmal compartment. Monoclonal antibodies used as single agents may be less effective if cleared rapidly from the cell surface since there may be less chance to fix complement or act as an opsonin. Alternatively, mAb conjugated to chemotherapeutic agents may need to access the lysosomal compartment in order to become active. One such mAb used clinically, binds to CD20 which is expressed on chronic lymphocytic leukemia cells. Interestingly, not all patients have a therapeutic response when treated with anti-CD20 (Rituximab). Conceivably, the difference may be due to the clearance and catabolism of the mAb amongst patient populations. Thus it would be of interest to determine the cellular fate of the mAb in established tumor cell lines and eventually apply this analysis to patient samples. A proof of concept study to track antibody drug conjugate trafficking has been performed utilizing the ImageStream imaging flow cytometer which acquires 6 channels of imagery simultaneously from cells in flow including bright field, dark field and 4 channels of fluorescent imagery at acquisition speeds of 300 cells per second. The Ramos Burkitt lymphoma cell line was incubated with fluorochrome labeled anti-CD20 for various times and then fixed and stained with fluorochrome labeled markers for endosomes and lysosomes. Cellular imagery was acquired and was analyzed using an algorithm that compares the similarity of the fluorescent imagery on a pixel by pixel basis between channels in a quantitative manner using the non-mean normalized Pearson´s correlation coefficient. The data demonstrated an initial capping of the anti-CD20, followed by association with the endosome marker and later with the lysosome marker. This was observed in sample sizes of 10,000 cells with a quantitative score which allows for statistical analysis. Thus this approach should be useful clinically in analyzing potential differences in intracellular fates of mAb between responder and non-responder populations.

Flow Instrumentation 3

96 MULTISPECTRAL CYTOMETY: A POWERFUL NEW TECHNOLOGY IN CELL ANALYSIS

J. Paul Robinson¹, Bartlomiej Rajwa², Kathy Rajheb³, James

T. Jones⁴, GéRald GréGori⁵, Valery P. Patsekin^{3 1}Purdue University, Basic Medical Sciences & Biomedical Engineering, West Lafayette, Indiana; ²Purdue University, Basic

Medical Sciences, Veterinary Medicine, West Lafayette, Indiana; ³Purdue University, Bindley Bioscience Center, W. Lafayette, Indiana; ⁴Purdue University, Institute-Interdisciplinary Engr Studies

- Biomed. Engr. Ctr., Engineering, West LaFayette, Indiana; ⁵Université de la Méditerranée, Marseille, Marsailes, France

We have developed a flow cytometer with the capability of collecting 32 channels of fluorescence spectra enabling a quantum leap in flow cytometry systems. Unlike previous instruments where single photomultiplier tubes collected wide bands of spectral bands, this instrument enables us to transform the entire spectrum of a particle of cell as a spectral parameter. This is a major change in technology and opens up many new possibilities including the use of multiple dyes with almost totally overlapping spectra as well as effectively removing the need for traditionally complex spectral compensation. There are many advantages of this technology that may not be obvious. The first is the vast reduction in hardware – this system utilizes a single multiplexed PMT with a single board for all the data collection electronics. Second, because we collect the entire spectrum, we can utize advanced algorithms to classify the spectrum in a variety of classes enabling us to classify cells or particles semi-automatically. Third, the opportunity now arises for very advanced databasing and multivariate analysis of data. This latter point is very important because the great majority of flow cytometry data, regardless of the number of variables collected is analyzed using univariate or bivariate techniques. When one has 32-40 variables, traditional analyses are no longer adequate. For flow cytometry to meet the 21st century demands of biotechnology and cell biology, minor advances in instrumentation will be insufficient. This presentation demonstrates the effectiveness of a truly next-generation approach to flow cytometry collection and analysis.

97 SPECTRAL ANALYSIS FLOW CYTOMETER: MODULAR INCLUSION OF HIGH RESOLUTION SPECTRAL ANALYSIS

Gregory Goddard¹, John Martin¹, Mark Naivar¹, Peter Goodwin¹, Steven Graves¹, Robert Habbersett¹, John P. Nolan¹, James Jett^{1 1}Los Alamos National Laboratory, National Flow Cytometry Resource, Los Alamos, New Mexico

Flow cytometry has established itself as an invaluable tool for general biomedical research with applications ranging from ligand-receptor studies, to high throughput screening and genotyping. While conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from a more comprehensive and flexible approach to spectral analysis including, but certainly not limited to: spectral deconvolution of overlapping emission spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, free versus bound dye resolution, and Raman spectroscopy. Sensitivity and reproducibility were investigated with a variety of dispersion methods and image sensor aspect ratios. Calibration of the prototype spectral analysis flow cytometer included wavelength characterization and calibration of the dispersive optics. Benchmarking of the initial system demonstrated a single particle/cell intensity sensitivity of 2160 MESF of R-Phycoerythrin. Subsequent improvements to the system resulted in a reduction of the detection limit to 1100 MESF of FITC. Single particle spectra taken with our instrument were validated against bulk solution fluorimeter and conventional flow cytometer measurements with coefficients of variation of integrated fluorescence intensity of standard fluorescent microspheres ranging from 2%-5%. It is demonstrated that the flow spectrometer has sufficient sensitivity and wavelength resolution to detect single cells and microspheres, including multi-fluorophore or quantum-dot labeled microspheres. The capability to use both standard mathematical deconvolution techniques for data analysis, coupled with the feasibility of integration with existing flow cytometers, will improve the accuracy and precision of ratiometric measurements, enable the analysis of more discrete emission bands within a given wavelength range, reduce electronic crosstalk, and allow more precise resolution of the relative contribution of individual fluorophores in multiply-tagged samples thereby enabling a range of new applications involving the spectral analysis of single cells and particles.This effort supported by NIH RR020064-01, and NIH RR001315-23 funding.

98 RAMAN FLOW CYTOMETRY

John P. Nolan¹, Dakota Watson¹, Daniel Gaskill¹, Mirianas Chachisvilis¹, Steven Graves², Lief Brown³, Stephen Doorn³, Hicham Fenniri^{4 1}La Jolla Bioengineering Institute, La Jolla, California; ²Los Alamos National Laboratory, Bioscience, Los Alamos, New Mexico; ³Los Alamos National Laboratory, Chemistry, Los Alamos, New Mexico; ⁴University of Alberta, Chemistry, Edmonton, Alberta, Canada

Flow cytometry is well-established as a method to analyze the optical properties of individual particles. Historically these optical properties have included fluorescence and Raleigh-type elastic light scatter. Raman scattering is an optical phenomenon that can reveal detailed information on chemical composition using relatively regions of the optical spectrum. This property makes Raman spectroscopy of interest for studies of cell physiology, as well as for microparticle-based and multiplexed assays. We have developed a flow cytometer capable of detecting and measuring Raman signals from particles in flow. The instrument features high NA collection optics coupled to a high throughput spectrograph via an optical fiber. The Raman spectra is dispersed onto an array of optical fibers coupled to individual PMTs or onto a CCD array. Nanoparticles exhibiting surface enhanced Raman scattering (SERS) can be differentiated on the basis of their spectra, demonstrating their potential as reporters or as encoding element for particle-based multiplexed analyses. We are developing this analytical platform to support highly multiplexed applications in disease diagnostics and drug discovery. The ability to make Raman spectral measurements of individual particles is an important new capability for flow based analysis and sorting.

99 FLUORESCENCE SENSITIVITY AND COMPENSATION IN MULTISPECTRAL FLOW IMAGING

David Basiji¹, William Ortyn¹, Cathleen Zimmerman¹, David Perry¹, David Coder¹, Keith Frost¹, Brian Hall¹, Thaddeus George¹, Richard Esposito¹, Richard Bauer¹, Philip Morrissey^{1 1}Amnis Corp., Seattle, Washington

The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity, multispectral imaging of cells in flow. Operation of the ImageStream system is similar to flow cytometry in that data from tens of thousands of cells can be collected in several minutes. The imagery can be used not only to quantify fluorescence intensity-based measurements similar to conventional flow cytometers, but also to measure the distribution of fluorescent signals and numerous morphologic and absorbance characteristics of each cell. These capabilities are well suited to study the influence of drug candidates and disease states on the location and movement of molecules within and between cells and the associated cell morphology. Effective analysis requires both high fluorescence sensitivity and accurate spectral compensation to properly isolate signals within cells. Here we provide a theoretical treatment of factors governing sensitivity and dynamic range in multispectral imaging and discuss their dependence on object size. We describe the compensation process which is conceptually similar to conventional flow-based compensation, but must be applied at the individual pixel level and requires the addition of several critical processing steps to eliminate the generation of artifacts. We develop an objective statistical metric for comparison of sensitivities between disparate instruments and show how alternative modes of multispectral imaging are applied to substantially improve fluorescence sensitivity. Finally, we show data collected on both the ImageStream multispectral imaging system and a recent generation 18 bit PMT-based flow cytometer to compare fluorescence sensitivity by application of the statistical metric.

Cell Physiology 3

103 COMPARISON OF FLUORESCEIN AND PHYCOERYTHRIN CONJUGATES FOR QUANTIFYING CD20 EXPRESSION ON NORMAL AND LEUKEMIC BCELLS

Gerald Marti¹, Lili Wang², Fatima Abbasi³, Adolfas Gaigalas⁴, Robert F. Vogt^{5 1}United States Food & Drug Administration CBER, Bethesda, Maryland; ²National Institute of Standards and Technology, Biochemical Science Division, Gaithersburg, Maryland; ³* CBER FDA. *Flow and Image Cytometry, Bethesda, Maryland; ⁴National Institute for Standards and Technology, Gaithersburg, Maryland; ⁵CDC, Division of Laboratory Sciences, Atlanta, Georgia

Quantitative fluorescence calibration has been advocated for the inter-laboratory data comparison and the quality control of clinical flow cytometers for more than a decade. At the present, numerous fluorescence quantification methods have been developed and instrument calibration kits have been produced. Due to the lack of follow-up support and consensus on resolving the differences with the use of these methods and materials, the quantification efforts have so far a limited clinical impact. We attempted to quantify CD20 receptor expression on B lymphocytes as a potential disease biomarker using two different and yet widely used methods. We measured CD20 expression on normal and B-CLL B-cells using both FITC and PE conjugates from the same monoclonal antibody (Mab). As a biological control and calibrator, CD4 expression on T-cells was also quantified using FITC and PE Mab. Calibration with QuantiBRITE PE-labeled microspheres and the use of unimolar PE conjugates provided direct measurements of antibody bound per cell (ABC) values for CD4 and CD20. Calibration for FITC conjugates was based on molecules of equivalent soluble fluorochrome (MESF) as determined by NIST RM 8640 microsphere standards. These MESF values were then converted to ABC values using the CD4 T-cell as a biologic calibrator to normalize FITC and PE results for CD20 expression. We demonstrated that CD4 expression on T-cells could be used as a biological calibrator to quantify CD20-FITC ABC with reasonable agreement between the two conjugates with two different fluorochromes. Issues regarding the accuracy of MESF microsphere calibrators and effective fluorochrome per protein ratios for FITC conjugates will require additional laboratory studies.

104 TRASTUZUMAB AND PERTUZUMAB DIFFERENTIALLY AFFECT HER2/NEU OVEREXPRESSING BREAST CANCER CELLS

Simone Diermeier¹, Arabel Vollmann¹, Andrea Sassen¹, Ferdinand Hofstaedter¹, Gero Brockhoff^{1 1}University of Regensburg, Institute of Pathology, Regensburg, Bavaria, Germany

Aim: Her2/neu (c-erbB2) overexpression in breast cancer indicates eligibility for Trastuzumab therapy. However, because Her2/neu overexpression alone does not assure responsiveness to this regimen there is a demand for an optimized Her2/neu based therapy. We evaluated the mechanism of action of Pertuzumab, a different antibody targeting Her2/neu, in comparison to Trastuzumab on the cellular level with respect to Her2/neu dimerization, shedding, EGFR/Her1 and Her2/neu internalization and cell proliferation in vitro. Methods: BT474 and SK-BR-3 breast cancer cell lines, both strongly overexpressing Her2/ neu, were treated with Trastuzumab or Pertuzumab. Flow cytometry was applied to investigate EGFR and Her2/neu internalization, receptor-interaction by FRET and cell proliferation. Shedding of the Her2/neu extracellular domain was evaluated by Protein Array Technology. Results: In both SK-BR-3 and BT474 cells Her2/neu homodimerization was inhibited by Pertuzumab, whilst Her2/neu homodimers seemed to be stabilized by Trastuzumab treatment. Trastuzumab seemed to inhibit cell proliferation of BT474 and SK-BR-3 more efficiently than Pertuzumab. Neither of the antibodies induces EGFR or Her2/neu internalization. Trastuzumab reduced Her2/neu-shedding in both cell lines, whereas Pertuzumab diminished it solely in SK-BR-3. Conclusion: Significant differences in the cellular mechanisms of action of Trastuzumab and Pertuzumab suggest a potential complementary antitumour effect if both antibodies are combined in clinical application.

105 APPLICATION OF RATIOMETRIC CELL ENUMERATION TO THE DEVELOPMENT OF FLOW CYTOMETRY CELL CITOTOXICITY ASSAYS

David Diaz¹, Alfredo Prieto¹, Hugo Barcenilla¹, Jorge Monserrat¹, Luis Chara¹, Julio Chevarria¹, Miguel Ángel Sánchez¹, Eduardo Reyes¹, Melchor Álvarez-Mon^{2 1}Alcala University, Immune system Diseases and Oncology Laboratory, CNB-CSIC R&D Associated Unit, Alcala de Henares, Spain; ²University Hospital “Príncipe de Asturias”, Immune system Diseases and Oncology Service, Alcala de Henares, Madrid, Spain

Traditional cell cytotoxicity assays are based on the labeling of target cells with 51 chromiun (⁵¹Cr). Target cell lysis by cytotoxic cells is estimated by measuring the gamma radiation emitted by the ⁵¹Cr released from lysed target cells and then percentages of specific lysis of the target cells can be calculated. Alternative methods based on release of fluorescent probes have been developed but to discard the excess of fluorescent probe, intensive washing is required damaging the target cells and reducing both sensitivity and reproducibility of the assay. Now, we are studying T cell cytotoxicity against autologous leukemic B-cell from patients with B-cell chronic lymphocytic leukemia (B-CCL). These resting target cells incorporate very low quantities of ⁵¹Cr in comparison with cell lines usually employed as targets in cytotoxicity assays. We tried to apply fluorometric assays to B-CLL and failed because B-CLL cells are fragile and suffered necrosis in the procedures used to wash the fluorescent probes. Therefore, we have to develop a new flow cytometry method to measure cytotoxicity. In previous works we have developed methods of cell enumeration of viable and apoptotic cells in culture and we decided to apply these methods to the enumeration of cells lysed in cell citotoxicity assays or in the context of drug toxicity. We designed a flow cytometry assay in which a fixed quantity of target cells is exposed to different concentrations of apoptogens (drugs or cytotoxic cells) and we enumerate the number of surviving B-CLL cells after culture. This method have the advantage that assays can be of longer duration than the assays based on release of radioactive or fluorescent labels. We applied this assay to test the efficacy of two in vitro methodologies to induce cytotoxicity against B-CLL cells. First we used microbeads as artificial APCs with two Abs against two T cell antigens CD3 and CD28. This procedure failed to induce cytotoxicity against B-CLL cells. It even induced helper activity that increased the survival of leukemic B-CLL cells in vitro. Second we used microbeads as a bridge between T cells and leukemic cells. The bead has Abs against a T cell antigen, CD28, and another against a B cell antigen, CD23. Anti-CD28 and anti-CD23 coated microbeads strongly stimulated the killing of the leukemic B-cells by autologous cytotoxic T cells. We have demonstrated the efficacy of these cytotoxic T cells to kill autologous leukemic B cells in vitro. However when the same assay is realized with PBMC from healthy controls anti-CD23 and anti-CD28 coated microbeads do not induce the lysis of non leukemic B cells. This can mean that in the B-CLL patients there are expanded clones of antileukemic lymphocytes that are not present in the healthy controls.

106 THE INVOLVEMENT OF LYSOPHOSPHATIDYLECHOLINE (LPC) IN LYMPHOCYTE APOPTOSIS IN ATHEROSCLEROSIS

Elena Afrimzon¹, Naomi Zurgil¹, Yana Shafran¹, Mordechai Deutsch^{1 1}Biophysical Interdisciplinary Schottenstein Center for Research and Technology of the Cellome, Physics, Bar Ilan University, Ramat-Gan, Israel

LPC, an intermediate metabolite of phosphatidylcholine, is one of the major bioactive lipids, which has been identified in atherosclerotic plaque, and a major component of oxidized LDL (oxLDL), which plays a key role in atherosclerosis. T-lymphocytes found in early and late atherosclerotic lesions, actively participate in lesion progression and rupture. The aim of the present study was to evaluate the apoptotic effect of LPC on human lymphocytes. The Optical LiveCell™ Array (Molecular Cytomics Inc.) technology, which enables quantitative measurements of individual, non-tethered living lymphocytes was employed to assess multiple functional apoptotic assays followed by post-fixation studies. Stimulation of activated peripheral blood lymphocytes, as well as Jurkat-T cell lines, with LPC, triggered apoptosis in association with an increase in rate of production of reactive oxygen species (ROS) and an elevation in Bax/Bcl-2 ratio. Real time kinetic of EGFP-Bid translocation was evident upon treatment of Jurkat-T cells that were transiently transfected with pd4eGFP-Bid plasmid. Additionally, LPC induced dose- and time-dependent changes in mitochondrial membrane potential, concurrently with the increase in ROS production rate, as measured at the individual cell resolution. In lymphocytes derived from unstable angina patients, exposure to LPC triggered neither an apoptotic manifestations nor an increase in ROS generation, and was associated with a significantly lower ratio of Bax/Bcl-2 expression. This data suggests that lymphocyte apoptosis induced by LPC may occur via several intracellular pathways, and may play an important role progression of atherosclerosis.

Immune Monitoring

A second and very promising strategy was pioneered by our laboratory. It uses microbeads to bridge leukemic cells and T cells. It uses one antibody against a leukemic cell antigen and other against costimulatory antigens of cytotoxic lymphocytes (anti-CD28). These microbeads link the leukemic cell and the cytotoxic T lymphocyte providing costimulatory signals for the T cell in the case it recognizes one antigen presented by the leukemic B cell. The application of methodologies of cell enumeration in coculture has demonstrated the efficacy of antiCD23 and anti-CD28 coated microbeads in the stimulation of cytotoxic T cells to kill autologous leukemic B-CLL cells. We are now developing methods of generation of antileukemic effector T cells in vitro. We have demonstrated the efficacy of these cytotoxic T cells to kill autologous leukemic B cells in vitro. We also want to identify and select the T cell subsets with the highest potential for growth and the strongest killing activity against leukemic cells.

110 STUDY OF CYTOKINE PRODUCTION BY NAÏVE, EFFECTOR, MEMORY AND REGULATORY T CELLS BY 8 COLORS MULTIPARAMETRIC FLOW CYTOMETRY

Jorge Monserrat¹, Hugo Barcenilla¹, Angela Hernández¹, Eduardo Reyes¹, Martin Villarroel¹, Miguel Ángel Sánchez¹, David Diaz¹, Alfredo Prieto¹, Manuel Leonardo Acuña¹, Melchor Álvarez-Mon^{2 1}Alcala University, Immune system Diseases and Oncology Laboratory, CNB-CSIC R&D Associated Unit, Alcala de Henares, Madrid, Spain; ²University Hospital “Príncipe de Asturias”, Immune system Diseases and Oncology Service, Alcala de Henares, Madrid, Spain

The best phenotypic criteria to define naïve, effector and memory T lymphocytes are subject of debate. Expression of CD45 isoforms (CD45RA y el CD45RO) has to be complemented with the concurrent expression of another markers as CD27, CD62L, CCR7 and CD31 to distinguish between truly naïve T cells and the different types of antigen experienced T cells: central memory T cells, non terminated effector memory T cells and terminated effector memory T cells. Regulatory T cells are another T cell subset with relevant immunoregulartory functions. Regulatory T cells have been defined as a subset of memory cells. However, the existence of naive regulatory T cells has been recently reported Objetive: By the use of multiparameter flow cyometry in eight colors we have studied the production by cytokines by the different subsets of naïve, effector and memory T lymphocytes and also the production by regulatory T cells. By this method we can study simultaneously five surface markers and intracellular expression of three cytokines. Studies of expression of TNFá, IFNã, IL-2, IL-4 and IL-10). We also studied intracellular expression of FOXP3. Materials and Methods: We have studied peripheral blood mononuclear cells (PBMC) from 6 healthy controls. PBMC were cultured for six hours in the presence or the absence of PMA (50 ng/ml) plus Ionomycin (1 ìg/ml). Monensin (2 ìM) was added to the cultures to retain the produced cytokines within the producer cells. We used for surface labeling antibodies against the antigens: CD3,CD4,CD8,CCR7,CD45RA, CD25 CD27 CD31 and for intracellular labeling antibodies against: TNFá, IFNã, IL-2, IL-4, IL-10 and FOXP3. Results: We have found significant differences in the intracellular production of TNFá, IFNã, IL-2 defined as CD3⁺CD8⁺/-CD45RA⁺/-CD27⁺/-CCR7⁺/-. The percentage of IL-2 producing cells is increased in CD4⁺ naive T cells but not in CD8⁺ naive T cells. The decrease in IL-2 production is correlated with an increase of IFNã and shift of surface markers indicative of transition to effector memory cells. This increase is more marked in T CD8 lymphocytes than in CD4 ones, and is associated to and increase in the expression of TNFá. We have found that there are naive regulatory T cells that express FOXP3 at levels lower than that found in classical memory T cells. Conclusion: The different stages of T cell activation are associated to different patterns of the production of IL-2, INFã y TNFá. The existence of a naive Regulatory T cell population is confirmed.

111 CD8⁺ T-CELL DYSFUNCTION IN AIDS: A CELLULAR DEFECT OR AN ABSENCE OF CD4⁺ T CELLS HELP?

Patrick J Autissier¹, Norman L Letvin¹, Igor J Koralnik¹, Joern E Schmitz^{1 1}Beth Israel Deaconess Medical Center, Medicine, Harvard Medical School, Boston, Massachusetts

HIV and/or SIV infection destroy CD4⁺ T lymphocytes and the result is a loss of immune competence. Loss of immune competence has been attributed to loss of CD4⁺ T lymphocytes by direct viral killing and an indirect impairment of the function of uninfected CD8⁺ T lymphocytes. CD8⁺ T-cells of infected individuals have been shown to lack perforin and normal indicators of maturation, and down modulate CD3 and CD28. However, it is possible that these CD8⁺ T-cells defects could be due to insufficient help. Ca2⁺ acts as an intracellular messenger that is involved in very early steps of cell activation. We evaluated Calcium-flux after anti-CD3 triggering in different subsets of CD8⁺ T lymphocytes of naïve and chronically SIV-infected rhesus monkeys. Using ionomycin activation, we did not see any differences in Ca-flux of CD8⁺ T lymphocytes of SIV- and SIV⁺ monkeys. However, after anti-CD3 triggering, we saw a Ca-flux defect in total CD8⁺ T lymphocytes. Interestingly this defect was associated with the CD4⁺ T-cell counts. The chronically SIV-infected monkeys with low CD4⁺ T-cell counts had a poor response, while the SIV-infected monkeys with high CD4⁺ T-cell counts had a good response. We then evaluated different subsets of CD8⁺ T lymphocytes for Ca-flux in infected monkeys with low CD4⁺ T cell counts. All subsets of CD8⁺ T lymphocytes had defective Ca-flux after CD3 stimulation. These data demonstrate that the SIV-infected monkeys with low CD4⁺ T-cell counts and high viral load have a Caflux defect in all subsets of CD8⁺ T lymphocytes. Since this defect was correlated to the CD4⁺ T-cell count of the monkeys, we suggest that this CD8⁺ T-cell impairment might be due to an absence of adequate CD4⁺ T-

cell help.

112 T CELLS HOMEOSTASIS IN CHILDREN WITH DOWN’S SYNDROME

Erika Roat¹<