The selective combination of physical, biochemical, and immunological prin­ ciples, along with new knowledge concerning the biology of cells and advance­ ments in engineering and computer sciences, has made possible the emergence of highly sophisticated and powerful methods for the analysis of cells and their constituents. This series on Cell Analysis is, therefore, aiming at providing the theoretical and practical background on how these methods work and what kind of information can be obtained. Cell Analysis will cover techniques on cell separation, cell identification and classification, characterization of orga­ nized cellular components, functional properties of cells, and cell interactions. Applications in cell biology, immunology, genetics, toxicology, specific diseases, diagnostics and therapeutics, and other areas will be covered whenever relevant results exist. Nicholas Catsimpoolas Boston, Massachusetts vii Contents Chapter I Quantification of Red Blood Cell Morphology James W. Bacus I. History .. II. Details of Red Cell Measurements. 3 III. Cell Sample Population Distributions. 11 IV. Discussion and Summary. 25 References. 30 Chapter 2 Laser Microirradiation and Computer Video Optical Microscopy in Cell Analysis Michael W. Berns and Robert J. Walter I. Introduction 33 II. Laser Microbeams 34 III. Computer-Enhanced Video Microscopy for Laser Microsurgery.

1 Quantification of Red Blood Cell Morphology.- I. History.- II. Details of Red Cell Measurements.- III. Cell Sample Population Distributions.- IV. Discussion and Summary.- References.- 2 Laser Microirradiation and Computer Video Optical Microscopy in Cell Analysis.- I. Introduction.- II. Laser Microbeams.- III. Computer-Enhanced Video Microscopy for Laser Microsurgery.- A. Contrast Enhancement.- B. Edge Enhancement.- C. Pseudocolor Enhancement.- IV. Chromosome Microsurgery.- A. Mitotic Organelles.- B. Cytoplasm.- V. Plant Cell Development (Chloroplast Irradiation).- VI. Developmental Cellular Neurobiology.- VII. Pattern Formation.- VIII. Conclusions.- References.- 3 Combination of Two Physical Parameters for the Identification and Separation of Lymphocyte Subsets.- I. Introduction.- II. Methods.- A. Preparation of Cell Suspensions.- B. Fractionation of Cell Suspensions by Free-Flow Preparative Electrophoresis.- C. Separation of Cells by Isopycnic Centrifugation in a Percoll Gradient.- D. Cell Enumeration and Size Distribution Analysis.- E. Construction of the Fingerprints.- F. Interpretation of the Fingerprints.- III. Applications.- A. Fingerprint Analysis of Murine Thymocytes.- B. Fingerprint Analysis of Murine Splenocytes.- C. Fingerprint Analysis of Murine Lymph Node Cells.- IV. Conclusion.- References.- 4 Electrical Sizing of Cells in Suspension.- I. Introduction.- II. Theory.- A. Electric Field.- B. Hydrodynamic Field.- C. Cell Volume.- D. Shape and Orientation.- E. Membrane Conductance.- F. Electrical Size.- III. Instrumentation.- A. The Transducer.- B. The Amplifiers.- C. The Selector.- D. Data Handling.- E. Auxiliary Units.- IV. Operation.- A. Cell Suspensions.- B. Orifice Dimensions.- C. Amplification.- D. Calibration.- V. Applications.- A. Fast Kinetics.- B. High Fields.- C. Membrane Permeability.- VI. The Future.- A. Sensitivity.- B. Multiparameter Systems.- References.- 5 Light Scattering Analysis of Single Cells.- I. Introduction.- II. Theoretical Considerations.- A. Scattering Theory.- B. Coated Sphere Model Tests.- C. Expected Detector Responses.- III. Recent Applications of Forward and 90° Scatter.- A. Human Peripheral Blood.- B. Osmolarity Effects.- C. Hematopoietic Stem Cells.- D. T-Lymphocyte Subclasses.- E. Non-Hodgkin's Lymphomas.- F. Cervical Cell Analysis with 90° Scatter.- G. Sputum Samples.- IV. Multiangle and Multiwavelength Scattering.- A. Multiangle Scattering.- B. Two-Color Light Scattering.- V. Fourier Transform Techniques.- VI. Polarization Studies-New Directions.- A. Polarization Transformations.- B. Circular Intensity Differential Scattering.- VII. Conclusion.- References.- 6 Methods for Measuring Leukocyte Locomotion.- I. Introduction.- II. Visual Assays.- A. Time-Lapse Filming.- B. Indirect (Non-Time-Lapse) Methods.- C. Analysis of Cell Movement.- D. The Orientation Assay.- E. The Candida Assay.- III. The Micropore Filter Assay.- A. Principle.- B. Apparatus.- C. Measurement of Leukocyte Locomotion in Filter Assays.- D. The Checkerboard Assay.- E. Automated Method.- IV. The Under-Agarose Assay.- V. Miscellaneous Techniques.- A. The Capillary-Tube Assay for Macrophage Locomotion.- B. The Agarose Microdroplet Assay.- VI. Factors Influencing Locomotion.- A. Cell-Substratum Interactions.- B. Composition of the Fluid Phase.- C. Cell-Cell Interactions.- References.- 7 Sizing of Cells by the Electrical Resistance Pulse Technique: Methodology and Application in Cytometric Systems.- I. Introduction.- A. Reasons for Measuring Size of Biological Cells.- B. A Short Review on the History of Electrical Sizing.- II. Basic Events and Problems in Electrically Sizing Transducers.- A. The Basic Coulter Effect.- B. Fundamental Relations of Particle Volume, Electrical Resistivity, Resistance Change, and Pulse Height.- C. Flow Conditions and Flow Line Coordination in the Sensitive Zone.- D. Behavior of Biological Particles in the Sizing Transducer.- III. Instrumentation.- A. General Conditions and Review.- B. Transducer.- C. Electronic Instrumentation.- D. Electronic Pulse Handling Devices as Alternatives to Hydrodynamic Focusing.- IV. Calibration Problems of Electrical Sizing Flow Cytometers.- A. Calibration in Long Orifices with Homogeneous Field.- B. Calibration with Short Orifices.- C. Calibration with Reference Particles.- V. Applications of the Metricell Cell Volume Analyzer.- A. Erythrocyte Volume Distribution Curves of Young Rats.- B. Red Blood Cell Aggregation in Humans.- C. Thrombocyte Volume Distributions Measured from Whole Blood.- D. Effect of Nerve Growth Factor on Pheochromocytoma Cells.- VI. Combination of Electrical Sizing with Multiparameter Optical Flow Analysis.- A. The Fluorescence-Volume (FLUVO) Transducer.- B. Cell Volume-Cell Absorption Transducer.- C. Electronic Evaluation.- D. Examples of Combined Electrical-Optical Cell Analysis.- VII. Sorting Activated by Electrical Sizing.- VIII. Size-Triggered Imaging in Flow.- A. Instrumentation for Size-Triggered Imaging.- B. Problems of Visualization Cell Structures.- C. Examples of Cell Imaging in Flow.- References.