Book contents
- Frontmatter
- Contents
- List of contributors
- List of abbreviations
- 1 Principles of flow cytometry
- 2 Introduction to the general principles of sample preparation
- 3 Fluorescence and fluorochromes
- 4 Quality control in flow cytometry
- 5 Data analysis in flow cytometry
- 6 Laser scanning cytometry: application to the immunophenotyping of hematological malignancies
- 7 Leukocyte immunobiology
- 8 Immunophenotypic analysis of leukocytes in disease
- 9 Analysis and isolation of minor cell populations
- 10 Cell cycle, DNA and DNA ploidy analysis
- 11 Cell viability, necrosis and apoptosis
- 12 Phagocyte biology and function
- 13 Intracellular measures of signalling pathways
- 14 Cell–cell interactions
- 15 Nucleic acids
- 16 Microbial infections
- 17 Leucocyte cell surface antigens
- 18 Recent and future developments: conclusions
- Appendix
- Index
- Plate section
3 - Fluorescence and fluorochromes
Published online by Cambridge University Press: 06 January 2010
- Frontmatter
- Contents
- List of contributors
- List of abbreviations
- 1 Principles of flow cytometry
- 2 Introduction to the general principles of sample preparation
- 3 Fluorescence and fluorochromes
- 4 Quality control in flow cytometry
- 5 Data analysis in flow cytometry
- 6 Laser scanning cytometry: application to the immunophenotyping of hematological malignancies
- 7 Leukocyte immunobiology
- 8 Immunophenotypic analysis of leukocytes in disease
- 9 Analysis and isolation of minor cell populations
- 10 Cell cycle, DNA and DNA ploidy analysis
- 11 Cell viability, necrosis and apoptosis
- 12 Phagocyte biology and function
- 13 Intracellular measures of signalling pathways
- 14 Cell–cell interactions
- 15 Nucleic acids
- 16 Microbial infections
- 17 Leucocyte cell surface antigens
- 18 Recent and future developments: conclusions
- Appendix
- Index
- Plate section
Summary
Introduction
The great analytical power of current cytometers stems from their ability to quantify simultaneously many separate parameters on thousands of cells within a few minutes. Laser scanning cytometers and flow cytometers measure, respectively, one or two light scattering parameters, and, depending on the instrument, up to 12 fluorescence parameters. Only a few cellular components (e.g. pyridine-and flavin-containing nucleotides) are intrinsically fluorescent (autofluorescent), so cells are usually stained with compounds (known as fluorochromes, fluorophores or fluors) that fluoresce when they report the presence or activity of a particular cellular component. Three main approaches are used for staining:
a fluorochrome-labelled antibody or other ligand is allowed to bind to complementary structures, either within or on the surface of cells
a fluorescent dye, e.g. one that binds to nucleic acids, is allowed to accumulate within cells
a cell-permeant precursor compound is converted by the activity, often enzymic, of some cell component into a form with distinct fluorescence.
Usually samples are stained with more than one fluorochrome and, depending on the number of fluorochromes used, the technique is known as two-colour, three-colour, etc. staining. At present, the main constraint on the number of different stains that can be measured simultaneously is the shortage of fluorochromes with distinct emission spectra and the difficulty of distinguishing those fluorochromes with emission spectra that overlap. To appreciate how fluorochromes can best be used in cytometric assays it is worth first considering how light and matter interact.
The interaction of light and matter
Light is a form of electromagnetic radiation with a wave motion defined by electric and magnetic vectors at right angles to each other and perpendicular to the direction of propagation.
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- Cytometric Analysis of Cell Phenotype and Function , pp. 45 - 73Publisher: Cambridge University PressPrint publication year: 2001
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