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What is Flow Cytometry?

Flow cytometry is a means of measuring certain physical and chemical characteristics of cells or particles as they travel in suspension one by one past a sensing point.  In one way flow cytometers can be considered to be specialised fluorescence microscopes.  The modern flow cytometer consists of a light source, collection optics, electronics and a computer to translate signals to data. 

In most modern cytometers the light source of choice is a laser which emits coherent light at a specified wavelength.  Scattered and emitted fluorescent light is collected by two lenses (one set in front of the light source and one set at right angles) and by a series of optics, beam splitters and filters, specific bands of fluorescence can be measured. 

We can measure physical characteristics such as cell size, shape and internal complexity and, of course, any cell component or function that can be detected by a fluorescent compound can be examined.  So the applications of flow cytometry are numerous, and this has led to the widespread use of these instruments in the biological and medical fields.  More about the applications of flow cytometry in this Institute can be found by clicking the appropriate link in the menu on the left.

In addition, many flow cytometers have the ability to sort, or physically separate, particles of interest from a sample, which can be particularly useful.  More information on this and the specialist applications at the FACS Lab can be found at the relevant pages.

There are many good books devoted to the theory of flow cytometry.  A few examples are:
  1. Flow Cytometry: A Practical Approach. Edited by MG Ormerod.
    (IRL Press, Oxford. 1994. ISBN 0-19 963461-0)

  2. Practical Flow Cytometry. 3rd Edition. Howard M Shapiro.
    (Alan R Liss, Inc. ISBN 0-471-30376-3)

  3. Flow Cytometry. First Principles. Alice Longobardi Givan.
    (Wiley-Liss, New York, 1992. ISBN 0-471-56095-2)

  4. Handbook of Flow Cytometry Methods. Edited by J Paul Robinson.
    (Wiley-Liss, New York, 1993. ISBN 0-471-59634-5)

Lasers and Fluorochromes

There are several laser types available that can be used in flow cytometers.  They differ in the gain medium that is used to amplify light.  Lasers can be gas lasers (e.g. Helium-Neon, Argon, Helium-Cadmium); solid state lasers (e.g. NdYAG); dye lasers or semi-conductors lasers.

The choice of fluorochrome to be used is influenced both by the application and the excitation wavelengths available.  The following table is by no means exhaustive, but lists the major fluorochromes, their excitation and emission wavelengths together with their common applications.  All these fluorochromes can be used at the FACS facility here.

To view the excitation and emission spectra of a range of commonly used fluorochromes go the Joe Trotter's excellent fluorescence Spectrum viewer (requires Netscape 4.7 or Internet Explorer 5.0 or higher).

 
FLUOROCHROME EX. EM. APPLICATION
Indo-1 (unbound) 335 490 Calcium Flux
Indo-1 (Bound to Calcium) 335 405 Calcium Flux
Hoechst 33342 350 470 DNA Analysis
DAPI 359 468 DNA Analysis
Alexa350 350 442 Phenotyping
PerCP 470 670 Phenotyping
R-Phycoerythrin 480 578 Phenotyping
Green Fluorescent Protein (GFP) 488 510 Reporter molecule
YO-PRO-1 488 510 Apoptosis analysis
Fluoroscein diacetate 488 530 Cell viability
Alexa488 488 530 Phenotyping
Sytox Green 488 530 DNA Analysis
SNARF-1 488 530-640 pH measurement
Fluo-3 488 530 Calcium flux
dsRED 488 588 Reporter molecule
PE-Cy5 (TriColor, Cychrome) 488 670 Phenotyping
PE-Cy7 488 670 Phenotyping
Propidium Iodide 495 637 DNA Analysis
Rhodamine 123 515 525 Membrane potential
Yellow Fluorescent Protein (YFP) 519 534 Reporter molecule
LDS-751 543 712 Nucleated cell detection
7-Aminoactinomycin D 546 655 DNA analysis
Alexa 546 546 573 Phenotyping
Cy3 550 565 Phenotyping
CMXRos (Mitotracker Red) 560 610 Mitochondrial membrane potential
Texas Red 596 615 Phenotyping
TO-PRO-3 643 661 DNA Analysis
Alexa 647 647 667 Phenotyping
APC-Cy7 647 774 Phenotyping
Allophycocyanin (APC) 650 660 Phenotyping
Page updated 09/11/2004

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