Fluorescence Activated Cell Sorter (FACS)
How a Flow Cytometer Operates
Cytometry is the quantitative analysis of single cells. Flow Cytometry is the characterization of individual cells as they pass at high speed through a laser beam. While a hematologist can count 200 cells in less than a minute by hand (hemocytometer) on a stage microscope, a flow cytometer can discriminate cells at speeds up to 70,000 cells/second. The Flow component is a fluidics system that precisely delivers the cells at the intersection of the laser beam and light gathering lens by hydrodynamic focusing (a single stream of cells is injected and confined within an outer stream at greater pressure). The laser acting as a light source develops parameters of light scatter as well as exciting the fluorescent molecules used to label the cell. Cells are characterized individually by their physical and/or chemical properties (ref. #6) which provide analytical parameters capable of accurate quantitation of the number of molecules/cell through Quantitative Flow Cytometry (QFCM). The physical (morphological) profile of a cell can be observed by combining forward light scatter (FS) and orthogonal or side light scatter (SSC). In forward light scatter the laser beam is interrupted by the cell and the light that passes around the cell is measured. Comparable to casting shadow puppets on a wall with a flashlight. This measurement is an indication of the cell's unique refractive index which depends on a cell's size, organelles, water and molecular contents. The refractive index (forward scatter) of a cell can change through cell cycle progression, activation, fixation, etc. Cellular side scatter is the light that is reflected 900 to the laser beam (all fluorescence is emitted and therefore collected at this angle) and is an indication of cytoplasmic density or cell surface granularity. One example of detectable changes in morphology by forward and side scatter is in the comparison of normal isolated B cells and activated blast stage B lymphocytes where the rough endoplasmic reticulum engorges as it produces antibodies causing a dramatic increase in both forward and side scatter. A rather simplistic example of the morphology associated with cells in advanced apoptosis is the comparison of the light scatter properties between a fully inflated basket ball (live) and one that has lost inflation (dead). The ball with less air will be smaller (less FS) and its puckered surface will scatter more light (greater SSC) as compared to the smoother surface of the fully inflated ball.
Cells express proteins (antigens) on their surface which are unique to that cell. Antibodies (immunoglobulins) are proteins that bind to cell surface antigen epitopes very specifically. By tagging each antibody with a colored fluorochrome it is easy to distinguish the cell type and quantity of antigens expressed by each cell. Employing dichroic splitting mirrors, band pass filters and compensation the colors can be resolved as illustrated in the drawing at the top of the page where each color is associated with a single antibody.
As each cell, tagged with a fluorescently labeled antibody, enters the laser light outer orbital electrons in the fluorochrome are excited at a specific excitation wavelength (FITC-494nmEX) to greater, and more unstable energy levels. As it transitions the width of the laser beam maximum peak fluorescence is achieved within approximately 10nsec as the excited outer orbital electrons return to their more stable ground state and emit a photon of light at a longer wavelength (FITC-520nmEM Stokes Shift) than that at which they were excited. Photomultiplier tubes (PMT's) detect these faint fluorescent signals and their sole role is to change discrete packets of light called photons (hv) into electrons and amplify them by producing as much as a 10 million electrons (ref. #6) for every photon captured. Cytoplasmic and nuclear membranes can be breached (permeabilized) to introduce antibodies against intracellular proteins or stain nucleic acids with intercalating dyes (propidium iodide, 7-AAD, etc.) or DNA base specific dyes (Hoescht). A short list of some of the information that can be discerned by multiparameter (multi-color) Flow Cytometry includes; Apoptosis (programmed cell death), Cell Type, DNA Content, Enzyme Activity, Intracellular Proteins, Cell Surface Antigens, Cytoplasmic Granularity, Surface Membrane Integrity, Intracellular [Ca++]-Signal Transduction, DNA Synthesis-Proliferation, Cell Surface Receptors, Intracellular Cytokines, Oxidative Metabolism, Intracellular pH, RNA Content, Cell Size.
A flow cytometer sorts cells by vibrating the solid flow stream with a quartz crystal tuned to an exact frequency to form stable drops. As the last drop breaks from the stream an electrical charge is conferred on the drop and charged deflection plates (+/-) redirect the drops to the left or right of the flow stream. Ideally each drop contains a single cell and are sorted according to the parameters selected (gating) into collecting tubes or a 96-well plate. The computer makes about 30,000 decisions/second in this process. If a drop contains any unwanted cells a coincidence abort is utilized to reject that drop and increase the purity, consequently yield is sacrificed for purity.
The above photo is an example of how sorting is used for production quality control in the food industry to sort dehulled peanuts. "The edible peanuts are individually inspected with an electric eye picking machine. The electric eye is a high-speed electronic color sorter that examines the peanuts from six different angles as they roll down a chute, and kicks out discolored or dark peanuts with a puff of air. The Lee County Peanut Company was the first sheller within the south-west to use this leading-edge technology, which has become today's industry standard." ( from Texas Co-Op Power a publication of the Bluebonnet Electric Cooperative, November 2006, Vol. 63, No. 5, pg. 19)
A brief history of the advancements in Flow Cytometry reveals contributions from several remarkable resources, a pattern that continues today. Presenting cells in an orderly manner to a collecting lens by hydrodynamic focusing can be credited to the work of Bernoulli in 1738(1), Euler in 1755 & 1759, and Reynolds in 1883. In 1953 Crosland-Taylor(2) employed all the results of these early fluidics pioneers and developed a hydrodynamic focused flow chamber. While blood cell counting was being developed by light scattering and dark field illumination techniques it wasn't until Wallace Coulter, an electrical engineer frustrated with optics problems proposed another method of cell detection. His idea was to pass single cells in a saline stream through a small orifice and measure the changes in conductance as he applied an electrical current through the flow stream, the cells providing a greater impedance to the flow of electrons across the fluid as compared to the salt stream alone. The first prototype for the Coulter orifice was made from a cigarette package's cellophane wrapper. Thus the Coulter Counter was developed to count and size cells.
Many technological advances are market driven. Advances in Flow Cytometry are no exception. Because the medical community wanted information on white cell counts it wasn't until Wallace Coulter developed a method for lysing red blood cells did the Coulter Counter market successfully. In establishing stable droplet formation it was shown as early as 1833 by Savart (3) that regular oscillations could produce standing waves in a jet stream of water. In 1965 Richard Sweet(4) of Stanford used the principle combined with electrostatic deflection to position charged ink droplets on paper, an advent of the ink-jet printer. This method was incorporated into the flow cytometer to sort cells. Monoclonal antibody technology first introduced by Kohler and Milstein (6) provided a diverse, highly specific and expanding repertoire of immunological reagents used to study immunophenotypic development as well as cell classification.
Early in the development of Flow Cytometry these instruments were assembled on large physics benches and operated by teams of scientists and engineers. Even today we see core facilities with dedicated operators providing a collegial environment where multiple lasers and sorters are utilized. Several contributions have transformed Flow Cytometry in the clinical arena. As antibody enumeration and the formation of an international consortium to establish and recognize universally accepted clusters of differentiation (CD) markers, the field of Flow Cytometry became an analytical tool providing diagnostic and prognostic information to the physician and patient. Advancement in the treatment of lymphomas and leukemias is in part credited to the ability to further subphenotype cell differentiation in the disease process, which translates into more successful intervention and greater life expectancy for the cancer patient.
One example of this as a diagnostic tool is in CD4 counts. When patients present themselves with HIV infection the CD4/8 ratio is monitored and typically is 1.4 to 2.0 in value. When the CD4 counts drop the patient has transitioned from an HIV infection to full blown AIDS. As a prognostic indicator (5) in breast tumor biopsies from patients with the same diagnostic disease process (early stages with no node involvement or metastasis) analyzed for s-phase, a correlation has been shown in those patients with high s-phase or greater tumor cell proliferation there may be a greater chance of relapse or reduced life expectancy after 5 years. Recently a "prognostic protein" (Cyclin E) whose expression levels were correlated with survival in patients with breast cancer was cited (8).
Each time an additional color is added to a cell assay the amount of information goes up dramatically. A two color assay generally provides four analytical areas, in addition to cell morphology by light scatter profiles. Add another color and the three color assay can discriminate twelve subsets. A four color assay can have twenty-four quantifiable subsets for each cell population. Other interesting applications of the flow cytometer include Operation Desert Storm where small portable flow cytometers were mounted on HUMVEE's and sent out before advancing ground troops to scour the air for the biological warfare agent anthrax. For years NASA has brought back blood samples from astronauts in space to evaluate what effects the microgravity of space and ionizing radiation (IR) has upon their immune systems and DNA. NASA plans to include a portable flow cytometer to accompany the astronaughts on their shuttle missions to perform real time analysis.
Manufacturers have made a significant contribution by providing smaller, more easily operated and less costly flow cytometers to clinical laboratories and hospitals. The field of flow cytometry remains one of collegial interaction limited only by one's imagination.
Ref. #1 Watson, J.V. (1999) The Early Fluidic and Optical Physics of Cytometry Cytometry (Communications in Clinical Cytometry) 38: p.2-14
Ref. #2 Crosland-Taylor (1953) A device for counting small particles suspended in fluid through a tube. Nature 171: p. 37-388
Ref. #3 Savart F. (1833) Memoire sur la Constitution des Veines liquides lancees par des orifices circulaires en mince paroi. Ann. Chim. Phys. 53: p. 337-374
Ref. #4 Sweet R.G. (1965) High frequency recording with electrostatically deflected Ink jets. Rev. Sci. instrum. 36: p.131-136
Ref. #5 Bagwell, C.B., Clark, G.M., Spyratos, F., Chassevent, A., Bendahl, P.O., Stal, O., Killander, D., Jourdan, M.I., Romaain, S., Hunsberger, B., and Baldertorp, B. (2001) Optimizing Flow Cytometric DNA Ploidy and S-Phase Fraction as Independent Prognostic Markers for Node-Negative Breast Cancer Specimens Cytometry 46:3 p.121-135
Ref. #6 Kohler, G. and Milstein, C. (1975) Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity. Nature 256: p. 495-49
Ref. #7 Melamed M.R., Mullaney, P.F., and Shapiro, H.M. (1990) An Historical Review of the Development of Flow Cytometers and Sorters from Melamed, M.R., Lindmo, T., & Mendelsohn, M.L., Ed.s: Flow Cytometry and Sorting, Second Ed. 1990, Wiley-Liss, Inc., New York, NY p. 1-9
Ref. #8 Keyomarsi, K., Tucker, S.L., Buchholz, T.A., Callister, M., Ding, Y., Hortobagyi, G.N., Bedrosian, I., Knickerbocker, C., Toyofuku, W., Lowe, M., Herliczek, T.W., and Bacus, S.S. (2002) Cyclin E and Survival in Patients with Breast Cancer. N. Engl. J. Med. 347:20 p. 1566-1575
Free On-Line Access
Shapiro, Howard M.: Practical Flow Cytometry, Fourth Edition 2005, Wiley-Liss, Inc., New York, NY
Dr. J. Paul Robinson has assembled some fascinating lectures (in PowerPoint or HTML formats) at this Purdue University education web site.
Purdue University Cytometry Laboatories General Education Initiatives
Purdue University CD-ROM Series for Cytometry & Microscopy
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