Flow cytometry instrumentation

A fluorescence-activated cell sorter (FACS) is a flow cytometry instrument used to separate and identify cells in a heterogeneous population. Cell mixtures to be sorted are first bound to fluorescent dyes such as fluorescein or phycoerythrin. The labeled cells are then pumped through the instrument and are excited by a laser beam. Cells that fluoresce are detected, and an electrostatic charge is applied. The charged cells are separated using voltage deflection. 

Van Dilla MA, Dean PN, Laerum OD, Melamed MR, eds. (1985). Flow Cytometry Instrumentation and Data Analysis. Academic Press, London. A venerable, but still current book with an emphasis on the physics and mathematics of flow systems and data analysis. It has some excellent (and readable) articles on some theoretical subjects. 

Fig. 8.7. Reprinted (A,D) from Dean PN (1987). Data analysis in cell kinetics. Gray JW and Darzynkiewicz Z (eds). Techniques in Cell Cycle Analysis. Clifton, NJ Humana Press, pp 207-253 and (B,C) from Dean PN (1985). Methods of data analysis in flow cytometry. Van Dilla MA, et al. (eds). Flow Cytometry Instrumentation and Data Analysis. London Academic Press, pp 195-221.

Flow cytometry instrumentation (e.g., FACScan, FACSCalibur, FACSArray, from BD Biosciences)... 

The difficulty of separation is highly dependent on peak spreading, as shown in Fig. 6.5. It is therefore critical to minimize the peak width as far as possible. This would be difficult for cell display methods if only single color fluorescent labeling were used, because the primary source of variability is biological. Flow cytometry instrumentation point spread functions generally contribute below 2 % to the overall coefficient of variance (CV = standard deviation/mean), but typical overall CVs for yeast display are approximately 50 - 100 % for the logarithmic fluorescence intensity. 

However, the flow cytometers are bulky and expansive, and are available only in large reference laboratories. In addition, the required sample volumes are quite large, usually in the 100 pL range. Many clinical applications require frequent blood tests to monitor patients status and the therapy effectiveness. It is highly desirable to use only small amount of blood samples Ifom patients for each test. Furthermore, it is highly desirable to have affordable and portable flow cytometry instruments for field applications, point-of-care applications and applications in resource-limited locations. To overcome these drawbacks and to meet the increasing needs for versatile cellular analyses, efforts have been made recently to apply microfluidics and lab-on-a-chip technologies to flow cytometric analysis of cells. 

Van Dilla, M. A., Dean, P. N., Laerum, O. D., Melamed, M. R., Flow Cytometry Instrumentation and Data Analysis, Aeademic Press, New York, 1985. 

Several additional instrumental techniques have also been developed for bacterial characterization. Capillary electrophoresis of bacteria, which requires little sample preparation,42 is possible because most bacteria act as colloidal particles in suspension and can be separated by their electrical charge. Capillary electrophoresis provides information that may be useful for identification. Flow cytometry also can be used to identify and separate individual cells in a mixture.11,42 Infrared spectroscopy has been used to characterize bacteria caught on transparent filters.113 Fourier-transform infrared (FTIR) spectroscopy, with linear discriminant analysis and artificial neural networks, has been adapted for identifying foodbome bacteria25,113 and pathogenic bacteria in the blood.5... 

The availability of a wide array of fluorescent probes and versatile, high-speed instruments has established flow cytometry as a preferred scientific tool with apphcations that seem hmited only by the imagination of the investigator. Excellent discussions of the theory and practice of flow cytometry may be found in the reviews cited herein, in the comprehensive textbook by Shapiro (2003), and at a dedicated web site for flow cytometry (Purdue University Cytometry Laboratories, 2005, http //www.cyto.purdue.edu/), which provides many useful links to additional technical resources. 

Chapter 1 in Melamed et al., Chapter 3 in Shapiro, and Chapter 1 in Darzynkiewicz are good historical reviews of flow cytometry. Alberto Cambrosio and Peter Keating (2000) have used flow cytometry as a model for looking at historical changes in the way scientists use instrumentation to view the world Of lymphocytes and pixels The techno-visual production of cell populations. Studies in History and Philosophy of Biological and Biomedical Sciences 31 233-270. 

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