Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Flow cytometry: retrospective, fundamentals and recent instrumentation


Flow cytometry is a complete technology given to biologists to study cellular populations with high precision. This technology elegantly combines sample dimension, data acquisition speed, precision and measurement multiplicity. Beyond the statistical aspect, flow cytometry offers the possibility to physically separate sub-populations. These performances come from the common endeavor of physicists, biophysicists, biologists and computer engineers, who succeeded, by providing new concepts, to bring flow cytometry to current maturity. The aim of this paper is to present a complete retrospective of the technique and remind flow cytometry fundamentals before focusing on recent commercial instrumentation.

This is a preview of subscription content, log in to check access.

Fig. 1


  1. Arndt-Jovin DJ, Jovin TM (1974) Computer-controlled multiparameter analysis and sorting of cells and particles. J Histochem Cytochem 22:622–625

  2. Ashcroft RG, Lopez PA (2000) Commercial high speed machines open new opportunities in high throughput flow cytometry (HTFC). J Immunol Methods 243:13–24

  3. Bonner WA, Hulett HR, Sweet RG, Herzenberg LA (1972) Fluorescence activated cell sorting. Rev Sci Instrum 43:404–409

  4. Brecher G, Schneiderman M, Williams GZ (1956) Evaluation of electronic red blood cell counter. Am J Clin Pathol 26:1439–1449

  5. Chapman GV (2000) Instrumentation for flow cytometry. J Immunol Methods 243:3–12

  6. Cho SH, Godin JM, Chen CH, Qiao W, Lee H, Lo YH (2010a) Review article: recent advancements in optofluidic flow cytometer. Biomicrofluidics 4:43001. doi:10.1063/1.3511706

  7. Cho SH, Qiao W, Tsai FS, Yamashita K, Lo YH (2010b) Lab-on-a-chip flow cytometer employing color-space-time coding. Appl Phys Lett 97:093704, 1–3 . doi:10.1063/1.3481695

  8. Coons AH, Kaplan MH (1950) Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med 91(1):1–13

  9. Coons AH, Creech HJ, Jones RN, Brliner E (1942) The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J Immunol 45:159

  10. Crissman HA, Steinkamp JA (1982) Rapid, one step staining procedures for analysis of cellular DNA and protein by single and dual laser flow cytometry. Cytometry 3:84–90. doi:10.1002/cyto.990030204

  11. Crosland-Taylor PJ (1953) A device for counting small particles suspended in a fluid through a tube. Nature 171:37–38

  12. Curbelo R, Schildkraut ER, Hirschfeld T, Webb RH, Block MJ, Shapiro HM (1976) A generalized machine for automated flow cytology system design. J Histochem Cytochem 24:388–395

  13. De Rosa SC, Roederer M (2001) Eleven-color flow cytometry. A powerful tool for elucidation of the complex immune system. Clin Lab Med 21:697–712, vii

  14. De Rosa SC, Herzenberg LA, Roederer M (2001) 11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nat Med 7:245–248. doi:10.1038/84701

  15. Dittrich W, Gohde W (1969) Impulse fluorometry of single cells in suspension. Z Naturforsch B 24:360–361

  16. Fulwyler MJ (1965) Electronic separation of biological cells by volume. Science 150:910–911

  17. Fulwyler MJ (1977) Hydrodynamic orientation of cells. J Histochem Cytochem 25:781–783

  18. Goddard G, Kaduchak G (2005) Ultrasonic particle concentration in a line-driven cylindrical tube. J Acoust Soc Am 117:3440–3447

  19. Goddard G, Martin JC, Graves SW, Kaduchak G (2006) Ultrasonic particle-concentration for sheathless focusing of particles for analysis in a flow cytometer. Cytometry A 69:66–74. doi:10.1002/cyto.a.20205

  20. Goddard GR, Sanders CK, Martin JC, Kaduchak G, Graves SW (2007) Analytical performance of an ultrasonic particle focusing flow cytometer. Anal Chem 79:8740–8746. doi:10.1021/ac071402t

  21. Gray JW, Dean PN, Fuscoe JC, Peters DC, Trask BJ, van den Engh GJ, Van Dilla MA (1987) High-speed chromosome sorting. Science 238:323–329

  22. Greimers R, Trebak M, Moutschen M, Jacobs N, Boniver J (1996) Improved four-color flow cytometry method using fluo-3 and triple immunofluorescence for analysis of intracellular calcium ion ([Ca2 +]i) fluxes among mouse lymph node B- and T-lymphocyte subsets. Cytometry 23:205–217. doi:10.1002/(SICI)1097-0320(19960301)23:3<205:AID-CYTO4>3.0.CO;2-H

  23. Gucker FT Jr, O’Konski CT (1947) A photoelectronic counter for colloidal particles. J Am Chem Soc 69:2422–2431

  24. Hulett HR, Bonner WA, Sweet RG, Herzenberg LA (1973) Development and application of a rapid cell sorter. Clin Chem 19:813–816

  25. Ibrahim SF, van den Engh G (2003) High-speed cell sorting: fundamentals and recent advances. Curr Opin Biotechnol 14:5–12

  26. Kamentsky LA, Melamed MR (1967) Spectrophotometric cell sorter. Science 156:1364–1365

  27. Kamentsky LA, Melamed MR, Derman H (1965) Spectrophotometer: new instrument for ultrarapid cell analysis. Science 150:630–631

  28. Leif SB, Leif RC, Auer R (1985) The EPICS C analyzer. An ergometrically designed flow cytometer computer system. Anal Quant Cytol Histol 7:187–191

  29. LePecq JB, Paoletti C (1967) A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization. J Mol Biol 27:87–106

  30. Loken MR, Parks DR, Herzenberg LA (1977) Two-color immunofluorescence using a fluorescence-activated cell sorter. J Histochem Cytochem 25:899–907

  31. Mansberg HP, Saunders AM, Groner W (1974) The Hemalog D white cell differential system. J Histochem Cytochem 22:711–724

  32. Mattern CF, Brackett FS, Olson BJ (1957) Determination of number and size of particles by electrical gating: blood cells. J Appl Physiol 10:56–70

  33. Moldavan A (1934) Photo-Electric Technique for the Counting of Microscopical Cells. Science 80:188–189. doi:10.1126/science.80.2069.188

  34. Mullaney PF, Van Dilla MA, Coulter JR, Dean PN (1969) Cell sizing: a light scattering photometer for rapid volume determination. Rev Sci Instrum 40:1029–1032

  35. Novo D, Wood J (2008) Flow cytometry histograms: transformations, resolution, and display. Cytometry A 73:685–692. doi:10.1002/cyto.a.20592

  36. Nunez R (2001) Flow cytometry: principles and instrumentation. Curr Issues Mol Biol 3:39–45

  37. Ornstein L, Ansley HR (1974) Spectral matching of classical cytochemistry to automated cytology. J Histochem Cytochem 22:453–469

  38. Parks DR, Roederer M, Moore WA (2006) A new “Logicle” display method avoids deceptive effects of logarithmic scaling for low signals and compensated data. Cytometry A 69:541–551. doi:10.1002/cyto.a.20258

  39. Perfetto SP, Ambrozak DR, Koup RA, Roederer M (2003) Measuring containment of viable infectious cell sorting in high-velocity cell sorters. Cytometry A 52:122–130. doi:10.1002/cyto.a.10033

  40. Perfetto SP, Chattopadhyay PK, Roederer M (2004) Seventeen-colour flow cytometry: unravelling the immune system. Nat Rev Immunol 4:648–655. doi:10.1038/nri1416

  41. Peters D, Branscomb E, Dean P, Merrill T, Pinkel D, Van Dilla M, Gray JW (1985) The LLNL high-speed sorter: design features, operational characteristics, and biological utility. Cytometry 6:290–301. doi:10.1002/cyto.990060404

  42. Petersen TW, van den Engh G (2003) Stability of the breakoff point in a high-speed cell sorter. Cytometry A 56:63–70. doi:10.1002/cyto.a.10090

  43. Reinherz EL, Kung PC, Goldstein G, Schlossman SF (1979) Separation of functional subsets of human T cells by a monoclonal antibody. Proc Natl Acad Sci USA 76:4061–4065

  44. Reynolds O (1883) An experimental investigation of the circumstances which determine whether the motion of water in parallel channels shall be direct or sinuous and of the law of resistance in parallel channels. Philosoph Trans R Soc 174:935–982

  45. Salzman GC, Crowell JM, Goad CA, Hansen KM, Hiebert RD, LaBauve PM, Martin JC, Ingram ML, Mullaney PF (1975a) A flow-system multiangle light-scattering instrument for cell characterization. Clin Chem 21:1297–1304

  46. Salzman GC, Crowell JM, Martin JC, Trujillo TT, Romero A, Mullaney PF, LaBauve PM (1975b) Cell classification by laser light scattering: identification and separation of unstained leukocytes. Acta Cytol 19:374–377

  47. Salzman GC, Wilder ME, Jett JH (1979) Light scattering with stream-in-air flow systems. J Histochem Cytochem 27:264–267

  48. Schmid I, Dean PN (1997) Introduction to the biosafety guidelines for sorting of unfixed cells. Cytometry 28:97–98

  49. Schmid I, Nicholson JK, Giorgi JV, Janossy G, Kunkl A, Lopez PA, Perfetto S, Seamer LC, Dean PN (1997) Biosafety guidelines for sorting of unfixed cells. Cytometry 28:99–117

  50. Shapiro HM (1977) Fluorescent dyes for differential counts by flow cytometry: does histochemistry tell us much more than cell geometry? J Histochem Cytochem 25:976–989

  51. Shapiro HM (2003) Practical flow cytometry. Fourth edition edn, Wiley-liss, New Jersey (USA)

  52. Shapiro HM, Perlmutter NG (2001) Violet laser diodes as light sources for cytometry. Cytometry 44:133–136

  53. Shapiro HM, Schildkraut ER, Curbelo R, Laird CW, Turner B, Hirschfeld T (1976) Combined blood cell counting and classification with fluorochrome stains and flow instrumentation. J Histochem Cytochem 24:396–401

  54. Shapiro HM, Schildkraut ER, Curbelo R, Turner RB, Webb RH, Brown DC, Block MJ (1977) Cytomat-R: a computer-controlled multiple laser source multiparameter flow cytophotometer system. J Histochem Cytochem 25:836–844

  55. Snow C (2004) Flow cytometer electronics. Cytometry A 57:63–69. doi:10.1002/cyto.a.10120

  56. Steen HB (1990) Light scattering measurement in an arc lamp-based flow cytometer. Cytometry 11:223–230. doi:10.1002/cyto.990110202

  57. Steinkamp JA, Fulwyler MJ, Coulter JR, Hiebert RD, Horney JL, Mullancy PF (1973) A new multiparameter separator for microscopic particles and biological cells. Rev Sci Instrum 44:1301–1310

  58. Steinkamp JA, Romero A, Horan PK, Crissman HA (1974) Multiparameter analysis and sorting of mammalian cells. Exp Cell Res 84:15–23

  59. Steinkamp JA, Orlicky DA, Crissman HA (1979) Dual-laser flow cytometry of single mammalian cells. J Histochem Cytochem 27:273–276

  60. Sweet RG (1965) High frequency recording with electrostatically deflected ink jets. Rev Sci Instrum 36:131–136

  61. van den Engh G, Stokdijk W (1989) Parallel processing data acquisition system for multilaser flow cytometry and cell sorting. Cytometry 10:282–293. doi:10.1002/cyto.990100307

  62. Van Dilla MA, Trujillo TT, Mullaney PF, Coulter JR (1969) Cell microfluorometry: a method for rapid fluorescence measurement. Science 163:1213–1214

  63. Ward M, Turner P, DeJohn M, Kaduchak G (2009) Fundamentals of Acoustic Cytometry. Current Protocols in Cytometry 1:1–12

  64. Watson JV (1999) The early fluidic and optical physics of cytometry. Cytometry 38:2–14; discussion 1

The latest specifications of commercial instrumentation can be found on the web pages of the manufacturers:

  1. Amnis Corporation:

  2. Apogee flow systems:

  3. Applied Biosystems by Life Technologies:

  4. Bay Bioscience:

  5. BD Biosciences:

  6. Beckman Coulter:

  7. Cyntellect:

  8. CytoBuoy b.v.:

  9. DVS Science:

  10. Fluid imaging technologies:

  11. Merck Millipore:

  12. Miltenyi Biotec:

  13. On-chip Biotechnologies:

  14. Partec:

  15. Stratedigm:

  16. i-cyt by Sony:

  17. Union Biometrica:

Download references

Conflict of interests

The authors state that they have no conflict of interest.

Author information

Correspondence to Julien Picot.

Additional information

Julien Picot and Coralie L. Guerin contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Picot, J., Guerin, C.L., Le Van Kim, C. et al. Flow cytometry: retrospective, fundamentals and recent instrumentation. Cytotechnology 64, 109–130 (2012).

Download citation


  • Flow cytometry
  • Retrospective
  • Fundamentals
  • Instrumentation
  • Analyzer
  • Cell-sorter