Flow Cytometry :: Applications

Cell sorters may be used in the analysis and separation of a wide range of biological particles. The instruments are particularly suited to collect a rare cell type with desirable properties from among a large number of non-relevant events. The following pictures demonstrate applications in microbiology, stem cell research, genome analysis, and marine biology.

Chromatic shifts in Hoechst 33342 bound to murine thymocytes

Flow cytometers typically measure scattering and fluorescence intensities from cells and other particles with an accuracy of 1-2%.  Accuracies of this order are necessary for measurements such as cell cycle, sperm sex selection, and flow karyotyping.  In all of these techniques, the measured fluorescence is the result of binding of fluorescent dyes to DNA.  A critical question for flow cytometric measurements, therefore, is whether the fluorescence intensity accurately represents the cellular dye content.  A natural extension of this inquiry is to investigate to what degree nonlinearities in the fluorescent intensity versus dye concentration affect the estimation of DNA content.

In this work, murine thymocytes were dyed with different concentrations of Hoechst 33342, a DNA-binding dye often used in cell cycle analysis and stem cell isolation.  This dye exhibits a concentration-dependent chromatic shift as the intracellular dye concentration increases. Flow cytometric bivariate dot plots of the Hoechst 33342 blue versus red fluorescence should follow the curve shown in this figure as the intracellular dye concentration increases.  A fit to these data is also shown to allow smooth interpolation between different dye concentrations.  As dye moves across the cellular membrane, the blue/red fluorescence ratio should follow this curve. A popular method for enriching and isolating hematopoietic stem cells uses this property, and the measurements presented in this work elucidate the mechanisms responsible for the different red/blue fluorescence ratios.

Blue autofluorescence in Pseudo-nitzschia multiseries

Many marine organisms such as the diatoms at right contain autofluorescent pigments such as chlorophyll, phycoerythrin, phycocyanin, allophycocyanin, and green fluorescent protein (GFP). Some of these pigments have remarkable roles in photosynthesis and are useful for the identification and enumeration of different functional groups of phytoplankton by flow cytometry.  In addition to enumerating phytoplankton populations, flow cytometry has led to other significant discoveries, including the description of new oceanic taxa and species.

Of particular scientific and economic interest are species that produce harmful algal blooms (HABs) such as Microcystis spp., dinoflagellates, and diatoms belonging to the genus Pseudo-nitzschia such as P. pungens, P. multiseries, P. pseudodelicatissima, and P. australis. Pseudo-nitzschia is a pennate diatom that can produce the neurotoxin domoic acid under toxic environmental conditions related to iron limitation and copper toxicity. This powerful neurotoxin can be transported upwards through the food web resulting in amnesic shellfish poisoning (ASP) in humans.

We used a flow cytometer to measure the emission spectrum of this marine diatom by coupling the output of one of the light paths to a scanning monochromator. At each event, the monochromator position was digitized along with the other fluorescent parameters. At the same time, we also collected cytometric information along a second light path. The cytometric information contained in the bivariate dot plots allowed us to filter out signals that resulted from debris in the culture.

Cell cycle analysis

Many cancerous precursors are characterized by changes in the numbers of copies of chromsomes, or ploidy abnormalities. Elevated 4N (G2-tetraploid) cell populations are unstable intermediates in the development of many human cancers. One can use flow cytometry and cell sorting to purify populations of spontaneously arising premalignant 4N cells from cell strains derived from Barrett's esophagus biopsies. In order to accurately quantify the number of 4N cells, however, one must be able to discrimnate these cells from clusters of cells. In order to accomplish this, an inFlux detection module was modified to measure peak DNA as well as integrated DNA signal to accurately estimate the number of 4N cells in a clinical biopsy.

For more information on Barrett's Esophagus, see:

http://cancerres.aacrjournals.org/cgi/content/full/63/14/4211