Fundamentals

What is Flow Cytometry?

Flow cytometry is a process in which individual cells or biological particles are labeled with fluorescent markers and pass in single file through a fluidic stream. While in the fluidic stream the cells are hit by a laser beam resulting in emitted scattered visible light and fluorescence detection. Physical and chemical properties of cells or particles are subsequently analyzed. Flow cytometric analysis has been performed on mammalian lymphocytes and other cellular elements, chromosomes, tumor cells, bacteria and fungi.

 

What applications are performed using a flow cytometer?

Chemical and physical characteristics of cells which can be measured by flow cytometry include cell size, cell shape, surface membrane receptors, DNA content, nuclear antigens, intracellular Ca2+, intracellular pH, and gene expression.  Flow cytometers can analyze a population of cells, at rates of 1000 to 30,000 cells per second depending on the type of flow cytometer. One powerful application of flow cytometry requires identification and subsequent characterization of a subpopulation of cells within heterogeneous populations. This is referred to as cell sorting.  In comparison to other technologies such as fluorescence microscopy, flow cytometry offers many advantages such as large sampling statistics, precision, and sensitivity. In comparison to RIA and ELIZA methodologies, flow cytometry has the ability to detect rare cells and select specific subsets within a mixed population.

 

What are the components of a flow cytometer?

The basic components of a flow cytometer/cell sorter are a laser light source, a fluidic sample chamber and optical assembly, and photodetectors and processors to convert light signals into subsequent digital signals. The instrument is interfaced with a computer system for the storage and analysis of digitized listmode data. 

 

What parameters are measured with flow cytometry?

Cells pass in a single file into the center of a flow cell or nozzle where they are surrounded by sheath fluid which is typically isotonic saline. As the cells pass through the stream they are hit by the laser beam.  Some light is scattered at very small angles (3-12o) and this is referred to as forward angle light scatter, which is a function of cell size and is detected with a photodiode. Light which is scattered at larger angles (90o), is proportional to the magnitude of cellular granularity or internal complexity, and this is referred to as right angle or side scatter and is detected with a photomutliplier tube (PMT). If the cells are stained with particular fluorescent marker(s), fluorescence emission is also detected 90o to the incident laser beam with a PMT.  The PMTs allow conversion of photons  to electrical impulses. Subsequently, signals are amplified and evaluated, digitized and fed to a pulse height analyzer (PHA) for storage. The listmode data is illustrated in the form of histograms or dot plot displays with the computer interface.