Principles of Flow Cytometry

Flow cytometry combines cell biology with the study of light waves. When a beam of light is bounced off a particle, the light scatters in two directions. The angles from the scattered light are measured, and the metrics create an image of objects between 0.5um to 40um in diameter. Flow cytometry metrics are used to measure several properties of biological cells, including size, granularity, cell receptors, and even biological activities, such as DNA and RNA replication and synthesis.

Process

• Flow cytometry techniques start by taking a biopsy of cells and passing them one-by-one through a beam of light in a cytometer. As the cells pass through the beam, the light scatters and measurements of the refractory angles are taken. It is conducted in conjunction with a fluorescent microscope, which is used to evaluate components of a cell through light emissions of fluorescent dyes.

Flow Chamber

• The purpose of the flow chamber is to act as a funnel to ensure that the cells are delivered to the beam of light in a single file line. The funnel is filled with a sheath fluid, and it is pushed through the funnel with the suspended cells. The funnel width is typically 10 micrometers, and the fluid is pushed into a collecting lens that is used to scatter the light.

Light Source

• Although the majority of labs use a laser, other light sources include an arc lamp or LED. An arc lamp is not capable of detecting weaker fluorescence, but it can be used with technology that uses strong fluorescent emitters, such as DNA analysis. Primarily, the light source is an argon laser that emits blue light at 488 nanometers.

Immunology

• Flow cytometry is used to measure a number of conditions in immunology and infection. Flow cytometry is used in diagnostics of HIV for both hereditary and acquired forms. It is also used to measure levels of white blood cell proliferation in cases of lymphoma and carcinomas. Additionally, immunology uses flow cytometry to measure the results of bone marrow transplant patients.

DNA Analysis

• Flow cytometry is used to measure the amount of DNA in the nucleus of the cell. By measuring DNA, the specific phase of the cell cycle can be identified. Flow cytometry can also detect abnormal number of chromosomes to identify genetic diseases. DNA analysis with flow cytometry is also used to detect Barrett's esophagus.