|  | Public Health Safety | Public Health

Essential Flow Cytometry Methods

Flow cytometry involves the principle of hydrodynamic focusing, using a light excitation source such as a laser, to measure and analyze the physical characteristics of individual particles. Flow cytometry is predominantly applied to cell analysis, and the properties analyzed include cell size, granularity and fluorescence intensity. Essential detection methods used in flow cytometry include forward-scattered light, side-scattered light and fluorescence.

  1. Flow Cytometer

    • A flow cytometer is comprise of fluidics, optics and an electronics system. The fluidics system is responsible for transporting the sample stream to the laser beam for interrogation. The optic system contains lasers and optical filters that illuminate samples and direct light signals to the relevant detectors. This detected light is converted to electronic signals by the electronic system, for interpretation by the computer.

    Forward-Scattered Light

    • Diffracted light scattered in a forward direction is detected approximately 20 degrees of the axis from the laser beam by a photodiode called the forward-scattered channel (FSC). Forward-scattered light is directly proportional to particle surface area, and is used as an indicator of cell size and to distinguish between living and dead cells. Due to these characteristics, FSC is applied to immunophenotyping as a trigger of signal processing.

    Side Scattered Light

    • Side-scattered light refers to light refracted at any cellular interface, which is measured at approximately 90 degrees from the laser beam axis by the side-scatter channel (SSC), then redirected to the relevant detector by a beam splitter. Side-scattered light is roughly equivalent to cell granularity or internal complexity, and is used to determine the granular content of cells. Side and forward-scattered light are used in combination to distinguish between different cell types.

    Fluorescence

    • Fluorescence provides both quantitative and qualitative information on intracellular molecules such as cytokines and DNA, and cell surface receptors, by using fluorochrome labels and measuring at different wavelengths. Each fluorochrome label has a specific wavelength rang at which light is absorbed called the absorption spectrum. Fluorescence functions as a photon of light is emitted by an excited electron subsequent to light absorption.

Public Health - Related Articles