Flow Cytometry and Cell Sorting

Determine a cell's size, complexity, and fluorescence intensity

The cell’s size, internal complexity, and fluorescence intensity are determined using an optical-to-electronic coupling system. These parameters can be used to separate cells into different populations one by one, through a technique called Fluorescence Activated Cell Sorting (FACS).

A flow cytometer incorporates a fluidics, optics, and computer system in order to characterise fluorescently tagged cells.

Flow cytometry

A flow cytometer is made up of three main systems: 

  • Fluidics
  • Optics
  • Electronics

The fluidics system funnels cells contained in a saline solution through a hydrodynamic focusing junction. This uses sheath fluid to constrict the sample flow and create a single-file stream of cells.

The optics system consists of a laser or lasers to illuminate the particles in the sample stream and dichroic mirrors to direct the resulting light signals towards the appropriate detectors. 

The electronics system converts the detected light signals into electronic signals that can be processed by the computer.

Physical characteristics such as the size, shape, and internal complexity of a cell can be determined by illuminating it with a laser beam and measuring the forward- and side-scattered light.

Light scattering occurs when a cell deflects incident laser light. The extent to which this occurs depends on the physical properties of a cell, namely its size, shape, and internal complexity. 

  • The cell’s size is proportional to the amount of forward-scattered light, since it is a measurement of mostly diffracted light.
  • The cell’s internal complexity is proportional to the amount of side-scattered light. It is a measurement of mostly refracted and reflected light that occurs at any interface within the cell where there is a change in refractive index. Side-scattered light is collected at approximately 90° to the laser beam by a collection lens and then redirected by dichroic mirror to the appropriate detector.

Fluorescence activated cell sorting (FACS)

Fluorescence is a phenomenon in which certain molecules, namely “fluorophores”, become luminous after having been exposed to light. Samples are initially prepared by adding fluorophores which bind with specific types of tissue. 

The fluorescence pattern, combined with front-scattered and side-scattered data from flow cytometry, can be used to identify which cells are present in a sample and to count their relative percentages. The cells can also be sorted, and this is known as fluorescence activated cell sorting (FACS). 

When a fluorophore is excited by laser light in a flow cytometer, the fluorescent signal it emits will be collected by the optics system. A combination of dichroic mirrors and filters partition the scattered and fluorescent light, directing it to the appropriate detectors. The scatter of the laser light and the fluorescent signal tell a computer which population each cell belongs to. In addition to quantitively measuring each cell’s size and internal complexity, the computer may sort fluid droplets into categories of ‘no cell’, ‘cell with no fluorescence, ‘cell with red fluorescence’, and ‘cell with both green and blue fluorescence’, etc.

Applications

Flow cytometry is a useful tool for investigating many aspects of cell functions. It is established as a very important clinical technique in the areas of blood cells, cancer diagnostics, prognosis, and monitoring of diseases. Flow cytometers are also employed in other areas of lifescience research, drug discovery, and agricultural science research to assess cell lifecycles, cell proliferation, and DNA content. 

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