• Flow cytometry is a technique in which cells suspended in a buffered solution are counted as they pass through the beam of a laser. Cell suspensions are diluted and the flow rate of the suspension carefully controlled to ensure that one cell passes through the flow cell at any given time. Light detectors positioned around the flow cell can then detect any perturbation to the laser beam as each cell passes through.
  • The path of light through a cell depends on the size and internal composition of the cell. As light interacts with the cell and its internal components, it deviates from its initial direction. This is known as scattering.
  • Flow cytometers have a number of different detectors which measure the change in the beam of light as the cell passes through. Different characteristics of the cell can be detected based on certain parameters.
    • Forward scatter (FSC) is detected as changes to light which passes relatively straight through the cell. The degree of forward scatter is proportional to the cell surface area or size.
    • Side scatter (SSC) is detected as light which deviates significantly from the straight passage through the cell. It is proportional to the degree of internal complexity of the cell (eg. presence or size of the nucleus, presence of “granules”)

Scatter

  •  Through the positioning of light detectors, the degree of forward and side scatter can be determined and used to characterize the cells which pass through the flow cell.

eg. In the example below, a sample of white blood cells has been passed through the flow cell. Every time a cell passes through the flow cell, the degree of forward scatter and side scatter is measured and a point is plotted, graphing side scatter against forward scatter. 

Pass through flow cell

  • Using their FSC / SSC characteristics, a population of cells can be divided into subpopulations.
  • Flow cytometers can be calibrated to exclude objects above (eg. clumps of cells) or below (eg. cellular debris) a certain size

Fluorescence

  • The narrow bandwidth of the lasers used to count the cells in flow cytometry lends the process easily to the use of fluorescence to further categorise the cells.
  • Fluorescence is the emission of radiation following excitation by a higher energy of radiation. For example, when fluorescein isothiocyanate (FITC) is excited by light of a wavelength of 495nm (blue light) it emits light at a wavelength of 519nm (green light).
  • Fluorescent dyes can be conjugated to monoclonal antibodies which bind to proteins characteristic of certain cell types. This allows cells to be fluorescently labeled according to the proteins on their surfaces
  • Many flow cytometers use an argon laser which produces light at 488nm. This not only excites FITC, but also several other fluorogens which emit light at different wavelengths. This means that several different labels can be detected if the correct filters are used in conjunction with the light detectors.

eg.

Excitation

Emission

Wavelength

Colour

Wavelength

Colour

Fluorescein isothiocyanate (FITC)

488 (some)

Blue

519

Green

Phycoerythrin (PE)

488

Blue

570

Yellow

Data display

  • The output of the detectors can be displayed in a number of different ways :
    • Single Channel – generally expressed as a number of “events” (ie. cells) over the signal from a single detector. eg. if DNA is stained with a fluorescent marker such as propidium iodide, a population of cells may appear as :

Single channel

  • 2 Channel – expressed as a plot of the output from one detector with a filter for a single wavelength, against that of a second detector with a filter for a different wavelength. eg. in the following plot, a population of immune cells have been labelled with antibodies against the membrane protein CD3 (conjugated to FITC) and CD19 (conjugated to PE).

2 channel

Cell sorting

  • In conventional laser flow cytometry, cells which pass through the flow cell go to waste. In FACS (fluorescence assisted cell sorting), the characteristics of the cells determined in the flow cell may be used as a criteria to divert the cell to a collection chamber.
  • In order to sort cells, a set of criteria (a “sorting gate”) needs to be established which divided the cells into discrete groups. The four quadrants in the previous figure could be used to establish a sorting gate for these populations of cells.
  • As the cells pass through the flow cell, signals from each of the detectors are passed through to the processor, which makes a decision based on the characteristics of the cell and how these fit into the established sorting gate.
  • Depending on the model of the FACS unit, cell segregation may be achieved by :
     
    • Mechanical Means eg. a collecting tube swings into the flow stream to intercept the cells and divert them to the appropriate collection vessel.
       
    • Electrostatic Means eg. the flow stream is broken into a series of discrete drops through vibration, with each drop containing a single cell. Depending on the characteristics of the cell, each drop is given a charge, and charged plates are used to divert the drops to the collection vessel.