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Flow Cytometry and Cell Sorting

Flow cytometry is a technique that can simultaneously measure multiple physical and biochemical properties of a heterogeneous sample of cells suspended in a stream of fluid. It is typically used in clinical laboratories for analysis of samples such as; blood, bone marrow and lymphatic fluid for basic research, clinical trials and the diagnosis of disorders including blood cancers.

The ability to analyse the properties of thousands of cells passing through the detector array per second makes the technique a powerful tool in the clinical laboratory. By using input mechanisms such as hydrodynamic focusing to ensure cells pass through the measurement point one at a time, physical characteristics such as quantity, size, volume, morphology and colour can be measured by monitoring the behaviour of light from a laser, and the scatter that is caused by the interaction with the cell. The amount of light scattered in a forward direction is proportional to the size of the cell, whereas side scatter is caused by the granularity and complexity of the cell.

Additionally, if the cells are labelled with fluorescent active antibodies and the laser wavelength chosen is within the excitation bandwidth of the fluorophore, the resultant fluorescence can identify antigens specific to diseases like leukaemia, lymphoma, polio virus etc. As this can be done at a rate of thousands of cells per second, with a detection limit of 1 in 50,000, the flow cytometry laboratory can respond to clinicians with an effective result in under 2 hours.

Supporting this, if needed for further analysis, the modern flow cytometer can be used to electrostatically sort the cells into separate populations based on the measured results.

The cytometer often uses different laser wavelengths simultaneously to perform multiple analyses. By pre-treating the sample with multiple fluorophores that emit at different wavelengths, the efficiency of the technique can be further enhanced.

The choice of laser is therefore critical to the success of the cytometer and its results. Since one of the primary measures obtained is the “amount” of forward and side scatter, any fluctuation in the power output from the laser will artificially cause the measurement of particle size, granularity and internal complexity of the cell to change. Furthermore, since the diagnosis of antigen content can very often rely on the comparison of intensity between two fluorophores, wavelength and power output stability is also critical.

Laser Quantum lasers incorporate a power output feedback mechanism that maintains power output stability to industry leading levels and are therefore ideal for use in this increasingly-used detection technique.

  • gem – OEM integration laser with power stability of <1% measured over 100 hours
  • ventus – Scientific grade laser with easy power control and power stability of <0.4%

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