Coherent’s Dr Matthias Schulze explains how flow cytometry supports research including immunology – New UV Wavelengths and Integrated Laser Engines Benefit Flow Cytometry
Clinical labs and research facilities are making increased use of multi-parameter flow cytometry in part due to the fight against the current Covid-19 pandemic.
Coherent is supporting faster instrument design and increasing the number of parameters in two ways: integrated multi-wavelength OEM subsystems and new laser wavelengths in the ultraviolet. All with output beam parameters optimised for common flow cytometry platforms.
Flow Cytometry Background and Trends
Flow cytometry is a widely used method to analyse (count) and/or sort cells, sperm and other bio-entities according to one or more distinct parameters. This entails forcing the cells to pass in single file through an interaction zone often using a flow conformation called hydrodynamic focusing. In this zone, the cells are then sequentially irradiated by one or more focused laser beams. The resultant scatter and (Stokes shifted) fluorescence is collected by optics and separated by cut-off and bandpass filters into discrete wavelength bands. Light in each band is then quantitatively measured using a photodetector such as an avalanche photodiode (APD). For example, in blood counting applications in clinical laboratories, the cells are treated with fluorescent labels (fluorochromes) that are bonded to antibodies targeted at specific antigens on the outer membrane of the cells. For every cell that passes through the interaction zone, analysis of the relative intensities in the wavelength bands can be used to count the population as a function of different parameters typically using multivariate parameter analysis. In addition, scatter at key angles can be measured to determine information about cell shape.
For some research applications such as oncology, immunology and drug discovery, flow cytometry can be used to sort cells, i.e., to selectively collect a target type of cell while discarding all the others. In a sorting instrument the flow passes through a nozzle prior to the interaction zone that ensures the cells are individually held in tiny droplets which pick up a small static charge from the nozzle. Electrostatic plate electrodes are then activated to create a field that deflects the charged droplets into a collection tube, according to the cell type registered by the instrument’s computer.
Although flow cytometry has a history spanning decades, it is currently a remarkably dynamic field. In research applications, a key trend is increasing the number of sorting parameters. In the clinical field, instrument builders are responding to a growing market demand for benchtop economical instruments driven by increasingly personalised medicine in the west and the demand for better health care supported by growing affluence in Asia. These trends are supported by laser manufacturers by the development of new wavelengths, particularly in the ultraviolet, and off the shelf multi-wavelength laser engines that simplify the design and manufacture of next generation cytometry instruments.
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