Flow cytometry

Flow cytometry is a method for the quantitative determination of cellular properties through the analysis of a light incident on a heterogenous population of biological samples.

 

Cells, or other particles, are individually passed through a light path at high speed while suspended in a tightly focused stream of fluid, causing an energy change in the incident light by scattering or fluorescent emission. The resulting light is detected and examined, allowing the various properties of thousands of cells to be determined or discriminated every second - such as size, shape, health, surface properties, proteins, by-products. This allows structures or components of the cell to be directly examined.

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This method enables quick, precise, non-invasive data collection of many different parameters simultaneously. It often utilizes multiple light sources and fluorophore labels during the same measurement, to enhance or better spatially restrict the signal by binding with specific constituent parts of the cell. It is even possible to discriminate the measured cells in real time, allowing complex liquids to be electrostatically filtered on a cellular level.

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There are numerous applications, particularly in the Life Sciences, though also in the fields of microbiology, food quality control, plant and animal cytology, among others. In immunology, for example, flow cytometry is used to identify, separate, and characterize different immune cell subtypes based on their size and morphology.

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Ultraviolet lasers are of increasing importance for Flow Cytometry applications, due in part to their decreasing cost and increasing availability, and in part due to an increase in applications requiring their use. The recent development of commercial ultraviolet fluorochromes – detection reagents used for the absorption of a specific wavelength, here in the UV – demonstrate their increasing relevance to this field.

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Historically, these UV requirements have been met by either DPSS lasers operating in the near-UV, most often at 375 nm, by HeCd lasers operating at 325 nm, or by argon-ion or krypton-ion laser systems. These laser sources are either not truly UV, or are large and require ongoing maintenance for operation. The availability of DPSS UV lasers, with solid state function and small footprint, offer a newly cost-effective and practical replacement for many of these applications.

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For instance, the detection of the fluorescent indicator, Indo-1, first introduced in 1985, allows for the ratiometric detection of calcium ion, Ca2+ - an important factor in the role of intercellular regulation. When excited at 349 nm, the emission peak of the fluorochrome shifts in the presence of Ca2+, allowing for the relative intensities of the two emission wavelength peaks to determine the concentration of the calcium ion present. Researchers are looking to develop new fluorescent probes at lower wavelengths, to allow further analysis on a wider range of simultaneously measured parameters by expanding the ‘colors’ available and increasing the need for suitable UV laser sources.

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There are several requirements when considering a suitable laser for flow cytometry:

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High power output will increase the signal strength measured, particularly for scattering effects, though this should be balanced with consideration of damaging the sample to be measured.

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Power stability and imperceptible power noise are also vital. For example, the magnitude of light scattering back towards the light source is used to determine the size of the cell and changes in the incident power level during the measurement will cause inaccuracy.

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Excellent beam quality and pointing stability are also key parameters for ensuring consistent and accurate analysis.

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Skylark design and manufacture single frequency continuous wave laser sources with unrivalled wavelength stability, narrow linewidths and long coherence lengths over a range of wavelengths within a small footprint. We currently offer single frequency lasers in the red area of the visible spectrum with our 640 Series Laser, along with our ultraviolet 349 NX Laser.

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