Flow cytometry: properly characterizing your laser’s power level, stability and profile
Thursday, April 23, 2020
The evolution of laser technology and design has contributed significantly to the advancement of cytometers and sorters in the last decade. Smaller equipment has been made possible by the development of smaller lasers with adequate power outputs and stability.
However, despite continuous advancements, one thing remains constant – the need to properly characterize your cytometer laser beam. Understanding the different properties of lasers and how they impact fluorescence and overall accuracy can help ensure that your cytometry equipment performs at optimal capacity.
While laser beams consist of several essential properties, the most crucial of these is the profile. Although the beam emitted from the laser is symmetric, its intensity can vary across its width. This results in a beam shape where photons are most concentrated in the middle while tapering off at the ends.
In simpler terms, the beam is more intense in the center than its edges. This phenomenon is known as a Gaussian or normal distribution. Since most of this energy is at the center of the beam, it is necessary to have the cell samples flow through this region to generate the most fluorescence.
Cells that pass through the edge of the beam, where the power is weakest, are exposed to fewer photons and, therefore, generate less fluorescence and contribute to inaccurate readings.
Since most cells in flow cytometry are smaller than 20 µm in diameter, the spot size, and power distribution across the beam width (profile) needs to be as small and precise as possible. To achieve this level of accuracy, consistent calibration and measurement using the right measurement equipment is critical.
In addition to the power distribution, another laser beam property that is crucial in flow cytometry is the actual output power level (usually measured in milliwatts). This characteristic, which can be adjustable on some cytometers, is simply a measure of how many photons are released from the laser per unit time.
Since cytometry depends largely on fluorescence, the more photons cells are exposed to, the higher the chance that it will be illuminated; therefore, the higher the possibility that a useful biological reading can be obtained.
Typical cytometer lasers range between 20 mW to 100 mW, although some devices are designed to output higher power levels.
Flow cytometry is among one of the most demanding laser applications in terms of accuracy and precision. Therefore, your laser’s characteristics, including the power distribution, power value and overall beam stability need to meet stringent specifications to achieve the required accuracy.
A robust beam profiler, which measures the spatial distribution of power across the beam diameter, is particularly crucial, especially when it comes to detecting cells that are in the order of microns.
The right power detector should also be used to ensure that the photon output of your beam meets the requirements of your application.
As we can see, characterizing your cytometry laser beam should be a top priority when dealing with cells that are not visible to the naked eye. Be sure to characterize and regularly calibrate your beams with the right profilers and power detectors to minimize costly errors due to inaccurate reading.