Flow cytometry is a laser-based technology used to analyze, characterize, and define cell expressions in a cell population. With the proper flow cytometry equipment, operators can analyze the makeup of heterogeneous cell populations to determine purity levels, gain more insight into the shape of individual cells, or analyze changes to a sample over time. As an example, scientists can measure fluorescent-labeled antibodies to determine their intensity.
As a laser-based process, flow cytometry relies on the accuracy and quality of its glass and optical components to accurately analyze samples. Laser light and optics create a controlled environment of illumination and light collection to derive results. Filters are particularly crucial for collecting and moving light along the guided path, with different filters available for specific fluorophores.
Some of the most common applications for flow cytometry include:
Adjacent dyes have spectrally wide emissions, which generates significant overlap. Dichroic filters split off fluorescent signals that are very close to each other that may otherwise confuse results. They’re commonly used in multicolor detection setups, which use multiple fluorescents simultaneously to gather information more efficiently. More simultaneous colors necessitate more filters.
Multichannel systems demand properly aligned emissions filters that can isolate and collect a specific fluorescent signal without collecting other signals within the sample. As multichannel systems become more complex, the need for filters that address existing fluorescent signals and cross-talk will also grow in complexity.
Emission filters are pieces of coated glass. The coats on both sides of the glass control which wavelengths are allowed to pass through unobstructed while simultaneously disrupting other wavelengths. Flow cytometry systems typically use bandpass filters, which determine the specific fluorophores detectors a system will measure. Other common filter types include longpass and shortpass varieties.
Today’s flow cytometers use multiple lasers to excite the fluorescing dyes and can measure up to 20 fluorophores, allowing technicians to gather more data simultaneously. However, high-quality multicolor detection is only possible with reliable interference filters. These components — made from emission filters and mirrors — differentiate the various fluorescence signals so the measured light is not confused or contaminated with other light.
Many optical instruments use polarized light to collect information about fluorescence and scatter from a sample, but polarized light creates polarization bias throughout the light source, filters, detectors, and other components. Dichroic mirrors have especially notable polarization bias because of their atypical angles of incidence.
Mirrors provide spectral resolution so operators can distinguish between scattered and fluorescent light. Dichroic mirrors have a one-sided coating that controls the light wavelength range it will permit to pass through. So, it’s important to find components with minimal possible sensitivity to polarization bias while still allowing for clear fluorophore readings.
At Evaporated Coatings, Inc. (ECI), we specialize in coating high-quality optical filters and mirrors for high-performance flow cytometry systems. We provide optical filter coatings for applications ranging from aerospace and telecommunications to biomedical lasers and laser safety. Our popular custom filters include:
We also provide metal mirror coatings, dielectric mirror coatings, and laser dielectric mirror coatings. Our standard and custom optical coatings are deposited through our in-house deposition systems to guarantee ideal optical performance.
Increasingly advanced flow cytometry systems rely on fine-tuned filters. The right optical coatings guarantee clear results and long-term reliability. To learn more about our optical coatings capabilities, please don’t hesitate to contact us or request a quote today.