Flow Cytometry Application Guide

Basics of Flow Cytometry

Flow cytometry is a high-throughput application that allows for the detection of multiple fluorochromes in a sample consisting of thousands to millions of cells in an extremely rapid fashion. These systems utilize very sensitive PMT-based detectors and often use comparatively narrow bandpass emission filters to capture the multiple fluorescences.

Total internal reflection fluorescence (TIRF) microscopy places unusually high demands on emission filters and dichroic mirrors. Unlike standard widefield setups, the reflected excitation beam travels back through the objective — meaning emission filters must block excitation energy they'd never see in other configurations, and dichroic mirrors must maintain flatness specifications that are difficult to achieve with standard manufacturing methods.

Filter Requirements for Flow Cytometry

All flow cytometers use multiple lasers to drive the individual fluorescences, as well as emission filters to block/attenuate the lasers while transmitting the appropriate emission wavelength ranges. However, these systems can differ in their overall optical configuration.

Some flow models are linear in their configuration, which allows the operator to use common dichroic mirrors and emission filters designed for use at 45 and 0° incidence (AOI), respectively. Other systems require the use of dichroic mirrors designed for use at other AOI (e.g. 11.25°), while still using common 0° AOI emission filters in front of the detectors.

Additional Resources

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Our applications team works with flow cytometry setups across most major commercial platforms. Tell us your instrument and fluorochromes and we'll point you in the right direction.

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