CytoFlex Flow Cytometer Application Notes

WHITE PAPER

Expanding the Useful Spectrum for Flow Cytometry: Increasing the Number of Parameters Available Without Adding Compensation Complexity

Introduction As our understanding of biological systems increases, so does our knowledge of the complexity of those systems. Flow cytometry with its multi-parametric, high throughput capabilities has enabled the discovery of varied and nuanced cell types in terms of phenotypic markers and biological functions. In the modern marker panel, researchers need a minimum of eight colors to define identity and additional parameters to interrogate function 1 . We can expect that these requirements will continue to grow as cell biology advances. The visible spectrum places limitations on the number of extrinsic parameters that can be assessed as well as the number of dyes with suitable properties and non-overlapping emission spectra. Expanding the available palette requires exploring options on the periphery of the visible spectrum (Figure 2). Lower wavelengths, toward the blue and violet end of the spectrum, pose complications from auto-fluorescence. The fluorescence emission arising from endogenous fluorophores is excited by UV/blue wavelengths of light, typically due to structures and compounds in mitochondria and lysosomes 2 . The emission characteristics of auto-fluorescence is similar to fluorescein and PE and will, therefore, interfere with the detection of FITC and GFP fluorescence. Excluding auto-fluorescence isn’t always possible and quenching it can affect experimental signals. Expanding toward the far red end of the spectrum has been hindered due to limitations of photomultiplier tube detectors (PMTs). However, with the introduction of photodiode detectors, high and stable quantum efficiency has been demonstrated from 400 nm to 1100 nm.

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In this whitepaper we demonstrate the biological applications of an Infrared laser in a commercially available flow cytometer, the CytoFLEX S Flow Cytometer, which utilizes avalanche photodiode technology along with innovations in light management to provide a sensitive yet small research flow cytometer. IRChannel QuantumEfficiency Using Avalanche Photodiode Detectors Infrared light, of wavelengths from 750 nm to 1000 nm, occupies the edge of the spectrum after the red and extends into the microwave area. Photocathode materials, such as used in PMTs, limit the useful working range of these detectors to approximately 750 nm. In contrast, the Avalanche Photodiode (APD) exhibits good quantum efficiencies up to 1100 nm 3 . APDs improve the detection sensitivity of red and near infrared wavelengths (Figure 1).

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Figure 1. Photomultiplier Tube versus Avalanche Photodiode Quantum Efficiency by Wavelength. The PMT has historically dominated use in flow cytometers. The quantum efficiency drops rapidly for wavelengths above 600 nm. The APD however exhibits stable quantum efficiency up to 1000 nm. The bars on the lower axis indicate the CytoFLEX platform excitation wavelengths at 355 nm, 375 nm, 405 nm, 488 nm, 561 nm, 638 nm, and 808 nm. Adapted from “A Comparison of Avalanche Photodiode and Photomultiplier Tube Detectors for Flow Cytometry” by Paul Wallace et al, 2008, Proceedings of SPI, Vol 6859.

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