CytoFlex Flow Cytometer Application Notes

First Commercially Available Flow Cytometer Incorporating Infrared Laser and Avalanche Photodiode Detectors CytoFLEX S Blue-Red-Violet-Infrared (B-R-V-I) Series from Beckman Coulter is the first flow cytometer offering IR laser option as a standard product. The fully activated instrument includes four fluorescent channels from the 488 nm (Blue) laser, three from the 638 nm (Red) laser, four from the 405 nm (Violet) laser, and two from the 808 nm (Infrared) laser. The instrument includes 13 band pass filters which can be repositioned as needed. Instruments with as few as four fluorescent channels activated are available with the ability to activate additional parameters later (Figure 3).

This instrument is based upon the unique assembly of technologies comprising the CytoFLEX platform, which contributes to the exquisite sensitivity of the instrument. Borrowing technology from the telecommunications industry, the Wavelength Division Multiplexer (WDM) deconstructs and measures multiple wavelengths of light. The WDM relies on fiber optics and band pass filters to separate the light wavelengths. Unlike more traditional instruments, multiple dichroic filters to direct the light path are not required. This makes it much easier to configure the fluorescence channels, but also increases efficiency by minimizing light loss due to refraction. The WDM utilizes Avalanche Photodiode detectors (APDs), versus Photomultiplier tubes (PMTs). One hallmark of the photodiode is the high quantum efficiency in excess of 80%, especially for wavelengths greater than 800 nm. Integrated optics also increases the efficiency of light movement through the system by focusing light onto the flow cell. With conventional analyzers, laser excitation sources are optimized by shaping and focusing light through a series of lenses and filters onto the flow cell. Each of these light interactions is an opportunity for light loss. All of these technologies work together to ensure efficient light management for optimal excitation and emission of fluorochrome-tagged cells. This increased quantum efficiency results in decreased measurement variation. Low measurement variation reduces the standard deviation in measurements. The impact of this on resolution can be understood by considering the Staining Index. The Staining Index characterizes the relative brightness of different fluorochromes under actual experimental conditions. It quantifies the variance of the positive and negative population, normalized by the data spread of the negative population. The data spread has two components, one is population or experimental variation and the other is measurement error. The smaller this normalization value, the larger the Staining Index. A large Staining Index indicates that it will be easier to differentiate positive populations from negative populations. Figure 3. Detector Configuration of the CytoFLEX S Blue-Red-Violet-Infrared Instrument. The instrument includes four fluorescent channels from the Blue (488 nm, 50 mW) solid state laser, three from the Red (638 nm, 50 mW) solid state laser, four from the Violet (405 nm, 80 mW) solid state laser, and two from the Infrared (808 nm, 60 mW) solid state laser.

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