Introduction
Applications of flow cytometry include:
- Cell sorting, cell counting
- Cell analysis: biomarker detection, cell viability, cell cycle status, redox state, ligand binding, protein phosphorylation
- Cancer diagnosis (especially for immunology and hematological diseases)
- Isolation of rare cells (adult stem cells, liquid biopsy)
- DNA enrichment for downstream sequencing
- Quality control of cell therapy products
- Detection of micro-organisms
- Drug discovery
Steps in the flow cytometry analysis
- Isolation of sample for analysis: for example, blood drawn from the patient or cultured cells isolated
- Sample suspension: centrifugation, enzymatic digestion or mechanical dissociation and filtration to produce single-cell suspension
- Fluorescent staining: target-specific antibodies that are attached to specific fluorophore labels for fluorescence detection
- Optical detection by flow cytometry
- Software-driven data analysis
Optics in flow cytometry
Lasers are used for illuminating samples in flow cytometry because they provide very focused, noise-free, and monochromatic illumination as the samples are passing the illumination spot in a narrow line. Rapid and powerful illumination by the lasers is needed as the cells pass by the laser at rates of 10,000 cells per second or in some instrument even at up to 80,000 cells per second, making the requirement for optical detection and analysis to be very rapid without compromising accuracy. Detection is done with photomultiplier tubes or avalanche photodiodes, and some newest instruments on the market can even have fluorescence cameras to visualize the location of fluorescent dyes in the cells. 3 separate detection technologies are used in combination to comprehensively characterize the sample properties:
1. Forward-scattered laser light: indicative of particle size
2. Side-scattered laser light: indicative of particle granularity and complexity
3. Fluorescence emission: detection of target biomarkers by fluorescently labeled antibodies
Flow cytometers can have from one to multiple laser lines for exciting different fluorescent labels each with own absorption and emission characteristics. The laser wavelengths are chosen based on the most commonly used antibody labels in biosciences: 405 nm, 445 nm, 488 nm, 532 nm, 561 nm, 633 nm, 640 nm, 660 nm, and 810 nm. Even with one laser, several different fluorescent labels with overlapping absorption spectra can be excited; however, having multiple laser wavelengths increases the versatility and flexibility of the system: multiple fluorescent targets can be detected within the same experiment, with more freedom for fluorophore selection.
In addition to wavelengths, extremely high laser uniformity and power stability are critical for flow cytometry analysis since any fluctuations of laser will artificially cause forward and side scattered light to change. This also affects fluorescence detection, since diagnosis of antigen expression levels often relies on comparison of fluorescence intensity between 2 fluorophores, which is heavily dependent on laser parameters.
Modulight is specialized to provide whole fluorescence sensing solution for flow cytometry, with all of the most important wavelengths in a compact package. Sensitive detection optics can be tailored to fit the final system’s requirements in terms of filtering and spatial configuration.
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