A powerful capability of spectral technology is Unmixing. This allows researchers to separate fluorophores into pure signals that measure the quantity of each fluorophore at each pixel to more accurately measure data for analysis.
Spectral Unmixing separates each spectral fingerprint for complete and optimal visualization of fluorochromes.
Uniform measurement of Fluorescent Emissions
A correction system adjusts the offset and sensitivity of each channel of the 32 channel PMT to ensure a uniform and accurate measurement of fluorescent emission from 420nm to 800nm. The corrective system brings a standardization to all 32 PMTs ensuring the user is getting the most reliable data with the ease of adjusting only one voltage. This saves time over conventional flow cytometry operation where users must calibrate each individual PMT.
Pre and Post Correction Profile. Each graph illustrates pre and post correction in the SP6800 32 channel PMT. The correction improves accuracy of spectral visualization.
Spectral emission of PE Cy5 pre and post correction. Corrections support accurate unmixing of closely overlapping fluorescence spectra.
Subtracting Auto-Fluorescence Improves Visualization
In conventional flow cytometry cellular auto-fluorescence produced by pyridine (NAD/NADH), flavin (FMN, FAD), and other intracellular oxidative reactions can cause fluorescent signal contamination of other fluorescent markers. Other common sources of auto-fluorescence include cell fixation and permeabilization. Using spectral technology, auto-fluorescent spectral fingerprints can be subtracted to allow researchers to see the true fluorescent population.
Unstained mouse splenocytes were analyzed with the SP6800 revealing three distinct auto-fluorescent populations. Using the spectral fingerprints obtained in analysis, the appropriate auto-fluorescence can be removed, increasing the precision and quality of results.
Auto-fluorescence can result in the appearance of additional cell populations leading to erroneous data interpretation. A. T cells expressing GFP and stained with an antibody conjugated to Alexa Fluor® 700 were analyzed with the SP6800. A double positive population is present in the uncorrected density plot. B. This double positive population disappears when the auto-fluorescent spectral fingerprint is subtracted. C. Liposomes from cells expressing GFP were analyzed. D. This uncovered a small positive GFP population after auto-fluorescence was subtracted.
Fluorochrome Panel Guide
Application data dye options and combinations. Select one from each group.