- Sony Biotechnology
- Sony Biotechnology
The ID7000 spectral cell analyzer builds on Sony’s experience with spectral analysis and simplifies many operations, even for complex experiments. With the true signal for each fluorochrome unaffected by autofluorescence or subjective adjustment of spillover, spectral analysis yields cleaner, unbiased data for every experiment.
In spectral analysis, signals from all detected channels are used to create one spectral emission signal, regardless of the number of fluorochromes analyzed. Unmixing, a powerful capability, then separates fluorophores into pure signals that measure the intensity of each fluorophore at each wavelength to more accurately measure data for analysis. The ID7000 uses the WLSM (Weighted Least Squares Method) fluorescence unmixing algorithm to separate the individual spectral fingerprints and enable scientists to analyze dim and rare phenotypic marker expression.
Unmixing delivers a more comprehensive picture of rare populations while decreasing the complexities associated with working with fluorescent proteins and fluorochromes excited by multiple lasers. Overall, spectral technology simplifies multicolor panel design by eliminating the use of bandpass filters and highly subjective, conventional compensation matrixes.
Compensation beads were stained with three different sets of fluorescent antibodies, and excited by the 405-nm, 561-nm, and 637-nm lasers as described below. Data was analyzed on the ID7000 spectral cell analyzer.
A. For the fluorochromes BV421/ eFluor™ 450/ BV480/ BV510/ eFluor™ 506/ Pacific Orange™ excited by the 405-nm laser, distinctly resolved emission peaks and populations were observed.
B. For the fluorochromes PE/ CF®568/ PE/Dazzle™ 594/ PE-Alexa Fluor® 610/ PE/FIRE™ 640/ PE-Cy™5/ PE-Cy™5.5/ PE-Alexa Fluor® 700 excited by the 561-nm laser, distinctly resolved emission peaks and populations were observed.
C. For the fluorochromes APC/ Alexa Fluor® 647/ Spark NIR™ 685/ APC-R700™/ Alexa Fluor® 700/ Zombie NIR™ excited by the 637-nm laser, distinctly resolved emission peaks and populations were observed.
Spectral analysis, while allowing researchers to see the full emission signal without using bandpass filters, also enables autofluorescence to be handled as a separate color. In conventional flow cytometry, cellular autofluorescence produced by pyridine (NAD/NADH), flavin (FMN, FAD), and other intracellular oxidative reactions can interfere with signals of other fluorescent markers. Other common sources of autofluorescence include cell fixation and permeabilization. Spectral technology subtracts one or more autofluorescent spectral fingerprints to allow researchers to see the true fluorescent populations.
Spectral emission curves from a sample stained with nine fluorochromes are shown, as analyzed with a 5-laser ID7000 system. The cellular autofluorescence spectral curve can be seen along with the fluorescence spectral curves due to the nine fluorochromes used. The ability to distinguish the cellular fluorescence contribution independently from the signal due to the fluorochromes yields unbiased data.
An unstained lysed whole blood sample was analyzed with the ID7000 cell analyzer to understand the contribution of cellular autofluorescence.
A. The figure shows spectral ribbon displays when studying the gated populations of lymphocytes, granulocytes, and eosinophils. Removing the signal due to this autofluorescence by using spectral fingerprints obtained in the analysis, increases the precision and quality of results.
B. This figure shows the parametric display for the same samples. In the first row (i) the plots show presence of additional spurious populations as a result of intrinsic cellular autofluorescence. In the second row (ii) the autofluorescence subtraction is applied to remove contributions of AF1, AF2, and AF3, and the spurious populations are eliminated.
Users acquire single-color control data before experimental samples so that the unmixing algorithm has emission spectra inputs that it can reference to unmix the experimental sample. Single-color control spectra are stored in the Spectral Reference Library. Unlike compensation controls, spectral references do not need to be acquired for every new experiment and can be reused. This allows users to create a personal or shared reagent library that simplifies experiment creation, saves time and valuable reagents, and increases efficiency.
The ID7000 system can be operated in a Normal or Standardization mode. Standardization mode sets the system to an optimized master specification that allows researchers to maintain instrument settings between experiments and across multiple instruments to support longitudinal studies and cross-site collaboration. Standardization mode adjusts the output of each channel so that the SSC and fluorescence detection sensitivities are the same between multiple instruments.
PMT voltage correlation coefficients are calculated for each laser detection deck during daily QC, resulting in standardized measurements across all lasers, detectors, and instruments. Standardization mode voltages can be adjusted synchronously or for each laser detection deck. The system supports standardization of Area and Height data. This unique capability allows scientists to obtain the best data at maximum PMT voltages and reduces the need to rerun single-color controls for every experiment. Standardization mode minimizes subjectivity and instrument variability to yield highly reliable, accurate, and reproducible results.