Spectral Flow Cytometers
Overview and technology
Spectral Flow Cytometry is a new generation of flow cytometers, originally created by Sony Biotechnology, that collects the entire emission spectrum of fluorophores instead of just a band of the emission spectrum for each detector – as in done in conventional flow cytometry. Traditionally each fluorophore emission was detected by a separate detector thus “grabbing” this part of the spectrum using a bandpass optical filter.
With this methodology, if multiple fluorescence markers are to be detected simultaneously, careful planning of the fluorescent panel as well as complex compensation matrices are required.
Sony Biotechnology has dramatically innovated the detection method of FACS analyzers by collecting the entire spectrum of emitted light for each fluorophore, thus creating a spectral fingerprint for each marker that is unique. This method significantly simplifies the data collection and analysis process – thus allowing a much wider range of panels to be used, especially for high count multi-color experiments. The separation of the different colors is performed by a spectral unmixing algorithm, instead of compensation, which increases the sensitivity and quality of the data.
Spectral flow cytometry is heavily used in the field of immunology, where there is a need for 10-40 markers simultaneously to analyze the different cell populations.
Spectral cytometry also enables the detection of the entire autofluorescent spectrum. For cells with high autofluorescence, this is used to either subtract the autofluorescence from the marker emission or as a fluorescent parameter of its own – to distinguish specific cell types and/or to indentify cell apoptosis and necrosis processes without additional markers.
Spectral cytometry increases simplicity, combination, and quantity of fluorescent panels, sensitivity, and quality of data over conventional flow cytometry.
Flow cytometer Vs FACS
specialized flow cytometers called fluorescence activated cell sorters (FACS) leverage the capability to identify different cell subsets to sort out and recover them for experimentation. Often, the terms FACS and cytometers are confused and used interchangeably. However, if your lab is focused on analytics of cell populations rather than cell manipulation, you have no reason to make the considerable investment required to purchase a FACS machine.
Here at Merkel we stand prepared to advise you on the device best suited to your needs, and provide you with full post-purchase support of your selected product – all at an affordable price.
What can you measure with flow cytometry?
- RNA levels – including IncRNA, miRNA, and mRNA transcripts.
- Protein expression levels – not just in the cytoplasm, but in the nucleus as well.
- Post translational protein modification – including cleavage and phosphorylation.
- Cell cycle stage – Go/G1, S phase, G2, Polyploidty and proliferation.
- Most incredibly, in addition to the above parameters, the flow cytometer can identify specific cell subsets, be it developmental stage (Embryonic stem cells to cardiomyocytes) or immune activation status.
Flow cytometry was first used back in the 1950s, but a lot of cell medium has flowed in front of the viewing aperture since then! Advances in the field have resulted in a modern flow cytometer which is very different than the jury-rigged device used 70 years ago. Modern flow cytometers can measure the parameters of over 10,000 cells per second and certain models can measure up to 30 detectable parameters at the same time in the same cell.
This incredible speed and ability to zero in on multiple parameters of individual cells provide the cell biology researcher with a powerful statistical tool. With it, you can analyze the characteristics of millions of cells, categorized by size, developmental stage and so forth.