Placing several fluorescent proteins together in a flow cytometry panel offers greater power and capability for experiments. However, handling autofluorescent signal with fluorescent proteins is out of reach for conventional flow cytometetry users. Sony spectral flow cytometry analyzers enable researchers to harness up to five near infra-red fluorescent proteins in a single experiment. Moreover, spectral technology lets users accurately identify autofluorescence, and eliminate it if needed.

In 2008 the Nobel Prize in Chemistry was given to Shimomura, Chalfie and Tsien for the discovery and development of green fluorescent protein (GFP). This endogenously fluorescent protein was originally cloned from the jellyfish Aequorea Victoria.Since that time it has been extensively modified to improve performance in mammalian systems and to expand the palette of fluorescent proteins. This has resulted in new fluorescent proteins that are excited and detected at different wavelengths, allowing more proteins to be detected simultaneously.

Flow cytometry is a popular and common method for the detection of fluorescent proteins. However, conventional flow cytometers rely on optical filters to separate overlapping signals, which does not allow some combinations of fluorescent proteins to be resolved or compensated correctly. In contrast, spectral flow cytometry captures has no optical filters and uses spectral unmixing to separate highly overlapping fluorochromes.

To expand the number of available fluorescent proteins, Telford, et al. examined members of the iRFP series, proteins isolated from bacterial phytochromes, excited by red and near infared (NIR) lasers. Conventional flow cytometry can only detect two of the five iRFP at the same time. Spectral flow cytometry can detect all five fluorescent proteins at once. Combining these new fluorescent proteins with spectral technology will expand the number of fluorescent proteins that can be studied together in the same experiment.


References

Tsien, Roger Y., et al.  “The Green Fluorescent Protein” Annu Rev Biochem. 67 (1998): 509-544. doi: 10.1146/annurev.biochem.67.1.509 PubMed

Telford WG, et al. “Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes.” PLoS One 10(3) (2015): e0122342. doi:10.1371/journal.pone.0122342 PubMed