Whole-body imaging

MOSCOW—For in vivo studies, researchers need fluorescent proteins that fall within the favorable optical window between visible light absorption by water (>1100 nm) and melanin and hemoglobin (<650 nm). The red fluorescent proteins (RFPs) best suited for this window, however, offer limited brightness. Recently, researchers at Evrogen and the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry addressed this problem.

 

As they report in Nature Methods, the researchers mutated TurboRFP, a pH-stable, high-brightness RFP they previously isolated from sea anemone, focusing their efforts on the amino acids surrounding the protein's chromophore. In the process, the researchers generated Katushka, an RFP with high brightness (about 0.67x EGFP) and far-red fluorescence (max 635 nm). Transfected into muscle cells of frogs, Katushka fluorescence was highly visible in both embryos and adult frogs, and exhibited fewer toxic effects than other fluorescent proteins.

 

The researchers then generated a monomeric version of Katushka—mKate—to facilitate the labeling of cellular proteins. mKate offered almost identical fluorescence spectra to Katushka and provided distinct labeling with fused in vivo to β-actin and α-tubulin. The Katushka and mKate also exhibited 2- to almost 6-fold greater photostability than commercially available fluorescent proteins.