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TitleUnderstanding the Fluorescence Change in Red Genetically Encoded Calcium Ion Indicators.
Publication TypeJournal Article
Year of Publication2019
AuthorsMolina, Rosana S., Yong Qian, Jiahui Wu, Yi Shen, Robert E. Campbell, Mikhail Drobizhev, and Thomas E. Hughes
JournalBiophys J
Volume116
Issue10
Pagination1873-1886
Date Published2019 May 21
ISSN1542-0086
Abstract

For over 20 years, genetically encoded Ca indicators have illuminated dynamic Ca signaling activity in living cells and, more recently, whole organisms. We are just now beginning to understand how they work. Various fluorescence colors of these indicators have been developed, including red. Red ones are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a red fluorescent protein that is circularly permuted and fused to a Ca-sensing domain. When Ca binds, a conformational change in the sensing domain causes a change in fluorescence. Three factors can contribute to this fluorescence change: 1) a shift in the protonation equilibrium of the chromophore, 2) a change in fluorescence quantum yield, and 3) a change in the extinction coefficient or the two-photon cross section, depending on if it is excited with one or two photons. Here, we conduct a systematic study of the photophysical properties of a range of red Ca indicators to determine which factors are the most important. In total, we analyzed nine indicators, including jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, and a new variant termed jREX-GECO1. We find that these could be separated into three classes that each rely on a particular set of factors. Furthermore, in some cases, the magnitude of the change in fluorescence was larger with two-photon excitation compared to one-photon because of a change in the two-photon cross section, by up to a factor of two.

DOI10.1016/j.bpj.2019.04.007
Alternate JournalBiophys. J.
PubMed ID31054773
PubMed Central IDPMC6531872
Grant ListF31 NS108593 / NS / NINDS NIH HHS / United States
U01 NS090565 / NS / NINDS NIH HHS / United States
U01 NS094246 / NS / NINDS NIH HHS / United States
U24 NS109107 / NS / NINDS NIH HHS / United States