Understanding the Fluorescence Change in Red Genetically Encoded Calcium Ion Indicators

Abstract

For over 20 years, genetically encoded Ca 2þ indicators have illuminated dynamic Ca 2þ 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 2þ -sensing domain. When Ca 2þ 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 2þ 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. a Excitable form of the chromophore, anionic (A) or neutral (N). b Estimated relative SD. cThe F n maximal ratios were measured directly by taking the ratio of total fluorescence signals normalized to the known relative protein concentration between the Ca 2þ -free and Ca 2þ -saturated samples.

Different categories of fluorescent proteins result in GEVIs with similar characteristics

“…The activity dependent change of fluorescence intensity in FP-biosensors can result from changes in the extinction coefficient, quantum yield, and/or change in the protonation equilibrium of the chromophore 50 (Molina et al, 2019). From existing studies, it seems that amino acid residues surrounding the chromophore have the most effect on amplitude of GEVI voltage sensitivity 13,18,19,23,24 .…”

Optically Activated, Customizable, Excitable Cells - A Kuhl Platform for Evolving Next Gen Biosensors

“…These prototypes will be expressed in cells that also express a benchmark sensor such as GCAMP7f. Finally, the most promising variants would then be photo physically characterized (Molina et al 2019; Barnett, Hughes, and Drobizhev 2017).…”

A Bright and Colorful Future for G-Protein Coupled Receptor Sensors

“…In our opinion, this is largely due to the complexity of the problem. While in FRET sensors a single photophysical process is responsible for the observed changes, in cpFPs a complex combination of effects needs to be taken into account (Barnett et al, 2017;Molina et al, 2019). In a cpFP sensor, the acid and basic forms of the chromophore exist in equilibrium.…”

“…Interconversion between the two forms can happen also in the excited state (Meech, 2009;Barnett et al, 2017), so that the excitation of the acid form can result in the emission of the basic one. In the case of multiphoton absorption, also the absorption cross-sections can depend on the barrel conformation (Molina et al, 2019). As a consequence of this complexity, the relation between intensity and lifetime is very difficult to predict.…”

“…Careful photophysical studies (Barnett et al, 2017;Molina et al, 2019) have shown that even for seemingly similar cpFP sensors the mechanisms that determine the fluorescence response can change considerably, and that good candidates for FLIM definitely exist. We expect that in the future more studies will be devoted to this specific aspect and cpFPs optimized for FLIM readouts will start to emerge.…”