<br/>Optogenetic tools, such as Genetically Encodable Fluorescent Calcium Indicators (GECIs), that modulate their fluorescence in response to changes in calcium concentration, are powerful tools for visualizing large-scale brain activity in model organisms at high resolution. These fluorescent indicators are vital for cellular imaging because they allow a non-invasive study of cells, tissues, and sub-cellular structures at a detail that was previously not possible. The most notable of these is the first Green Fluorescent Protein (GFP) based Ca2+ indicator, GCaMP, created by fusing circularly permuted (cp) GFP scaffold with Calmodulin (CaM) and RS20 calcium sensing and binding domains from myosin light chain kinase. Applications of calcium sensors have revealed several limitations in the currently available GCaMP series of indicators, including fluorescence brightness and signal-to-noise ratio (SNR) for deep tissue imaging, slow kinetics in response to neuronal firing, and lack of spatial resolution. To address these fundamental challenges, we have optimized the mVenus fluorescent protein to create a new generation of indicators that will enable deeper imaging into tissues using two-photon excitation with 1030-nm lasers.<br/><br/>Using red-shifted mVenus fluorescent protein, we hope to create a new scaffold for indicators that researchers can use with 2-photon microscopy to investigate the physiological activity of organs, understand the signaling patterns deep within the tissue, and use to study the various disease states at the resolution and depth that is currently not possible. In my talk, I will present the spectral properties of mVenus based indicators, and discuss their 2P data in contrary to commonly used GFP based indicators.

Genetically encoded fluorescent indicators for 2-photon imaging

2019 Fall National Meeting

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