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Authors & Affiliations
Ryan Fink, Shosei Imai, Martin Kubitschke, Robert Campbell, Olivia Masseck
Abstract
Fluorescent calcium imaging is an essential tool for measuring cellular activity, particularly as it applies to neurons and reporting action potentials. Genetically encoded calcium indicators are incredibly versatile and particularly useful for both in vivo and in vitro research. Depending on the application and use of indicators, the desired properties can drastically change from being very bright, to a very strong signal, to having fast dynamics, or any combination of the three. However, there are several disadvantages to using existing red shifted calcium indicators, those limitations being their dimness in comparison to green calcium indicators such as the various generations of GCaMP and very often show false positive signals due to photoswitching when stimulated by blue light. To address the primary limitations of the current generation of red shifted genetically encoded calcium indicators, we have created a library of indicators with a circularly permutated form of mScarlet3. Being published only last year, mScarlet3 has a quantum yield of 0.75 and is around twice as bright as its predecessor, which was already the brightest red fluorescent protein (Gadella et al. 2023). By using a combination of screening methods, we explore the characteristics of this new generation of red-shifted calcium indicators and compare them to existing ones.