CaBLAM! A high-contrast bioluminescent Ca 2+ indicator derived from an engineered Oplophorus gracilirostris luciferase
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Abstract
Measuring ongoing cellular activity is essential to understanding the dynamic functions of biological organisms. The most popular current approach is imaging fluorescence-based genetically encoded Ca 2+ indicators (GECIs). While fluorescent probes are useful in many contexts, bioluminescence-based GECIs—probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein—have several distinct advantages. Because bioluminescent (BL) GECIs do not use the bright extrinsic excitation light required for fluorescence, BL GECIs do not photobleach, do not suffer from nonspecific autofluorescent background, and do not cause phototoxicity. Further, BL GECIs can be applied in contexts where directly shining photons on an imaging target is not possible. Despite these advantages, the use of BL GECIs has to date been limited by their small changes in bioluminescence intensity, high baseline signal at resting Ca 2+ concentrations, and suboptimal Ca 2+ affinities. Here, we describe a new BL GECI, CaBLAM ( Ca 2+ B io L uminescence A ctivity M onitor), that displays much higher dynamic range than previous BL GECIs and has a Ca 2+ affinity suitable for capturing physiological changes in cytosolic Ca 2+ concentration. With these improvements, CaBLAM captures single-cell and subcellular resolution activity at high frame rates in cultured neurons and in vivo , and allows multi-hour recordings in mice and behaving zebrafish. This new advance provides a robust alternative to traditional fluorescent GECIs that can enable or enhance imaging across many experimental conditions.
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Only cultures where neurons were evenly distributed across the entire coverslip
part of a sentence missing
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for single
typo?
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As these experiments did not include synaptic blockers, the true indicatorkinetics are likely faster than measured
true; adding synaptic blockers improved kinetics but also dramatically increased day-to-day repeatability of the GCaMP-series sensors. this would probably be worth doing for a large-scale screen.
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CTZ
Acronym not previously defined
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(A)
In A and B, is the stimulus the same or increasing in the number of pulses? Trying to understand why the response seems to grow over time.
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(A)
For maximum clarity, it may be useful to use d prime or SNR as the key metric for comparison because both metrics are designed to measure what most users would care about: how detectable is the Ca2+ signal regardless of baseline brightness.
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diffusion of furimazine (Fz) m
it may be helpful to contextualize the role of furimazine before this, as it may not be familiar to readers without BL assay experience
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(C)
Stylistic note: the label makes it seem like "CaBLAM" and "GCaMP8s" in D is a trace on the plot along with ROI1,2,3. Maybe it can be left out?
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