CaBLAM: a high-contrast bioluminescent Ca2+ indicator derived from an engineered Oplophorus gracilirostris luciferase
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Abstract
Monitoring intracellular calcium is central to understanding cell signaling across nearly all cell types and organisms. Fluorescent genetically encoded calcium indicators (GECIs) remain the standard tools for in vivo calcium imaging, but require intense excitation light, leading to photobleaching, background autofluorescence and phototoxicity. Bioluminescent GECIs, which generate light enzymatically, eliminate these artifacts but have been constrained by low dynamic range and suboptimal calcium affinities. Here we show that CaBLAM (‘calcium bioluminescence activity monitor’), an engineered bioluminescent calcium indicator, achieves an order-of-magnitude improvement in signal contrast and a tunable affinity matched to physiological cytosolic calcium. CaBLAM enables single-cell and subcellular activity imaging at video frame rates in cultured neurons and sustained imaging over hours in awake, behaving animals. These capabilities establish CaBLAM as a robust and general alternative to fluorescent GECIs, extending calcium imaging to regimes where excitation light is undesirable or infeasible.
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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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