CaBLAM! A high-contrast bioluminescent Ca 2+ indicator derived from an engineered Oplophorus gracilirostris luciferase
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Abstract
Ca 2+ plays many critical roles in cell physiology and biochemistry, leading researchers to develop a number of fluorescent small molecule dyes and genetically encodable probes that optically report changes in Ca 2+ concentrations in living cells. Though such fluorescence-based genetically encoded Ca 2+ indicators (GECIs) have become a mainstay of modern Ca 2+ sensing and imaging, bioluminescence-based GECIs—probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein—have several distinct advantages over their fluorescent counterparts. Bioluminescent tags do not photobleach, do not suffer from nonspecific autofluorescent background, and do not lead to phototoxicity since they do not require the extremely bright extrinsic excitation light typically required for fluorescence imaging, especially with 2-photon microscopy. Current BL GECIs perform poorly relative to fluorescent GECIs, producing small changes in bioluminescence intensity due to high baseline signal at resting Ca 2+ concentrations and suboptimal Ca 2+ affinities. Here, we describe the development of a new bioluminescent GECI, “CaBLAM,” which displays much higher contrast (dynamic range) than previously described bioluminescent GECIs and has a Ca 2+ affinity suitable for capturing physiological changes in cytosolic Ca 2+ concentration. Derived from a new variant of Oplophorus gracilirostris luciferase with superior in vitro properties and a highly favorable scaffold for insertion of sensor domains, CaBLAM allows for single-cell and subcellular resolution imaging of Ca 2+ dynamics at high frame rates in cultured neurons and in vivo . CaBLAM marks a significant milestone in the GECI timeline, enabling Ca 2+ recordings with high spatial and temporal resolution without perturbing cells with intense excitation light.
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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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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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