CaBLAM! A high-contrast bioluminescent Ca(2+) indicator derived from an engineered Oplophorus gracilirostris luciferase. Lambert, G. G., Crespo, E. L., Murphy, J., Boassa, D., Luong, S., Celinskis, D., Venn, S., Hu, J., Sprecher, B., Tree, M. O., Orcutt, R., Heydari, D., Bell, A. B., Torreblanca-Zanca, A., Hakimi, A., Lipscombe, D., Moore, C. I., Hochgeschwender, U., & Shaner, N. C. October, 2023. Journal Abbreviation: bioRxiv Pages: 2023.06.25.546478 Publication Title: bioRxiv : the preprint server for biologydoi abstract bibtex 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 a much higher contrast (dynamic range) than previously described bioluminescent GECIs coupled with 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. 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.
@misc{lambert_cablam_2023,
address = {United States},
title = {{CaBLAM}! {A} high-contrast bioluminescent {Ca}(2+) indicator derived from an engineered {Oplophorus} gracilirostris luciferase.},
doi = {10.1101/2023.06.25.546478},
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 a much higher contrast (dynamic range) than previously described bioluminescent GECIs coupled with 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. 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.},
language = {eng},
author = {Lambert, Gerard G. and Crespo, Emmanuel L. and Murphy, Jeremy and Boassa, Daniela and Luong, Selena and Celinskis, Dmitrijs and Venn, Stephanie and Hu, Junru and Sprecher, Brittany and Tree, Maya O. and Orcutt, Richard and Heydari, Daniel and Bell, Aidan B. and Torreblanca-Zanca, Albertina and Hakimi, Ali and Lipscombe, Diane and Moore, Christopher I. and Hochgeschwender, Ute and Shaner, Nathan C.},
month = oct,
year = {2023},
pmid = {37425712},
pmcid = {PMC10327125},
note = {Journal Abbreviation: bioRxiv
Pages: 2023.06.25.546478
Publication Title: bioRxiv : the preprint server for biology},
}
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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 a much higher contrast (dynamic range) than previously described bioluminescent GECIs coupled with a Ca(2+) affinity suitable for capturing physiological changes in cytosolic Ca(2+) concentration. 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A high-contrast bioluminescent Ca(2+) indicator derived from an engineered Oplophorus gracilirostris luciferase.","year":2023}