CaBLAM! A high-contrast bioluminescent Ca2+ indicator derived from an engineered Oplophorus gracilirostris luciferase.

Ca²⁺ 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 Ca²⁺ concentrations in living cells. Though such fluorescence-based genetically encoded Ca²⁺ indicators (GECIs) have become a mainstay of modern Ca²⁺ 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 Ca²⁺ concentrations and suboptimal Ca²⁺ 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 Ca²⁺ affinity suitable for capturing physiological changes in cytosolic Ca²⁺ 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 Ca²⁺ dynamics at high frame rates in cultured neurons and in vivo. CaBLAM marks a significant milestone in the GECI timeline, enabling Ca²⁺ recordings with high spatial and temporal resolution without perturbing cells with intense excitation light.