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

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 Ca²⁺ 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 Ca²⁺ concentrations, and suboptimal Ca²⁺ affinities. Here, we describe a new BL GECI, CaBLAM (<underline>Ca</underline> ²⁺ <underline>B</underline>io<underline>L</underline>uminescence <underline>A</underline>ctivity <underline>M</underline>onitor), that displays much higher dynamic range than previous BL GECIs and has a Ca²⁺ affinity suitable for capturing physiological changes in cytosolic Ca²⁺ 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.