For decades, genetically encoded Ca<sup>2+</sup> indicators (GECIs), have been under development for the purpose of visualizing intracellular Ca<sup>2+</sup> dynamics. Fluorescent GECIs are limited for the purpose of deep tissue long-term whole-body imaging due to their requirement for external illumination. Bioluminescent GECIs overcome these restraints but are somewhat compromised in terms of spatiotemporal resolution compared to fluorescent probes. To address this, we developed a bimodal Ca<sup>2+</sup> indicator by combining a single fluorescent protein based Ca<sup>2+</sup> indicator and a split luciferase. The novel design of this bimodal indicator enables Ca<sup>2+</sup> imaging in the same specimen in both fluorescent and bioluminescent mode. The ability to switch between fluorescent and bioluminescent modes with a single indicator should benefit applications where micro and macro scale observation of cells or tissues is desirable. Use of such a probe enables trans-scale imaging, where macro scale imaging of a group of cells or tissue can be combined with fluorescent imaging of single cells.
We report five new spectral variants of bright luminescent protein made by concatenation of the brightest luciferase,
NanoLuc, with various color hues of fluorescent proteins. These proteins, which we call enhanced Nano-lanterns (eNLs),
allow five-color live-cell imaging without external light illumination as well as detection of single molecules.
Furthermore, eNL-based Ca<sup>2+</sup> indicators could be used to image long-term Ca<sup>2+</sup> dynamics in iPS-derived cardiomyocytes.