Optogenetic techniques allow intracellular manipulation of Ca++ by illumination of light-absorbing probe molecules such as channelrhodopsins and melanopsins. The consequences of optogenetic stimulation would optimally be recorded by non-invasive optical methods. However, most current optical methods for monitoring Ca++ levels are based on fluorescence excitation that can cause unwanted stimulation of the optogenetic probe and other undesirable effects such as tissue autofluorescence. Luminescence is an alternate optical technology that avoids the problems associated with fluorescence. Using a new bright luciferase, we here develop a genetically encoded Ca++ sensor that is ratiometric by virtue of bioluminescence resonance energy transfer (BRET). This sensor has a large dynamic range and partners optimally with optogenetic probes. Ca++ fluxes that are elicited by brief pulses of light to cultured cells expressing melanopsin and to neurons-expressing channelrhodopsin are quantified and imaged with the BRET Ca++ sensor in darkness, thereby avoiding undesirable consequences of fluorescence irradiation.

The intrinsic fluorescence of samples confounds the use of fluorescence-based sensors. This is of particular concern in high-throughput screening (HTS) applications using large chemical libraries containing intrinsically fluorescent compounds. To overcome this problem, we developed a bioluminescence resonance energy transfer (BRET) Ca 2+ sensor, CalfluxCTN. We demonstrated that it reliably reported changes in intracellular Ca 2+ concentrations evoked by an agonist and an antagonist of the human muscarinic acetylcholine receptor M 1 (hM 1 R) even in the presence of the fluorescent compound fluorescein, which interfered with a standard fluorescent HTS sensor (Fluo-8). In an HTS using a chemical library containing fluorescent compounds, CalfluxCTN accurately identified agonists and antagonists that were missed or miscategorized using Fluo-8. Moreover, we showed that a luciferase substrate that becomes activated only when inside cells generated long-lasting BRET signals in HTS, enabling results to be reliably compared among replicate samples for hours. Thus, the use of a self-luminescent sensor instead of a fluorescent sensor could facilitate the complete screening of chemical libraries in a high-throughput context and enable analysis of autofluorescent samples in many different applications.