RESUMO
Significance: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. Aim: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. Approach: We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array recordings in primary neurons. Results: Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. Conclusions: Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer, the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.
RESUMO
Significance: Optical imaging has accelerated neuroscience in recent years. Genetically encoded fluorescent activity sensors of calcium, neurotransmitters, and voltage are commonly used for optical recording of neuronal activity. However, fluorescence imaging is limited to superficial regions for in vivo activity imaging, due to photon scattering and absorbance. Bioluminescence imaging offers a promising alternative for achieving activity imaging in deeper brain regions without hardware implanted within the brain. Bioluminescent reporters can be genetically encoded and produce photons without external excitation. The use of enzymatic photon production also enables prolonged imaging sessions without the risk of photobleaching or phototoxicity, making bioluminescence suitable for non-invasive imaging of deep neuronal populations. Aim: To facilitate the adoption of bioluminescent activity imaging, we sought to develop a low cost, simple in vitro method that simulates in vivo conditions to optimize imaging parameters for determining optimal exposure times and optical hardware configurations to determine what frame rates can be captured with an individual lab's imaging hardware with sufficient signal-to-noise ratios without the use of animals prior to starting an in vivo experiment. Approach: We developed an assay for modeling in vivo optical conditions with a brain tissue phantom paired with engineered cells that produce bioluminescence. We then used this assay to limit-test the detection depth versus maximum frame rate for bioluminescence imaging at experimentally relevant tissue depths using off-the-shelf imaging hardware. Results: We developed an assay for modeling in vivo optical conditions with a brain tissue phantom paired with engineered cells that produce bioluminescence. With this method, we demonstrate an effective means for increasing the utility of bioluminescent tools and lowering the barrier to adoption of bioluminescence activity imaging. Conclusions: We demonstrated an improved method for optimizing imaging parameters for activity imaging in vivo with bioluminescent sensors.
