Calcium signaling participates in almost every aspect of cell functioning, specifically in neurons. Genetically encoded calcium indicators (GECIs) developed from fluorescent proteins (FPs) provide a robust reliable readout of neuronal activity including spike number, timing, frequency, and levels of synaptic input. Extending the color palette of GECIs toward near-infrared (NIR) spectral range will facilitate deep-tissue imaging, allow functional imaging from multiple cell populations expressing various multicolor neuronal indicators, and enable to integrate NIR GECIs into optogenetic experiments. Reliable combination of GECIs with optogenetic modulation in all-optical electrophysiology setups has been difficult to achieve in practice due to spectral overlap between activation light of opsin actuators and excitation light of available GECIs. Building upon our molecular evolution technologies and extensive experience in engineering and characterization of various FPs and FP-based biosensors, we propose to generate two new classes of GECIs that are excited and fluoresce in the NIR spectrum by using novel NIR FPs of a miRFP series developed from bacterial phytochromes. Unlike other NIR FPs designed from phytochromes, miRFPs are monomeric and bright in mammalian cells, including neurons. The first class of the planned NIR GECIs will be based on the ratiometric FRET changes between NIR FP donor and NIR FP acceptor (Aim 1). The second class of GECIs will be based on the intensity changes of the single NIR FPs (Aim 2). To perform sensitive and specific measurements of neural activity, the NIR GECIs will be combined with the modern adaptive optics imaging technologies allowing calcium measurements in vivo with enhanced spatial and temporal resolutions at depth. We will apply the adaptive optics correction via direct wavefront sensing to NIR GECI two-photon imaging in vivo (Aim 3). This will allow non-invasive detection of neural activity at synaptic resolution throughout mouse cortex (1 mm depth) and at cellular resolution further into subcortical structures (to 1.6 mm depth). The large spectral separation of NIR GECIs from visible GECIs and opsin actuators will also allow multicolor functional imaging in a large number of neurons in brain and elucidation of the input/output interactions of neural circuits. The proposed research will provide highly demanded deep-tissue optical probes allowing a comprehensive view of neural activity at cellular and whole-brain levels.
|Effective start/end date||8/1/17 → 7/31/21|
- National Institute of Neurological Disorders and Stroke: $254,891.00
- Cell Biology