Multiphoton laser scanning microscopy has revolutionized neuroscience since it is less invasive than traditional electrophysiology methods for probing neuronal processes. In most cases, such imaging is limited to the observation of a single fluorescent species at modest depths (the depth penetration of standard 2-photon microscopy in brain tissue is limited a few 100s of microns). Recent proposals to extend this depth penetration have made use of 3-photon excitation. But to avoid heating due to water absorption, long wavelengths were employed, providing access to only deep red fluorescent markers. We propose to improve the versatility of multiphoton microscopy by enhancing its multiplexing capacity and its depth penetration. To do this, we will develop a novel laser design that can emit light at different colors simultaneously, that are chosen to enable 2- or 3-photon imaging using non-degenerate (as opposed to traditional degenerate) multiphoton imaging. The key novelty here is a tuneable laser that emits, on demand, a pair of colors across the wavelength ranges that avoid water absorption but whose multiple combinations of energies sum up to excite a variety of popular fluorescent sensors, such as channelrhodopsins, halorhodopsins, archearhodopsin, and GCaMPs etc, across the entire visible spectrum. Our laser's power and wavelengths will enable achieving deep tissue (up to ~2mm) imaging. Multiplexing will be performed by detecting fluorescence from all the non- degenerate multiphoton combinations available from our multicolour tunable high energy laser. We will demonstrate the proof-of-concept of multiplexed deep tissue imaging using labelled mouse-brain tissue. This would create an opportunity to image deep structures such as the thalamus and hippocampus (depth>1000 ?m) with minimal tissue damage, which have not been able to be achieved by current in vivo imaging methods. Success in proposed goals would create new avenues for neuroscience research, such as, for example, enabling imaging of large heterogeneous neuronal ensembles of transgenic mouse lines that can genetically label distinct neuronal populations, or facilitating circuit interrogation experiments involving glutamate uncaging that currently require multiple costly ultrafast lasers. Moreover, since the novel laser source is all-fiber in nature, the microscope is readily adaptable for facilitating future endoscopc in vivo imaging.
Multiplexing strategies are key to acquiring the large sets of imaging data required to build connectomic maps of the living brain. To enable this in a non-invasive manner, we propose to develop a novel 3 photon non-degenerate multicolor microscopy technique that allows for multiplexed noninvasive imaging of neurons deep in the brain.
| Rishøj, L; Jones, M; Demas, J et al. (2016) Polymer-clad silica fibers for tailoring modal area and dispersion. Opt Lett 41:3587-90 |
