Wide-field scan-less multi-photon endoscopy using spatio-temporal pulse delivery and temporal focusing

Johns Hopkins University, Baltimore MD

Investigators

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Abstract

Project Summary/ Abstract Multiphoton, depth sectioning micro-endoscopes are of paramount importance in capturing neural activity with cellular resolution. Imaging speed and the field-of-view in the current two-photon endoscopes has remained limited by the resonant scanning devices that are required at the distal end. Temporal focusing is multi-photon imaging technology which has enabled wide-field, scan-free, two-photon microscopy at over 200 frames per second. However miniaturization of this technology into a micro-endoscope has remained a challenge for a number of reasons including fiber damage at large pulse energies and the need for EMCCD for fluorescence imaging. The focus of this proposal is to develop a novel, high energy pulse delivery technology by using coherent imaging fiber bundles in conjunction with a two-dimensional spectral disperser at the proximal end of the fiber bundle and demonstrate its application to multi-photon endoscopy. First (Aim 1), we will use a two- dimensional spectral disperser in order to decompose high energy laser pulses into ? spectral slices, each with a fraction (1/?) of the total bandwidth, mapped to ? different cores in a multi-core fiber. This spectral decomposition results in pulses with ?ps durations, which feature orders of magnitude larger dispersion and nonlinear length compared with the 100-fs pulses commonly used in two-photon microscopy. This reduction in nonlinearity and dispersion will allow us to deliver significantly higher peak powers to the sample than conventionally possible with a multi-core fiber. At the distal end, these pulses will be coherently combined by the micro-objective using adaptive phase control at the proximal end in order to form a temporally focused illumination. This approach enables a significant increase in the pulse energy that can be delivered to the sample when using a fiber bundle allowing for practical multi-photon endoscopy with a fiber bundle. Second (Aim 2), we will investigate design of a miniaturized distal assembly that includes an achromatic micro-objective, a pair of miniaturized tube lenses, and a dichroic prism. Coherent imaging fiber bundles will be used in order to excite and collect the fluorescence signal. By using a fiber bundle this approach moves the multitude of optical components needed for temporal focusing (e.g. grating, EMCCD) from the distal to the proximal end resulting in a dramatically reduced distal size and improved performance. Third (Aim 3), we will utilize pseudo-random structured illumination and compressed sensing image recovery in conjunction with the developed technologies in order to reduce the impacts of tissue scatter and thus improve the imaging depth of the micro-endoscope. Ultimately, this technology will enable a compact, temporally focused micro-endoscope for high-speed imaging of neural activity in behaving animals.