Multiphoton microscopes are of paramount importance in capturing neural activity with cellular resolution. However, the imaging speed and field-of-view of traditional two-photon microscopes is limited by raster scanning technologies. Temporally-focused two-photon (TFTP) microscopy is a wide-field scan-free approach to increase the speed of two-photon microscopy. In conventional TFTP microscopy, wide-field depth sectioning is obtained by compressing a spatially pre-chirped pulse at the focal plane of the objective. Unfortunately, the greater imaging speed of TFTP microscopes comes at the expense of poor imaging depth in tissue due to scattering of the short-wavelength fluorescence photons en-route to the imaging camera. Here we demonstrate a compressive high-speed two-photon microscope based on wide-field temporally-focused structured illumination, which eliminates the loss of image contrast from scattering of the fluorescence signal by leveraging a single-pixel detector. Specifically, we illuminate the sample with a rapid sequence of randomly structured temporally-focused wide-field illumination pulses and integrate the net two-photon fluorescence response on a single photomultiplier tube (PMT). Notably, the longer wavelength structured illumination is significantly less susceptible to scattering and the use of integrated measurements on a single PMT provides immunity to fluorescence scattering since these measurements are solely concerned with the net fluorescence. Furthermore, our approach provides greater speed than point scanning two-photon microscopes through the use of wide-field illumination and compressive image acquisition. Experimentally we demonstrate this system operating over a 200×250-μm field-of-view and at a compression rate of 10%, which provides an order of magnitude increase in speed over a comparable point scanning architecture.
Milad Alemohammad, Jaewook Shin, Jasper R. Stroud, and Mark A. Foster, "Compressive temporal focusing microscopy (Conference Presentation)," Proc. SPIE 10505, High-Speed Biomedical Imaging and Spectroscopy III: Toward Big Data Instrumentation and Management, 1050504 (Presented at SPIE BiOS: January 29, 2018; Published: 15 March 2018); https://doi.org/10.1117/12.2289967.5752179948001.
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