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.
High-speed continuous imaging systems are constrained by analog-to-digital conversion, storage, and transmission. However, real video signals of objects such as microscopic cells and particles require only a few percent or less of the full video bandwidth for high fidelity representation by modern compression algorithms. Compressed Sensing (CS) is a recent influential paradigm in signal processing that builds real-time compression into the acquisition step by computing inner products between the signal of interest and known random waveforms and then applying a nonlinear reconstruction algorithm. Here, we extend the continuous high-rate photonically-enabled compressed sensing (CHiRP-CS) framework to acquire motion contrast video of microscopic flowing objects. We employ chirp processing in optical fiber and high-speed electro-optic modulation to produce ultrashort pulses each with a unique pseudorandom binary sequence (PRBS) spectral pattern with 325 features per pulse at the full laser repetition rate (90 MHz). These PRBS-patterned pulses serve as random structured illumination inside a one-dimensional (1D) spatial disperser. By multiplexing the PRBS patterns with a user-defined repetition period, the difference signal y_i=phi_i (x_i - x_{i-tau}) can be computed optically with balanced detection, where x is the image signal, phi_i is the PRBS pattern, and tau is the repetition period of the patterns. Two-dimensional (2D) image reconstruction via iterative alternating minimization to find the best locally-sparse representation yields an image of the edges in the flow direction, corresponding to the spatial and temporal 1D derivative. This provides both a favorable representation for image segmentation and a sparser representation for many objects that can improve image compression.
We demonstrate an ultrahigh-rate imaging system applied to very high speed microscopic flows. Chirp processing of ultrafast laser pulses in optical fiber is employed to create pseudorandom spectral patterns at a rate of one unique pattern per pulse. These spectral patterns then serve as structured illumination of the object flows inside a 1D spatial disperser before digitization at a rate of one sample per optical pulse with a fast single pixel photodetector. Diffraction-limited microscopic imaging of flows up to 31.2 m/s is achieved at up to 19.8 and 39.6 Gigapixel/sec rates from a 720 MHz acquisition rate.
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