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.
The process of multiple scattering has inherent characteristics that are attractive for high-speed imaging with high spatial resolution and a wide field-of-view. A coherent source passing through a multiple-scattering medium naturally generates speckle patterns with diffraction-limited features over an arbitrarily large field-of-view. In addition, the process of multiple scattering is deterministic allowing a given speckle pattern to be reliably reproduced with identical illumination conditions. Here, by exploiting wavelength dependent multiple scattering and compressed sensing, we develop a high-speed 2D time-stretch microscope. Highly chirped pulses from a 90-MHz mode-locked laser are sent through a 2D grating and a ground-glass diffuser to produce 2D speckle patterns that rapidly evolve with the instantaneous frequency of the chirped pulse. To image a scene, we first characterize the high-speed evolution of the generated speckle patterns. Subsequently we project the patterns onto the microscopic region of interest and collect the total light from the scene using a single high-speed photodetector. Thus the wavelength dependent speckle patterns serve as high-speed pseudorandom structured illumination of the scene. An image sequence is then recovered using the time-dependent signal received by the photodetector, the known speckle pattern evolution, and compressed sensing algorithms. Notably, the use of compressed sensing allows for reconstruction of a time-dependent scene using a highly sub-Nyquist number of measurements, which both increases the speed of the imager and reduces the amount of data that must be collected and stored. We will discuss our experimental demonstration of this approach and the theoretical limits on imaging speed.