Consequences of the Nyquist-Shannon sampling criterion in Mesoscopic Multiphoton Microscopy to avail full-field sub-micron resolution resolvability

With a limited effective voxel rate, to date, each laser-scanning mesoscopic multiphoton microscope (MPM), despite securing an ultra-large field of view (FOV) and an ultra-high optical resolution simultaneously, experiences a fundamental issue with digitization; i . e ., inability to satisfy the Nyquist-Shannon sampling criterion to resolve the optics-limited sub-micron resolution over the whole FOV. Such a system either neglects the criterion degrading the digital resolution to twice the pixel size, or significantly reduces the imaging area and/or the imaging speed to respect the digitization. Here we introduce a Nyquist figure of merit parameter to assess this issue, further to comprehend a maximum aliasing-free FOV and a cross-over excitation wavelength for a laser scanning MPM system. Based on our findings we demonstrate an ultra-high voxel rate acquisition in a custom-built mesoscopic MPM system to exceed the Nyquist-rate for a >3800 FOV-resolution ratio while not compromising the imaging speed as well as the photon-budget.