Nyquist-exceeding high voxel rate acquisition in mesoscopic multiphoton microscopy for full-field submicron resolution resolvability

Abstract

Summary The Nyquist-Shannon criterion has never been realized in a laser-scanning mesoscopic multiphoton microscope (MPM) with a large field-of-view (FOV)-resolution ratio, especially when employing a high-frequency resonant-raster-scanning. With a high optical resolution nature, a current mesoscopic-MPM either neglects the criterion and degrades the digital resolution to twice the pixel size, or reduces the FOV and/or the raster-scanning speed to avoid aliasing . We introduce a Nyquist figure-of-merit (NFOM) parameter to characterize a laser-scanning MPM in terms of its optical-resolution retrieving ability. Based on NFOM, we define the maximum aliasing-free FOV, and subsequently, a cross-over excitation wavelength, below which the FOV becomes NFOM-constrained irrespective of an optimized optical design. We validate our idea in a custom-built mesoscopic-MPM with millimeter-scale FOV yielding an ultra-high FOV-resolution ratio of >3,000, while securing up-to a 1.6 mm Nyquist-satisfied aliasing-free FOV, a ∼400 nm lateral resolution, and a 70 M/s effective voxel-sampling rate, all at the same time.

Rapid digital pathology of H&E-stained fresh human brain specimens as an alternative to frozen biopsy

“…It is noted that the number of slow-axis-lines was set to 6000 in order to exceed the limiting condition of the Nyquist-Shannon criterion considering a theoretical resolution of 429 nm (for 2-photon process, 1070 nm, 0.95 NA). See our previous publication 37 for a detailed point spread function (PSF) analysis. Nevertheless, it is feasible to lower the number of slow-axis-lines so as to enlarge the pixel-size up-to the limiting Nyquist-requirement, which would further boost the cumulative imaging speed.…”

“…Here we report an FS-alternative ITA-capable technique called Training-Free True-H&E Rapid Fresh digital-Pathology (the-RFP) that uses physical-sectioning-free whole-mount specimens rapidly stained with the gold standard H&E dyes, with no additional interpretation training to a pathologist. The-RFP is assisted by a mesoscale Nonlinear Optical Gigascope (mNLOG) with a high Nyquist-figure-of-merit (NFOM) 37 . In the domain of digital surgical pathology, our mNLOG platform, for the first time to the best of our knowledge, provides a truly WSI-competing whole specimen superficial imaging (WSSI) digital ITA solution enabling multicolor imaging of a 1 cm 2 area in <120 s with a total of 86 G bits or 3.6 Gigapixels (24-bit) preserving a submicron digital resolution with no requirement of post-acquisition data/image processing.…”

Construction of a high-NFOM multiphoton microscope with large-angle resonant raster scanning

“…This protocol illustrates a step-by-step guideline to construct a kilohertz resonant-scanning MPM with a large FOV/digital-resolution ratio of more than 3000, while simultaneously securing a high (≥1) Nyquist figure-of-merit ( Borah et al., 2021 ). This protocol is useful if you wish not to compromise with raster-scanning speed yet target an optical-zoom-free submicron digital resolution across a millimeter-scale FOV.…”

“…We further provide a free version of our custom acquisition software to assist with a smooth and easy construction process. For complete details on the use and execution of this protocol, please refer to Borah et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Borah et al. (2021) .…”