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

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.

Single-laser-based simultaneous four-wavelength excitation source for femtosecond two-photon fluorescence microscopy.

Multicolor labeling of biological samples with large volume is required for omic-level of study such as the construction of nervous system connectome. Among the various imaging method, two photon microscope has multiple advantages over traditional single photon microscope for higher resolution and could image large 3D volumes of tissue samples with superior imaging depth. However, the growing number of fluorophores for labeling underlines the urgent need for an ultrafast laser source with the capability of providing simultaneous plural excitation wavelengths for multiple fluorophores. Here, we propose and demonstrate a single-laser-based four-wavelength excitation source for two-photon fluorescence microscopy. Using a sub-100 fs 1,070-nm Yb:fiber laser to pump an ultrashort nonlinear photonic crystal fiber in the low negative dispersion region, we introduced efficient self-phase modulation and acquired a blue-shifted spectrum dual-peaked at 812 and 960 nm with 28.5% wavelength conversion efficiency. By compressing the blue-shift near-IR spectrum to 33 fs to ensure the temporal overlap of the 812 and 960 nm peaks, the so-called sum frequency effect created the third virtual excitation wavelength effectively at 886 nm. Combined with the 1,070 nm laser source as the fourth excitation wavelength, the all-fiber-format four-wavelength excitation source enabled simultaneous four-color two-photon imaging in Brainbow AAV-labeled (TagBFP, mTFP, EYFP, and mCherry) brain samples. With an increased number of excitation wavelengths and improved excitation efficiency than typical commercial femtosecond lasers, our compact four-wavelength excitation approach can provide a versatile, efficient, and easily accessible solution for multiple-color two-photon fluorescence imaging in the field of neuroscience, biomolecular probing, and clinical applications with at least four spectrally-distinct fluorophores.

Additive-color multi-harmonic generation microscopy for simultaneous label-free differentiation of plaques, tangles, and neuronal axons.

Multicolor fluorescence imaging has been widely used by neuroscientists to simultaneously observe different neuropathological features of the brain. However, these optical modalities rely on exogenous labeling. Here, we demonstrate, for the first time, a label-free additive-color multi-harmonic generation microscopy to elucidate, concurrently with different hues, Alzheimer's disease (AD) neuropathological hallmarks: amyloid ß (Aß) plaques and neurofibrillary tangles (NFT). By treating third harmonic generation (THG) and second harmonic generation (SHG) as two primary colors, our study can simultaneously label-free differentiate AD hallmarks by providing different additive colors between Aß plaques, NFT, and neuronal axons, with weaker THG presentation from NFT in most places of the brain. Interestingly our pixel-based quantification and Pearson's correlation results further corroborated these findings. Our proposed label-free technique fulfills the unmet challenge in the clinical histopathology for stain-free slide-free differential visualization of neurodegenerative disease pathologies, with a sub-femtoliter resolution in a single image field-of-view.