Multiphoton microscopy applied in bone tissue is susceptible to optical aberrations caused by heterogeneity in refractive index. Optical clearing can be applied to alleviate some of these aberrations, but it is invasive and causes deviations from normal tissue biology. We recover diffraction limited imaging by means of a high spatial frequency digital micromirror device (DMD), and binary wavefront modulation. A genetic algorithm optimizes the DMD pattern by evaluating the intensity of the Second Harmonic Generation point spread function measured in the bone sample. We present a five-fold GFP intensity improvement, and a 29% spatial resolution increase within an ex vivo mouse sample.
Mitochondria are extremely important organelles in the regulation of bone marrow and brain activity. However, live imaging of these subcellular features with high resolution in scattering tissues like brain or bone has proven challenging. In this study, we create a next-generation two-photon fluorescence microscope that leverages low-order wavefront correction by Shack-Hartmann wavefront sensor based on different metrics to achieve fast imaging of subcellular organelles of highly scattering living mice. Metrics include maximum intensity, minimum full width at half maximum (FWHM), and maximum energy of the point spread function (PSF), enabling accuracy and robustness of sensorless correction of the system. Using AO increases the fluorescence intensity and FWHM of the PSF and achieves fast imaging of subcellular organelles with 400nm resolution through 85 μm of highly scattering tissue. This study demonstrates a promising tool for imaging mitochondria and other organelles in optically distorting biological environments, which could facilitate the study of a variety of diseases connected to mitochondrial morphology and activity in a range of biological tissues.
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