Methanol immersion reduces spherical aberration of water dipping lenses at long wavelengths used in multi-photon laser scanning microscopy

Dipping objectives were tested for multi-photon laser scanning microscopy, since their large working distances are advantageous for thick specimens and the absence of a coverslip facilitates examination of living material. Images of fluorescent bead specimens, particularly at wavelengths greater than 850 nm showed defects consistent with spherical aberration. Substituting methanol for water as the immersion medium surrounding the beads corrected these defects and produced an increase in fluorescence signal intensity. The same immersion method was applied to two representative biological samples of fixed tissue: mouse brain labeled with FITC for tubulin and mouse gut in which the Peyer’s patches were labeled with Texas Red bilosomes. Tissue morphology was well preserved by methanol immersion of both tissues; the two-photon-excited fluorescence signal was six times higher than in water and the depth of penetration of useful imaging was doubled. No modification of the microscope was needed except the provision of a ring to retain a sufficient depth of methanol for imaging

Combined conjugate and pupil adaptive optics in widefield microscopy

Traditionally, adaptive optics (AO) systems for microscopy have focused on AO at the pupil plane, however this produces poor performance in samples with both spatially-variant aberrations, such as non-flat sample interfaces, and spatially-invariant aberrations, such as spherical aberration due to a difference between the sample index of refraction and the sample for which the objective was designed. Here, we demonstrate well-corrected, wide field-of-view (FOV) microscopy by simultaneously correcting the two types of aberrations using two AO loops. Such an approach is necessary in wide-field applications where both types of aberration may be present, as each AO loop can only fully correct one type of aberration. Wide FOV corrections are demonstrated in a trans-illumination microscope equipped with two deformable mirrors (DMs), using a partitioned aperture wavefront (PAW) sensor to directly control the DM conjugated to the sample interface and a sensor-less genetic algorithm to control the DM conjugated to the objective’s pupil

Full volume super-resolution imaging of thick mitotic spindle using 3D AO STED microscope

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing. Stimulated emission depletion (STED) nanoscopy is one of a suite of modern optical microscopy techniques capable of bypassing the conventional diffraction limit in fluorescent imaging. STED makes use of a spiral phase mask to enable 2D super-resolution imaging whereas to achieve full volumetric 3D super-resolution an additional bottle-beam phase mask must be applied. The resolution achieved in biological samples 10 μm or thicker is limited by aberrations induced mainly by scattering due to refractive index heterogeneity in the sample. These aberrations impact the fidelity of both types of phase mask, and have limited the application of STED to thicker biological systems. Here we apply an automated adaptive optics solution to correct the performance of both STED masks, enhancing robustness and expanding the capabilities of this nanoscopic technique. Corroboration in terms of successful high-quality imaging of the full volume of a 15μm mitotic spindle with resolution of 50nm x 50nm x 150nm achieved in all three dimensions is presented

Differentiable model-based adaptive optics for two-photon microscopy

Aberrations limit scanning fluorescence microscopy when imaging in scattering materials such as biological tissue. Model-based approaches for adaptive optics take advantage of a computational model of the optical setup. Such models can be combined with the optimization techniques of machine learning frameworks to find aberration corrections, as was demonstrated for focusing a laser beam through aberrations onto a camera [arXiv:2007.13400]. Here, we extend this approach to two-photon scanning microscopy. The developed sensorless technique finds corrections for aberrations in scattering samples and will be useful for a range of imaging application, for example in brain tissue

Numerically Enhanced Stimulated Emission Depletion Microscopy with Adaptive Optics for Deep-Tissue Super-Resolved Imaging

Copyright © 2019 American Chemical Society. In stimulated emission depletion (STED) nanoscopy, the major origin of decreased signal-to-noise ratio within images can be attributed to sample photobleaching and strong optical aberrations. This is due to STED utilizing a high-power depletion laser (increasing the risk of photodamage), while the depletion beam is very sensitive to sample-induced aberrations. Here, we demonstrate a custom-built STED microscope with automated aberration correction that is capable of 3D super-resolution imaging through thick, highly aberrating tissue. We introduce and investigate a state of the art image denoising method by block-matching and collaborative 3D filtering (BM3D) to numerically enhance fine object details otherwise mixed with noise and further enhance the image quality. Numerical denoising provides an increase in the final effective resolution of the STED imaging of 31% using the well established Fourier ring correlation metric. Results achieved through the combination of aberration correction and tailored image processing are experimentally validated through super-resolved 3D imaging of axons in differentiated induced pluripotent stem cells growing under an 80 μm thick layer of tissue with lateral and axial resolution of 204 and 310 nm, respectively

Coherent beam control through inhomogeneous media in multi-photon microscopy

Multi-photon fluorescence microscopy has become a primary tool for high-resolution deep tissue imaging because of its sensitivity to ballistic excitation photons in comparison to scattered excitation photons. The imaging depth of multi-photon microscopes in tissue imaging is limited primarily by background fluorescence that is generated by scattered light due to the random fluctuations in refractive index inside the media, and by reduced intensity in the ballistic focal volume due to aberrations within the tissue and at its interface. We built two multi-photon adaptive optics (AO) correction systems, one for combating scattering and aberration problems, and another for compensating interface aberrations. For scattering correction a MEMS segmented deformable mirror (SDM) was inserted at a plane conjugate to the objective back-pupil plane. The SDM can pre-compensate for light scattering by coherent combination of the scattered light to make an apparent focus even at a depths where negligible ballistic light remains (i.e. ballistic limit). This problem was approached by investigating the spatial and temporal focusing characteristics of a broad-band light source through strongly scattering media. A new model was developed for coherent focus enhancement through or inside the strongly media based on the initial speckle contrast. A layer of fluorescent beads under a mouse skull was imaged using an iterative coherent beam control method in the prototype two-photon microscope to demonstrate the technique. We also adapted an AO correction system to an existing in three-photon microscope in a collaborator lab at Cornell University. In the second AO correction approach a continuous deformable mirror (CDM) is placed at a plane conjugate to the plane of an interface aberration. We demonstrated that this “Conjugate AO” technique yields a large field-of-view (FOV) advantage in comparison to Pupil AO. Further, we showed that the extended FOV in conjugate AO is maintained over a relatively large axial misalignment of the conjugate planes of the CDM and the aberrating interface. This dissertation advances the field of microscopy by providing new models and techniques for imaging deeply within strongly scattering tissue, and by describing new adaptive optics approaches to extending imaging FOV due to sample aberrations