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.

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 utilising both a high power depletion laser (increasing risk of photodamage), while the depletion beam is very sensitive to sample-induced aberrations. Here we demonstrate a custom-built 3D STED microscope with automated aberration correction that is capable of 3D super-resolution imaging through thick, highly aberrating, tissue. We introduce and investigate image denoising by block-matching and collaborative filtering (BM3D) to numerically enhance fine object details otherwise mixed with noise. Numerical denoising provides an increase in the final effective resolution of the STED imaging of 31% using the well-established Fourier ring correlation metric. Experimental validation of the proposed method is achieved through super-resolved 3D imaging of axons in differentiated induced pluripotent stem cells growing under a 80µm thick layer of tissue with lateral and axial resolution of 256nm and 300nm, respectively..

Utilizing the refractive index as the endogenous contrast agent to noninvasively study transparent cells is a working principle of emerging quantitative phase imaging (QPI). In this contribution, we propose the Variational Hilbert Quantitative Phase Imaging (VHQPI)-end-to-end purely computational add-on module able to improve performance of a QPI-unit without hardware modifications. The VHQPI, deploying unique merger of tailored variational image decomposition and enhanced Hilbert spiral transform, adaptively provides high quality map of sample-induced phase delay, accepting particularly wide range of input single-shot interferograms (from off-axis to quasi on-axis configurations). It especially promotes high space-bandwidth-product QPI configurations alleviating the spectral overlapping problem. The VHQPI is tailored to deal with cumbersome interference patterns related to detailed locally varying biological objects with possibly high dynamic range of phase and relatively low carrier. In post-processing, the slowly varying phase-term associated with the instrumental optical aberrations is eliminated upon variational analysis to further boost the phase-imaging capabilities. The VHQPI is thoroughly studied employing numerical simulations and successfully validated using static and dynamic cells phase-analysis. It compares favorably with other single-shot phase reconstruction techniques based on the Fourier and Hilbert-Huang transforms, both in terms of visual inspection and quantitative evaluation, potentially opening up new possibilities in QPI. Optical imaging is a central aspect of biological research, biomedical examination, and medical diagnosis. Optical microscope is indispensable in biological and medical research facilitating a "seeing is believing" paradigm 1. Despite its rich history and well-established position, significant efforts are contemporarily put on developing new imaging modalities enabling enhanced resolution, contrast, depth, speed and information content. Among a suite of modern microscopy techniques, quantitative phase imaging (QPI) 2-4 stands out as a vividly blossoming label-free approach. It provides unique means for imaging cells and tissues merging beneficial features identified with microscopy 1 , interferometry and holography 5 , and numerical computations. Using refractive index as the endogenous contrast agent 6,7 QPI numerically converts recorded interference pattern into a nanoscale-precise subcellular-specific map of optical delay introduced by examined transparent specimen 7-9. This non-phototoxic non-destructive imaging technique brings biology and metrology closer as it generates quantitative maps of analyzed live bio-structure (related to cell mass, volume, surface area, and their evolutions in time). Therefore, QPI enables upgrading phase-contrast based 6 visualization of the sample to stain-free non-invasive measurement of sample-induced optical phase delay (related to refractive index and/or thickness variations). Impressive details can be imaged, e.g., via super-resolut...

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