Engineering a better light sheet in an axicon-based system using a flattened Gaussian beam of low order.

Lasers are fundamental tools in research and development. The shape of an incident laser beam directly affects the results, when it propagates through complex structured meso-aspheric optical elements. In conic-based systems utilizing elements such as axicons, the impact of secondary lobes is mostly overlooked, although the intensity distributions at the central spot and the side-lobes directly affect the beam properties. We investigate the interaction of two axicons (160° and 170°) with incident beams approximated by Gaussian, high-order Flattened-Gaussian, and low-order Flattened-Gaussian functions. We demonstrate that replacing an incident Gaussian beam with a low-order Flattened-Gaussian beam reduces the secondary lobes and significantly improves the uniformity of the intensity profile. We practically applied this effect in engineering a conic-aspheric-based static light-sheet microscope producing markedly improved results.

3D histopathology of human tumours by fast clearing and ultramicroscopy.

Here, we describe a novel approach that allows pathologists to three-dimensionally analyse malignant tissues, including the tumour-host tissue interface. Our visualization technique utilizes a combination of ultrafast chemical tissue clearing and light-sheet microscopy to obtain virtual slices and 3D reconstructions of up to multiple centimetre sized tumour resectates. For the clearing of tumours we propose a preparation technique comprising three steps: (a) Fixation and enhancement of tissue autofluorescence with formalin/5-sulfosalicylic acid. (b) Ultrafast active chemical dehydration with 2,2-dimethoxypropane and (c) refractive index matching with dibenzyl ether at up to 56 °C. After clearing, the tumour resectates are imaged. The images are computationally post-processed for contrast enhancement and artefact removal and then 3D reconstructed. Importantly, the sequence a-c is fully reversible, allowing the morphological correlation of one and the same histological structures, once visualized with our novel technique and once visualized by standard H&E- and IHC-staining. After reverting the clearing procedure followed by standard H&E processing, the hallmarks of ductal carcinoma in situ (DCIS) found in the cleared samples could be successfully correlated with the corresponding structures present in H&E and IHC staining. Since the imaging of several thousands of optical sections is a fast process, it is possible to analyse a larger part of the tumour than by mechanical slicing. As this also adds further information about the 3D structure of malignancies, we expect that our technology will become a valuable addition for histological diagnosis in clinical pathology.