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.

Deconvolution of light sheet microscopy recordings.

We developed a deconvolution software for light sheet microscopy that uses a theoretical point spread function, which we derived from a model of image formation in a light sheet microscope. We show that this approach provides excellent blur reduction and enhancement of fine image details for image stacks recorded with low magnification objectives of relatively high NA and high field numbers as e.g. 2x NA 0.14 FN 22, or 4x NA 0.28 FN 22. For these objectives, which are widely used in light sheet microscopy, sufficiently resolved point spread functions that are suitable for deconvolution are difficult to measure and the results obtained by common deconvolution software developed for confocal microscopy are usually poor. We demonstrate that the deconvolutions computed using our point spread function model are equivalent to those obtained using a measured point spread function for a 10x objective with NA 0.3 and for a 20x objective with NA 0.45.

Outlook on optimizing ultramicroscopy imaging technique through optical characterization.

Here, we present an optically optimized system for static ultramicroscopy imaging technique. The unit for generating an ultra-thin light sheet employs aspheric and meso-optical elements (meso-aspheric system). An analytical as well as an experimental comparison between the light sheet produced by the standard system (using a rectangular slit aperture and one cylindrical lens) and the one produced by our latest optimized system, which converts a symmetrical Gaussian beam into an ultra-thin light sheet is presented. Using the new light sheet in combination with our objective equipped with a modulator unit to compensate the refractive index mismatch between air and mediums with indices of 1.45-1.56, we present high resolution images of various biological samples that were chemically cleared using different methods. They demonstrate a marked improvement in quality, contrast and resolution.

Inhibition of CD34+ cell migration by matrix metalloproteinase-2 during acute myocardial ischemia, counteracted by ischemic preconditioning.

Background. Mobilization of bone marrow-origin CD34+ cells was investigated 3 days (3d) after acute myocardial infarction (AMI) with/without ischemic preconditioning (IP) in relation to stromal-derived factor-1 (SDF-1α)/ chemokine receptor type 4 (CXCR4) axis, to search for possible mechanisms behind insufficient cardiac repair in the first days post-AMI.  Methods. Closed-chest reperfused AMI was performed by percutaneous balloon occlusion of the mid-left anterior descending (LAD) coronary artery for 90min, followed by reperfusion in pigs. Animals were randomized to receive either IP initiated by 3x5min cycles of re-occlusion/re-flow prior to AMI (n=6) or control AMI (n=12). Blood samples were collected at baseline, 3d post-AMI, and at 1-month follow-up to analyse chemokines and mobilized CD34+ cells. To investigate the effect of acute hypoxia, SDF-1α and matrix metalloproteinase (MMP)-2 in vitro were assessed, and a migration assay of CD34+ cells toward cardiomyocytes was performed.  Results. Reperfused AMI induced significant mobilisation of CD34+ cells (baseline: 260±75 vs. 3d: 668±180; P<0.001) and secretion of MMP-2 (baseline: 291.83±53.40 vs. 3d: 369.64±72.89; P=0.011) into plasma, without affecting the SDF-1α concentration. IP led to the inhibition of MMP-2 (IP: 165.67±47.99 vs. AMI: 369.64±72.89; P=0.004) 3d post-AMI, accompanied by increased release of SDF-1α (baseline: 23.80±12.36 vs. 3d: 45.29±11.31; P=0.05) and CXCR4 (baseline: 0.59±0.16 vs. 3d: 2.06±1.42; P=0.034), with a parallel higher level of mobilisation of CD34+ cells (IP: 881±126 vs. AMI: 668±180; P=0.026), compared to non-conditioned AMI. In vitro, CD34+ cell migration toward cardiomyocytes was enhanced by SDF-1α, which was completely abolished by 90min hypoxia and co-incubation with MMP-2.  Conclusions. Non-conditioned AMI induces MMP-2 release, hampering the ischemia-induced increase in SDF-1α and CXCR4 by cleaving the SDF-1α/CXCR4 axis, with diminished mobilization of the angiogenic CD34+ cells. IP might influence CD34+ cell mobilization via inhibition of MMP-2.

Time course of endothelium-dependent and -independent coronary vasomotor response to coronary balloons and stents. Comparison of plain and drug-eluting balloons and stents.

OBJECTIVES: This study sought to determine the time dependency of the endothelium-dependent and -independent vascular responses after percutaneous coronary intervention (PCI) with drug-eluting (DEB) or plain balloons, bare-metal (BMS), and drug-eluting (DES) stents, or controls. BACKGROUND: Long-term endothelial dysfunction after DES implantation is associated with delayed healing and late thrombosis. METHODS: Domestic pigs underwent PCI using DEB or plain balloon, BMS, or DES. The dilated and stented segments, and the proximal reference segments of stents and control arteries were explanted at 5-h, 24-h, 1-week, and 1-month follow-up (FUP). Endothelin-induced vasoconstriction and endothelium-dependent and -independent vasodilation of the arterial segments were determined in vitro and were related to histological results. RESULTS: DES- and BMS-treated arteries showed proneness to vasoconstriction 5 h post-PCI. The endothelium-dependent vasodilation was profoundly (p < 0.05) impaired early after PCI (9.8 ± 3.7%, 13.4 ± 9.2%, 5.7 ± 5.3%, and 7.6 ± 4.7% using plain balloon, DEB, BMS, and DES, respectively), as compared with controls (49.6 ± 9.5%), with slow recovery. In contrast to DES, the endothelium-related vasodilation of vessels treated with plain balloon, DEB, and BMS was increased at 1 month, suggesting enhanced endogenous nitric oxide production of the neointima. The endothelium-independent (vascular smooth muscle-related) vasodilation decreased significantly at 1 day, with slow normalization during FUP. All PCI-treated vessels exhibited imbalance between vasoconstriction-vasodilation, which was more pronounced in DES- and BMS-treated vessels. No correlation between histological parameters and vasomotor function was found, indicating complex interactions between the healing neoendothelium and smooth muscle post-PCI. CONCLUSIONS: Coronary arteries treated with plain balloon, DEB, BMS, and DES showed time-dependent loss of endothelial-dependent and -independent vasomotor function, with imbalanced contraction/dilation capacity.