Search

Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy (mesoSPIM) initiative, an open-hardware project for building and operating a light sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample (www.mesospim.org).

Here, we describe a new immersion-based clearing method suitable for optical clearing of thick adult human brain samples while preserving its lipids and lipophilic labels such as 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). This clearing procedure is simple, easy to implement, and allowed for clearing of 5 mm thick human brain tissue samples within 12 days. Furthermore, we show for the first time the advantageous effect of the Periodate-Lysine-Paraformaldehyde (PLP) fixation as compared to the more commonly used 4% paraformaldehyde (PFA) on clearing performance.

Optical clearing techniques and light sheet microscopy have transformed fluorescent imaging of rodent brains, and have provided a crucial alternative to traditional confocal or bright field techniques for thin sections. However, clearing and labeling human brain tissue through all cortical layers and significant portions of a cortical area, has so far remained extremely challenging, especially for formalin fixed adult cortical tissue. Here, we present MASH (Multiscale Architectonic Staining of Human cortex): a simple, fast and low-cost cytoarchitectonic labeling approach for optically cleared human cortex samples, which can be applied to large (up to 5 mm thick) formalin fixed adult brain samples. A suite of small-molecule fluorescent nuclear and cytoplasmic dye protocols in combination with new refractive index matching solutions allows deep volume imaging. This greatly reduces time and cost of imaging cytoarchitecture in thick samples and enables classification of cytoarchitectonic layers over the full cortical depth. We demonstrate application of MASH to large archival samples of human visual areas, characterizing cortical architecture in 3D from the scale of cortical areas to that of single cells. In combination with scalable light sheet imaging and data analysis, MASH could open the door to investigation of large human cortical systems at cellular resolution and in the context of their complex 3-dimensional geometry.

PurposeNeurodegenerative diseases such as Alzheimer’s disease cause changes and disruption to cortical microstructure and architecture. Diffusion MRI (dMRI) could potentially be sensitive to such changes. There is a growing interest in modeling of human cortical areas using a combination of quantitative MRI and 3D microscopy. The purpose of this study was to quantitatively characterize the cytoarchitecture of human cortical tissue from 3D fluorescence microscopy to simulate diffusion MRI (dMRI) signal in the cortex to better understand its diffusion signal characteristics.MethodsDiffusion of water molecules and dMRI signal were simulated by an indirect geometry based method and a direct voxel based method in microstructural details extracted from microscopy of cortex. Additionally, residence times of diffusing spins inside voxel volumes were considered to set effective resolution limits. Mean diffusivity (MD) and kurtosis (MK) were calculated for variable cell and neurite densities, sizes and diffusion times under realistic values for permeability and free diffusion.ResultsBoth simulation methods could efficiently and accurately simulate dMRI signals with fractional anisotropy, diffusion coefficient and kurtosis in agreement with previous reports. Simulated MD and MK showed changes with increasing diffusion times specific to cortical cell density and sizes, with MK showing the highest sensitivity. Intra-voxel residence times with increasing diffusion times showed that the effective dMRI resolution approaches the thickness of cortical layers.ConclusionsMonte Carlo simulations based on 3D microscopy data enable estimating changes in MD and MK over diffusion times and are sensitive to cortical cytoarchitecture and its possible changes in neurodegenerative disease. When considering layer-specific cortical dMRI, effective resolution due to residence times is an important concern.

Over the course of the past decade, tissue clearing methods have reached a high level of sophistication with a wide variety of approaches now available[1][1]. To image large cleared samples, light-sheet microscopes have proven to be ideal due to their excellent optical sectioning capability in

We developed a platform for intravital bladder two-photon microscopy (TPM) in healthy wild-type mice. It was our aim to make intravital bladder TPM simple and reproducible for researchers with access to TPM. In this study, the intact murine bladder was examined by means of autofluorescence (AF), second-harmonic generation (SHG), and different i.v. injected fluorescent dyes. All bladder layers with containing structures, such as cells, nerves, and vessels were detected in different colour-coded spectral channels, and shown in high-resolution images and real-time movies. The presented method opens up avenues for many future studies and applications, such as to reveal mechanisms and physiology in the natural state of the bladder. Researcher can use intravital bladder TPM to investigate events, such as migration and metabolism of (inflammatory) cells, nanoparticle uptake, or micro contractions in response to stimuli.

A strong association between functional bladder disorders and bladder sensation is well-known, with a relationship between malfunctioning detrusor muscle and abnormal sensation arising from the suburothelium and the lamina propria (LP), has been suggested. However, the exact underlying pathophysiology of these bladder disorders is not completely understood. Therefore, it is important to gain knowledge on sensory innervation of the urinary bladder in order to understand the neural network function in healthy and diseased bladder. In the present study we aim at the development of a computer-assisted method for 3D-tracking of sensory innervation in the murine bladder mucosa using two-photon laser scanning microscopy (TPLSM). TPLSM was performed on 10 fixed, stained (CGRP) bladder samples in both the trigone and dome. Nerve tracking was performed in subvolumes (6.3 +/- 2.9 10(6) mu m(3); median +/- IQR) of 22 stacks with determining total nerve length, nerve segment lengths, curviness, straightness, and locations of branching and ending points in the lamina propria (LP). The results show that the highest concentration of afferent fibres was found at the urothelium-LP interface. Nerve curviness, a presumed indicator of nerve activity, showed an equal value throughout the complete LP. We found a significantly higher median nerve segment length in the LP of the trigone and significantly more curved nerves in the dome of the bladder. This indicates an adaptation to, or an involvement in the detection of, bladder volume changes. Conclusively, we successfully developed a computer-assisted method for 3D tracking of sensory nerve fibres in the LP of the murine bladder wall. (C) 2017 Elsevier B.V. All rights reserved.

Purpose: We developed 2-photon laser scanning microscopy analysis of the native murine bladder. Materials and Methods: Bladder tissue from wild-type mice was imaged by 2-photon laser scanning microscopy autofluorescence and second harmonic generation microscopy. Bladder wall layers and structures were analyzed using differences in color, size, shape and morphology. Results: Autofluorescence of the urothelium, nerve structures and muscles was visible in the green spectral channel due to autofluorochromes such as NAD(P)H and elastin. Second harmonic generation of collagen was seen in the blue spectral channel. Imaging from the mucosal side revealed umbrella cells at 0 and 30 mm, of which the high cellular NAD(P)H content allows autofluorescence detection. Below that a network-like connective tissue layer was visualized up to 50 mm that contained vessels with a diameter of 10 to 40 mm and nerves with a diameter of 1 to 6 mm. Imaging from the adventitial side revealed a radiant collagen layer covered with nerves and macrophages at 0 to 20 mm. Below at 20 to 25 mm we visualized a thick muscle layer containing elastic fibers and macrophages. Findings were also represented in 3-dimensional reconstructions, providing information on structure localization, orientation and interconnection. Conclusions: Two-photon laser scanning microscopy imaging using autofluorescence of the murine bladder is a promising technique to provide new insight into structures and morphology. It opens avenues to identify structural changes in bladder pathology.

Age : the official journal of the American Aging Association 38(1), 17 (2016). doi:10.1007/s11357-016-9878-1, Published by Springer Science+Business Media, New York, NY

The mucosa of the gastrointestinal tract is a dynamic tissue composed of numerous cell types with complex cellular functions. Study of the vital intestinal mucosa has been hampered by lack of suitable model systems. We here present a novel animal model that enables highly resolved three-dimensional imaging of the vital murine intestine in anaesthetized mice. Using intravital autofluorescence 2-photon (A2P) microscopy we studied the choreographed interactions of enterocytes, goblet cells, enteroendocrine cells and brush cells with other cellular constituents of the small intestinal mucosa over several hours at a subcellular resolution and in three dimensions. Vigorously moving lymphoid cells and their interaction with constituent parts of the lamina propria were examined and quantitatively analyzed. Nuclear and lectin staining permitted simultaneous characterization of autofluorescence and admitted dyes and yielded additional spectral information that is crucial to the interpretation of the complex intestinal mucosa. This novel intravital approach provides detailed insights into the physiology of the small intestine and especially opens a new window for investigating cellular dynamics under nearly physiological conditions. Electronic supplementary material The online version of this article (doi:10.1007/s00418-011-0905-0) contains supplementary material, which is available to authorized users.

Almost 600 million people are suffering from lower urinary tract dysfunctions worldwide, especially the elderly. The underlying causes are not fully understood. Therefore, this dissertation aims at increasing the knowledge of bladder tissue morphology, and more specifically also the nervous supply of the bladder wall. For this purpose, the organ architecture of the bladder of a mouse model has been investigated in a three dimensional setting. Moreover, age-related changes of bladder morphology have been found, when comparing old and young mice. This dissertation shows the successful development and application of different and novel microscopic techniques, such as a computer-assisted model for bladder nerve tracing and intravital bladder imaging.

The melatonin metabolite N(1)-acetyl-5-methoxykynuramine (AMK) has previously been shown to interact with various free radicals. Using the ABTS cation radical [ABTS = 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)] as an electron abstracting reactant, which does not destroy the aromate, we found that the reactive intermediate derived from AMK strongly interacts with the benzene rings of other AMK molecules to form di- and oligomers. Since oligomerization is rather unlikely at physiological concentrations, we investigated reactions with other putative reaction partners. The incubation of tyrosine or several of its structural analogs with AMK in the presence of the ABTS cation radical led to numerous products, amongst which were compounds not detected when one of the educts was incubated with the ABTS cation radical alone. With tyrosine and most of its analogs, the number of products formed in the presence of AMK and ABTS cation radical was relatively high and included numerous oligomers. To optimize the yield of products of interest as well as their separation from other compounds, especially oligomers, we investigated the interaction with 4-ethylphenol, which represents the side chain of tyrosine lacking the carboxyl and amino residues of the amino acid, which otherwise can undergo additional reactions. A prominent product was chromatographically separated and analyzed by mass spectrometry [(+)-ESI-MS, (-)-ESI-MS, (+)-HRESI-MS], (1)H-NMR, and H,H-COSY correlations. The substance was identified as N-{3-[2'-(5''-ethyl-2''-hydroxyphenylamino)-5'-methoxyphenyl]-3-oxopropyl} acetamide. This chemically novel compound represents an adduct in which the amino nitrogen of AMK is attached to the C-2 atom of 4-ethylphenol, which corresponds to the C-3 atom in the benzene ring of tyrosine. This finding suggests that, upon interaction of AMK with an electron-abstracting radical, the kynuric intermediate may modify proteins at superficially accessible tyrosine residues. In fact, protein modification by an unidentified melatonin metabolite has been observed in an earlier study. The possibility of protein AMKylation may be of interest with regard to an eventual interference with tyrosine nitration or, more importantly, with tyrosine phosphorylation.

We describe MASH (Multiscale Architectonic Staining of Human cortex): a simple, fast and low-cost cytoarchitectonic labeling and optical clearing approach for human cortex samples, which can be applied to large formalin fixed adult brain samples. A suite of small-molecule fluorescent nuclear and cytoplasmic dyes in combination with new refractive index matching solutions allows deep volume imaging. This enables highly scalable human neocortical cytoarchitecture characterization with a large 3D scope.