Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy

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

Reducing the stigma of academic mental health can save lives

My academic mental health advocacy at my local campus has led me to meet students and staff from all academic levels who feel that they have to hide who they really are. To raise mental health and stigma awareness and support others, I started a personal blog to share my story and co-founded Flourish Maastricht to promote mental health at my own institution

Monte Carlo Simulation of Diffusion MRI in geometries constructed from two-photon microscopy of human cortical grey matter

Purpose Neurodegenerative 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.Methods Diffusion 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.Results Both 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.Conclusions Monte 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

Scalable Labeling for Cytoarchitectonic Characterization of Large Optically Cleared Human Neocortex Samples

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

Reactions of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) with the tyrosine side-chain fragment, 4-ethylphenol

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

Age-related changes in murine bladder structure and sensory innervation: a multiphoton microscopy quantitative analysis

Our study aimed to examine and quantify age-related structural alterations in the healthy mouse bladder using ex vivo two-photon laser scanning microscopy (TPLSM). Freshly dissected bladders from 25-, 52-, and 85-week-old C57bl/6J mice were examined, and morphological analyses and quantification of cell layers and nerves were performed. The numbers of stretched, curled, branched, and total number of nerves in volume units of the stained muscle layer were quantified. We observed differences in the bladder wall architecture and innervation with age. Especially in 85-week-old mice, age-related changes were found, including detachment of urothelial cells and an increase in connective tissue, intermingled with the smooth muscle fibers in the muscle layer (collagen-smooth muscle ratio of 1.15 ± 0.29). In 25- and 52-week-old mice, the collagen-smooth muscle ratios were 0.20 ± 0.04 and 0.31 ± 0.11, respectively, and a clear separation of collagen and muscle was observed. The overall number of nerves and the number of curled nerves were significantly higher in the 85-week-old mice (74.0 ± 13.0 and 25.9 ± 4.8, respectively), when comparing to 25-week-old mice (26.0 ± 2.7 and 6.7 ± 1.2, respectively) and 52-week-old mice (43.8 ± 4.3 and 22.1 ± 3.3, respectively). Significant age-related alterations in bladder morphology and innervation were found, when comparing freshly dissected bladder tissue from 25-, 52-, and 85-week-old mice. The higher number of curled nerves might be an indication of an increased neurotransmitter release, resulting in a higher nerve activity, with a part of the nerves being possibly mechanically impaired. This study shows that two-photon laser scanning microscopy of healthy aging male mice is a useful method to investigate and quantify the age-related changes in the bladder wall

Murine Bladder Imaging by 2-Photon Microscopy: An Experimental Study of Morphology

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

High-resolution bladder morphology in the intact mouse bladder:an intravital two-photon study

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

hFRUIT: An optimized agent for optical clearing of DiI-stained adult human brain tissue

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