ABSTRACT
The ability to image human tissue samples in 3D, with both cellular resolution and a large field of view (FOV), can improve fundamental and clinical investigations. Here, we demonstrate the feasibility of light-sheet imaging of ~5 cm3 sized formalin fixed human brain and up to ~7 cm3 sized formalin fixed paraffin embedded (FFPE) prostate cancer samples, processed with the FFPE-MASH protocol. We present a light-sheet microscopy prototype, the cleared-tissue dual view Selective Plane Illumination Microscope (ct-dSPIM), capable of fast 3D high-resolution acquisitions of cm3 scale cleared tissue. We used mosaic scans for fast 3D overviews of entire tissue samples or higher resolution overviews of large ROIs with various speeds: (a) Mosaic 16 (16.4 µm isotropic resolution, ~1.7 h/cm3), (b) Mosaic 4 (4.1 µm isotropic resolution, ~ 5 h/cm3) and (c) Mosaic 0.5 (0.5 µm near isotropic resolution, ~15.8 h/cm3). We could visualise cortical layers and neurons around the border of human brain areas V1&V2, and could demonstrate suitable imaging quality for Gleason score grading in thick prostate cancer samples. We show that ct-dSPIM imaging is an excellent technique to quantitatively assess entire MASH prepared large-scale human tissue samples in 3D, with considerable future clinical potential.
Subject(s)
Prostate , Prostatic Neoplasms , Male , Humans , Prostate/diagnostic imaging , Microscopy/methods , Brain/diagnostic imaging , Prostatic Neoplasms/diagnostic imaging , FormaldehydeABSTRACT
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.
Subject(s)
Brain/cytology , Tissue Fixation/methods , Affinity Labels/chemistry , Animals , Brain/anatomy & histology , Carbocyanines/chemistry , Fluorescent Dyes/chemistry , Formaldehyde/chemistry , Humans , Lipids/chemistry , Lysine/chemistry , Mice , Periodic Acid/chemistry , SwineABSTRACT
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 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 ).
Subject(s)
Microscopy, Fluorescence/instrumentation , Animals , Chick Embryo , Microscopy, Fluorescence/methods , SoftwareABSTRACT
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.
Subject(s)
Neocortex/cytology , Optics and Photonics/methods , Staining and Labeling/methods , Adult , Humans , Imaging, Three-Dimensional , Microscopy, Confocal , Microtomy , Neocortex/anatomy & histologyABSTRACT
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 sub-urothelium 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±2.9106µm3; 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.
Subject(s)
Image Processing, Computer-Assisted , Sensory Receptor Cells/physiology , Urinary Bladder/innervation , Urothelium/innervation , Animals , Mice , Microscopy , Microscopy, Confocal , Mucous MembraneABSTRACT
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.
Subject(s)
Aging , Microscopy, Fluorescence, Multiphoton/methods , Muscle, Smooth/ultrastructure , Sensory Receptor Cells/ultrastructure , Urinary Bladder/innervation , Animals , Male , Mice , Mice, Inbred C57BL , Muscle, Smooth/innervation , Urinary Bladder/ultrastructureABSTRACT
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 µm, 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 µm that contained vessels with a diameter of 10 to 40 µm and nerves with a diameter of 1 to 6 µm. Imaging from the adventitial side revealed a radiant collagen layer covered with nerves and macrophages at 0 to 20 µm. Below at 20 to 25 µm 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.
Subject(s)
Microscopy, Confocal , Urinary Bladder/anatomy & histology , Animals , MiceABSTRACT
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.
Subject(s)
Enterocytes/ultrastructure , Intestinal Mucosa/cytology , Intestine, Small/cytology , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Microvilli/ultrastructure , Anesthesia , Animals , Enterocytes/physiology , Female , Imaging, Three-Dimensional/instrumentation , Imaging, Three-Dimensional/methods , Intestinal Mucosa/physiology , Intestine, Small/physiology , Mice , Mice, Inbred BALB C , Microscopy, Confocal/instrumentation , Microscopy, Electron, Transmission , Microscopy, Fluorescence/instrumentation , Microvilli/physiologyABSTRACT
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.