SnapShot: Tissue Clearing.

Tissue clearing has become an important tool for the investigation of biological systems in three dimensions. However, many pioneering techniques were based on serendipitous discoveries. Next-generation clearing methods have been (re)designed with a better understanding of the chemistry and physics required to equalize the refractive index throughout a sample which prevents the random bending of light that clouds biological tissues.

Clarifying Tissue Clearing.

Biological specimens are intrinsically three dimensional; however, because of the obscuring effects of light scatter, imaging deep into a tissue volume is problematic. Although efforts to eliminate the scatter by "clearing" the tissue have been ongoing for over a century, there have been a large number of recent innovations. This Review introduces the physical basis for light scatter in tissue, describes the mechanisms underlying various clearing techniques, and discusses several of the major advances in light microscopy for imaging cleared tissue.

Deep 3D histology powered by tissue clearing, omics and AI.

To comprehensively understand tissue and organism physiology and pathophysiology, it is essential to create complete three-dimensional (3D) cellular maps. These maps require structural data, such as the 3D configuration and positioning of tissues and cells, and molecular data on the constitution of each cell, spanning from the DNA sequence to protein expression. While single-cell transcriptomics is illuminating the cellular and molecular diversity across species and tissues, the 3D spatial context of these molecular data is often overlooked. Here, I discuss emerging 3D tissue histology techniques that add the missing third spatial dimension to biomedical research. Through innovations in tissue-clearing chemistry, labeling and volumetric imaging that enhance 3D reconstructions and their synergy with molecular techniques, these technologies will provide detailed blueprints of entire organs or organisms at the cellular level. Machine learning, especially deep learning, will be essential for extracting meaningful insights from the vast data. Further development of integrated structural, molecular and computational methods will unlock the full potential of next-generation 3D histology.

Colorimetric histology using plasmonically active microscope slides.

The human eye can distinguish as many as 10,000 different colours but is far less sensitive to variations in intensity1, meaning that colour is highly desirable when interpreting images. However, most biological samples are essentially transparent, and nearly invisible when viewed using a standard optical microscope2. It is therefore highly desirable to be able to produce coloured images without needing to add any stains or dyes, which can alter the sample properties. Here we demonstrate that colorimetric histology images can be generated using full-sized plasmonically active microscope slides. These slides translate subtle changes in the dielectric constant into striking colour contrast when samples are placed upon them. We demonstrate the biomedical potential of this technique, which we term histoplasmonics, by distinguishing neoplastic cells from normal breast epithelium during the earliest stages of tumorigenesis in the mouse MMTV-PyMT mammary tumour model. We then apply this method to human diagnostic tissue and validate its utility in distinguishing normal epithelium, usual ductal hyperplasia, and early-stage breast cancer (ductal carcinoma in situ). The colorimetric output of the image pixels is compared to conventional histopathology. The results we report here support the hypothesis that histoplasmonics can be used as a novel alternative or adjunct to general staining. The widespread availability of this technique and its incorporation into standard laboratory workflows may prove transformative for applications extending well beyond tissue diagnostics. This work also highlights opportunities for improvements to digital pathology that have yet to be explored.

Inactivation of face-selective neurons alters eye movements when free viewing faces.

During free viewing, faces attract gaze and induce specific fixation patterns corresponding to the facial features. This suggests that neurons encoding the facial features are in the causal chain that steers the eyes. However, there is no physiological evidence to support a mechanistic link between face-encoding neurons in high-level visual areas and the oculomotor system. In this study, we targeted the middle face patches of the inferior temporal (IT) cortex in two macaque monkeys using an functional magnetic resonance imaging (fMRI) localizer. We then utilized muscimol microinjection to unilaterally suppress IT neural activity inside and outside the face patches and recorded eye movements while the animals free viewing natural scenes. Inactivation of the face-selective neurons altered the pattern of eye movements on faces: The monkeys found faces in the scene but neglected the eye contralateral to the inactivation hemisphere. These findings reveal the causal contribution of the high-level visual cortex in eye movements.

Tissue clearing and its applications in neuroscience.

State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.

Eye drift during fixation predicts visual acuity.

Visual acuity is commonly assumed to be determined by the eye optics and spatial sampling in the retina. Unlike a camera, however, the eyes are never stationary during the acquisition of visual information; a jittery motion known as ocular drift incessantly displaces stimuli over many photoreceptors. Previous studies have shown that acuity is impaired in the absence of retinal image motion caused by eye drift. However, the relation between individual drift characteristics and acuity remains unknown. Here, we show that a) healthy emmetropes exhibit a large variability in their amount of drift and that b) these differences profoundly affect the structure of spatiotemporal signals to the retina. We further show that c) the spectral distribution of the resulting luminance modulations strongly correlates with individual visual acuity and that d) natural intertrial fluctuations in the amount of drift modulate acuity. As a consequence, in healthy emmetropes, acuity can be predicted from the motor behavior elicited by a simple fixation task, without directly measuring it. These results shed new light on how oculomotor behavior contributes to fine spatial vision.

Optical sectioning of unlabeled samples using bright-field microscopy.

The bright-field (BF) optical microscope is a traditional bioimaging tool that has been recently tested for depth discrimination during evaluation of specimen morphology; however, existing approaches require dedicated instrumentation or extensive computer modeling. We report a direct method for three-dimensional (3D) imaging in BF microscopy, applicable to label-free samples, where we use Köhler illumination in the coherent regime and conventional digital image processing filters to achieve optical sectioning. By visualizing fungal, animal tissue, and plant samples and comparing with light-sheet fluorescence microscopy imaging, we demonstrate the accuracy and applicability of the method, showing how the standard microscope is an effective 3D imaging device.

Self-loops in evolutionary graph theory: Friends or foes?

Evolutionary dynamics in spatially structured populations has been studied for a long time. More recently, the focus has been to construct structures that amplify selection by fixing beneficial mutations with higher probability than the well-mixed population and lower probability of fixation for deleterious mutations. It has been shown that for a structure to substantially amplify selection, self-loops are necessary when mutants appear predominately in nodes that change often. As a result, for low mutation rates, self-looped amplifiers attain higher steady-state average fitness in the mutation-selection balance than well-mixed populations. But what happens when the mutation rate increases such that fixation probabilities alone no longer describe the dynamics? We show that self-loops effects are detrimental outside the low mutation rate regime. In the intermediate and high mutation rate regime, amplifiers of selection attain lower steady-state average fitness than the complete graph and suppressors of selection. We also provide an estimate of the mutation rate beyond which the mutation-selection dynamics on a graph deviates from the weak mutation rate approximation. It involves computing average fixation time scaling with respect to the population sizes for several graphs.

Connecting structure and function from organisms to molecules in small-animal symbioses through chemo-histo-tomography.

Our understanding of metabolic interactions between small symbiotic animals and bacteria or parasitic eukaryotes that reside within their bodies is extremely limited. This gap in knowledge originates from a methodological challenge, namely to connect histological changes in host tissues induced by beneficial and parasitic (micro)organisms to the underlying metabolites. We addressed this challenge and developed chemo-histo-tomography (CHEMHIST), a culture-independent approach to connect anatomic structure and metabolic function in millimeter-sized symbiotic animals. CHEMHIST combines chemical imaging of metabolites based on mass spectrometry imaging (MSI) and microanatomy-based micro-computed X-ray tomography (micro-CT) on the same animal. Both high-resolution MSI and micro-CT allowed us to correlate the distribution of metabolites to the same animal's three-dimensional (3D) histology down to submicrometer resolutions. Our protocol is compatible with tissue-specific DNA sequencing and fluorescence in situ hybridization for the taxonomic identification and localization of the associated micro(organisms). Building CHEMHIST upon in situ imaging, we sampled an earthworm from its natural habitat and created an interactive 3D model of its physical and chemical interactions with bacteria and parasitic nematodes in its tissues. Combining MSI and micro-CT, we present a methodological groundwork for connecting metabolic and anatomic phenotypes of small symbiotic animals that often represent keystone species for ecosystem functioning.

Perforating cutaneous vessels: A key feature of acupoints - Anatomical evidence from five-Shu acupoints in the upper limbs.

Acupuncture has been proven an effective clinical treatment for numerous pathological conditions and malfunctions. However, substantial anatomical evidence for acupuncture points (APs) and meridians is still lacking, so the location of APs is relatively subjective and understanding of the biological mechanisms of acupuncture is limited. All these problems hinder the clinical applications and worldwide acceptance of acupuncture. Our long-term microsurgery experience has indicated that Perforating Cutaneous Vessels (PCVs) are highly relevant to APs but the anatomical evidence is insufficient. To address this lack, two specimens of fresh adult human upper limbs were dissected using an advanced vascular perfusion-fixation method and then examined. The results show that all 30 five-Shu APs in the upper limbs have corresponding PCVs. Both specimens showed a 100% coincidence rate between APs and PCVs, indicating that PCVs could be critical anatomical features of APs. This study also provides an anatomical basis for locating APs objectively via preliminary detection of PCVs. The findings could lead to a better theoretical understanding of mechanisms of acupuncture and the essence of meridians.

Idiopathic Slow Transit Constipation: Pathophysiology, Diagnosis, and Management.

Slow transit constipation (STC) has an estimated prevalence of 2-4% of the general population, and although it is the least prevalent of the chronic constipation phenotypes, it more commonly causes refractory symptoms and is associated with significant psychosocial stress, poor quality of life, and high healthcare costs. This review provides an overview of the pathophysiology, diagnosis, and management options in STC. STC occurs due to colonic dysmotility and is thought to be a neuromuscular disorder of the colon. Several pathophysiologic features have been observed in STC, including reduced contractions on manometry, delayed emptying on transit studies, reduced numbers of interstitial cells of Cajal on histology, and reduced amounts of excitatory neurotransmitters within myenteric plexuses. The underlying aetiology is uncertain, but autoimmune and hormonal mechanisms have been hypothesised. Diagnosing STC may be challenging, and there is substantial overlap with the other clinical constipation phenotypes. Prior to making a diagnosis of STC, other primary constipation phenotypes and secondary causes of constipation need to be ruled out. An assessment of colonic transit time is required for the diagnosis and can be performed by a number of different methods. There are several different management options for constipation, including lifestyle, dietary, pharmacologic, interventional, and surgical. The effectiveness of the available therapies in STC differs from that of the other constipation phenotypes, and prokinetics often make up the mainstay for those who fail standard laxatives. There are few available management options for patients with medically refractory STC, but patients may respond well to surgical intervention. STC is a common condition associated with a significant burden of disease. It can present a clinical challenge, but a structured approach to the diagnosis and management can be of great value to the clinician. There are many therapeutic options available, with some having more benefits than others.

Tensor image registration library: Deformable registration of stand-alone histology images to whole-brain post-mortem MRI data.

BACKGROUND: Accurate registration between microscopy and MRI data is necessary for validating imaging biomarkers against neuropathology, and to disentangle complex signal dependencies in microstructural MRI. Existing registration methods often rely on serial histological sampling or significant manual input, providing limited scope to work with a large number of stand-alone histology sections. Here we present a customisable pipeline to assist the registration of stand-alone histology sections to whole-brain MRI data. METHODS: Our pipeline registers stained histology sections to whole-brain post-mortem MRI in 4 stages, with the help of two photographic intermediaries: a block face image (to undistort histology sections) and coronal brain slab photographs (to insert them into MRI space). Each registration stage is implemented as a configurable stand-alone Python script using our novel platform, Tensor Image Registration Library (TIRL), which provides flexibility for wider adaptation. We report our experience of registering 87 PLP-stained histology sections from 14 subjects and perform various experiments to assess the accuracy and robustness of each stage of the pipeline. RESULTS: All 87 histology sections were successfully registered to MRI. Histology-to-block registration (Stage 1) achieved 0.2-0.4 mm accuracy, better than commonly used existing methods. Block-to-slice matching (Stage 2) showed great robustness in automatically identifying and inserting small tissue blocks into whole brain slices with 0.2 mm accuracy. Simulations demonstrated sub-voxel level accuracy (0.13 mm) of the slice-to-volume registration (Stage 3) algorithm, which was observed in over 200 actual brain slice registrations, compensating 3D slice deformations up to 6.5 mm. Stage 4 combined the previous stages and generated refined pixelwise aligned multi-modal histology-MRI stacks. CONCLUSIONS: Our open-source pipeline provides robust automation tools for registering stand-alone histology sections to MRI data with sub-voxel level precision, and the underlying framework makes it readily adaptable to a diverse range of microscopy-MRI studies.

MSIr: Automatic Registration Service for Mass Spectrometry Imaging and Histology.

Mass spectrometry imaging (MSI) is a powerful tool that can be used to simultaneously investigate the spatial distribution of different molecules in samples. However, it is difficult to comprehensively analyze complex biological systems with only a single analytical technique due to different analytical properties and application limitations. Therefore, many analytical methods have been combined to extend data interpretation, evaluate data credibility, and facilitate data mining to explore important temporal and spatial relationships in biological systems. Image registration is an initial and critical step for multimodal imaging data fusion. However, the image registration of multimodal images is not a simple task. The property difference between each data modality may include spatial resolution, image characteristics, or both. The image registrations between MSI and different imaging techniques are often achieved indirectly through histology. Many methods exist for image registration between MSI data and histological images. However, most of them are manual or semiautomatic and have their prerequisites. Here, we built MSI Registrar (MSIr), a web service for automatic registration between MSI and histology. It can help to reduce subjectivity and processing time efficiently. MSIr provides an interface for manually selecting region of interests from histological images; the user selects regions of interest to extract the corresponding spectrum indices in MSI data. In the performance evaluation, MSIr can quickly map MSI data to histological images and help pinpoint molecular components at specific locations in tissues. Most registrations were adequate and were without excessive shifts. MSIr is freely available at https://msir.cmdm.tw and https://github.com/CMDM-Lab/MSIr.

Chloroacetate esterase reaction combined with immunohistochemical characterization of the specific tissue microenvironment of mast cells.

This study provides a combined histochemical method for detecting enzyme activity of chloroacetate esterase simultaneously with immunolabeling of the components of a specific tissue microenvironment on formalin-fixed, paraffin-embedded specimens. Chromogenic detection of the molecular targets within and outside the mast cells provides novel options in determining the histoarchitectonics of organ-specific mast cell populations, studying the functional significance of chloroacetate esterase and specifying the immune landscape of the tissue microenvironment.

Methods to expose subsurface objects of interest identified from 3D imaging: The intermediate sample preparation stage in the correlative microscopy workflow.

The correlative imaging workflow is a method of combining information and data across modes (e.g. SEM, X-ray CT, FIB-SEM), scales (cm to nm) and dimensions (2D-3D-4D), providing a more holistic interpretation of the research question. Often, subsurface objects of interest (e.g. inclusions, pores, cracks, defects in multilayered samples) are identified from initial exploratory nondestructive 3D tomographic imaging (e.g. X-ray CT, XRM), and those objects need to be studied using additional techniques to obtain, for example, 2D chemical or crystallographic data. Consequently, an intermediate sample preparation step needs to be completed, where a targeted amount of sample surface material is removed, exposing and revealing the object of interest. At present, there is not one singular technique for removing varied thicknesses at high resolution and on a range of scales from cm to nm. Here, we review the manual and automated options currently available for targeted sample material removal, with a focus on those methods which are readily accessible in most laboratories. We summarise the approaches for manual grinding and polishing, automated grinding and polishing, microtome/ultramicrotome, and broad-beam ion milling (BBIM), with further review of other more specialist techniques including serial block face electron microscopy (SBF-SEM), and ion milling and laser approaches such as FIB-SEM, Xe plasma FIB-SEM, and femtosecond laser/LaserFIB. We also address factors which may influence the decision on a particular technique, including the composition, shape and size of the samples, sample mounting limitations, the amount of surface material to be removed, the accuracy and/or resolution of peripheral parts, the accuracy and/or resolution of the technique/instrumentation, and other more general factors such as accessibility to instrumentation, costs, and the time taken for experimentation. It is hoped that this study will provide researchers with a range of options for removal of specific amounts of sample surface material to reach subsurface objects of interest in both correlative and non-correlative workflows.

Practical strategies for robust and inexpensive imaging of aqueous-cleared tissues.

Lightsheet microscopy offers an ideal method for imaging of large (mm-cm scale) biological tissues rendered transparent via optical clearing protocols. However the diversity of clearing technologies and tissue types, and how these are adapted to the microscope can make tissue mounting complicated and somewhat irreproducible. Tissue preparation for imaging can involve glues and or equilibration in a variety of expensive and/or proprietary formulations. Here we present practical advice for mounting and capping cleared tissues in optical cuvettes for macroscopic imaging, providing a standardised 3D cell that can be imaged routinely and relatively inexpensively. We show that acrylic cuvettes cause minimal spherical aberration with objective numerical apertures less than 0.65. Furthermore, we describe methods for aligning and assessing the light sheets, discriminating fluorescence from autofluorescence, identifying chromatic artefacts due to differential scattering and removing streak artefacts such that they do not confound downstream 3D object segmentation analyses, with mouse embryo, liver and heart imaging as demonstrated examples.

Evaluating the optimal tissue thickness for mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization.

RATIONALE: Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) utilizes a 2970 nm mid-IR laser to desorb samples with depth resolutions (Z) on the order of micrometers. Conventionally, 5-20 µm thick tissue sections are used to characterize different applications of the IR-MALDESI source, but an optimal thickness has not been systematically investigated. METHODS: Mouse liver was sectioned to various thicknesses and analyzed using IR-MALDESI mass spectrometry imaging (MSI). Height profiles of tissue sections of various cryosectioned thicknesses were acquired to affirm tissue thickness. Tissue sections of each thickness were measured using a Keyence microscope. Paraffin wax was cryosectioned, mounted on microscope slides, and measured using a chromatic confocal sensor system to determine the cryostat sectioning accuracy. RESULTS: Analyzing sectioned tissues at higher thickness (>10 µm) leads to lower ion abundance, a decrease in signal over long analysis times, and more frequent instrument cleaning. Additionally, increasing tissue thickness above the optimum (7 µm) does not result in a significant increase in lipid annotations. CONCLUSIONS: This work defines an optimal sample thickness for IR-MALDESI-MSI and demonstrates the utility of optimizing tissue thickness for MSI platforms of comparable Z resolution.

An introduction to lesions and histology of scleractinian corals.

Stony corals (Scleractinia) are in the Phylum Cnidaria (cnidae referring to various types of stinging cells). They may be solitary or colonial, but all secrete an external, supporting aragonite skeleton. Large, colonial members of this phylum are responsible for the accretion of coral reefs in tropical and subtropical waters that form the foundations of the most biodiverse marine ecosystems. Coral reefs worldwide, but particularly in the Caribbean, are experiencing unprecedented levels of disease, resulting in reef degradation. Most coral diseases remain poorly described and lack clear case definitions, while the etiologies and pathogenesis are even more elusive. This introductory guide is focused on reef-building corals and describes basic gross and microscopic lesions in these corals in order to serve as an invitation to other veterinary pathologists to play a critical role in defining and advancing the field of coral pathology.

Basic Concepts in Nail Pathology.

ABSTRACT: Understanding nail pathology is complex for general pathologists and even for those dermatopathologists who do not receive many nail samples in their laboratories. In this article, we attempt to review some of the primary entities in nail pathology with the aid of modern interpretations of nail histology and embryology. We also provide diagrams that can aid in comprehending this field of pathology.