Imaging Of Whole Organs Research Articles

A novel m-xylylene-diamine/glucose based-supramolecular eutectogels with tissue clearing for three dimensional histological imaging

Hydrogel-based tissue clearing technologies have shown significant promise for deep-tissue imaging and subcellular-level optical 3D reconstruction of whole organs. This study proposes a novel approach utilizing a deep eutectic solvent (DES) formulated with glucose and m-xylylene-diamine (MXDA) to create a highly efficient tissue-clearing hydrogel system named the passive hydrogel clearing system (PHCS). PHCS achieved efficient tissue clearing through a single-step tissue gelation process. The resulting hydrogel-tissue complex exhibited thermoreversible properties, transitioning into a sol state upon heating and vice versa upon cooling. Notably, PHCS enabled media embedding, facilitating immunofluorescence histopathology. Additionally, the system demonstrated compatibility with various fluorescent probes, particularly lipophilic dyes. Our study successfully employed PHCS for the reconstruction of vascular structures within the intestine, enabling the generation of a 3D pathology model. These findings suggest that PHCS is a promising novel method for fabricating hydrogels for tissue clearing and holds great potential for application as a mounting medium for morphological imaging.

Fluorescent Water-Soluble Polycationic Chitosan Polymers as Markers for Biological 3D Imaging.

Over the last decades, various tissue-clearing techniques have broken the ground for the optical imaging of whole organs and whole-organisms, providing complete representative data sets of three-dimensional biological structures. Along with advancements in this field, the development of fluorescent markers for staining vessels and capillaries has offered insights into the complexity of vascular networks and their impact on disease progression. Here we describe the use of a modified water-soluble chitosan linked to cyanine dyes in combination with ethyl cinnamate-based optical tissue clearing for the 3D visualization of tissue vasculature in depth. The water-soluble fluorescent Chitosans have proven to be an optimal candidate for labeling both vessels and capillaries ex vivo thanks to their increased water solubility, high photostability, and optical properties in the near-infrared window. In addition, the nontoxicity of these markers broadens their applicability to in vitro and in vivo biological applications. Despite the availability of other fluorescent molecules for vascular staining, the present study, for the first time, demonstrates the potential of fluorescent chitosans to depict vessels at the capillary level and opens avenues for advancing the diagnostic field by reducing the complexity and costs of the currently available procedures.

Considerations for Microscopic Imaging of Whole Organs and Animals.

Considerations for Microscopic Imaging of Whole Organs and Animals.

How innovations in methodology offer new prospects for volume electron microscopy

Detailed knowledge of biological structure has been key in understanding biology at several levels of organisation, from organs to cells and proteins. Volume electron microscopy (volume EM) provides high resolution 3D structural information about tissues on the nanometre scale. However, the throughput rate of conventional electron microscopes has limited the volume size and number of samples that can be imaged. Recent improvements in methodology are currently driving a revolution in volume EM, making possible the structural imaging of whole organs and small organisms. In turn, these recent developments in image acquisition have created or stressed bottlenecks in other parts of the pipeline, like sample preparation, image analysis and data management. While the progress in image analysis is stunning due to the advent of automatic segmentation and server‐based annotation tools, several challenges remain. Here we discuss recent trends in volume EM, emerging methods for increasing throughput and implications for sample preparation, image analysis and data management.

Cryo-fluorescence micro-optical sectioning tomography for volumetric imaging of various whole organs with subcellular resolution

SummaryOptical visualization of complex microstructures in the entire organ is essential for biomedical research. However, the existing methods fail to accurately acquire the detailed microstructures of whole organs with good morphological and biochemical preservation. This study proposes a cryo-fluorescence micro-optical sectioning tomography (cryo-fMOST) to image whole organs in three dimensions (3D) with submicron resolution. The system comprises a line-illumination microscope module, cryo-microtome, three-stage refrigeration module, and heat insulation device. To demonstrate the imaging capacity and wide applicability of the system, we imaged and reconstructed various organs of mice in 3D, including the healthy tongue, kidney, and brain, as well as the infarcted heart. More importantly, imaged brain slices were performed sugar phosphates determination and fluorescence in situ hybridization imaging to verify the compatibility of multi-omics measurements. The results demonstrated that cryo-fMOST is capable of acquiring high-resolution morphological details of various whole organs and may be potentially useful for spatial multi-omics.

Independently paced Ca2+ oscillations in progenitor and differentiated cells in an ex vivo epithelial organ.

ABSTRACTCytosolic Ca2+ is a highly dynamic, tightly regulated and broadly conserved cellular signal. Ca2+ dynamics have been studied widely in cellular monocultures, yet organs in vivo comprise heterogeneous populations of stem and differentiated cells. Here, we examine Ca2+ dynamics in the adult Drosophila intestine, a self-renewing epithelial organ in which stem cells continuously produce daughters that differentiate into either enteroendocrine cells or enterocytes. Live imaging of whole organs ex vivo reveals that stem-cell daughters adopt strikingly distinct patterns of Ca2+ oscillations after differentiation: enteroendocrine cells exhibit single-cell Ca2+ oscillations, whereas enterocytes exhibit rhythmic, long-range Ca2+ waves. These multicellular waves do not propagate through immature progenitors (stem cells and enteroblasts), of which the oscillation frequency is approximately half that of enteroendocrine cells. Organ-scale inhibition of gap junctions eliminates Ca2+ oscillations in all cell types – even, intriguingly, in progenitor and enteroendocrine cells that are surrounded only by enterocytes. Our findings establish that cells adopt fate-specific modes of Ca2+ dynamics as they terminally differentiate and reveal that the oscillatory dynamics of different cell types in a single, coherent epithelium are paced independently.

Three-dimensional label-free histological imaging of whole organs by microtomy-assisted autofluorescence tomography

SummaryThree-dimensional (3D) histology is vitally important to characterize disease-induced tissue heterogeneity at the individual cell level. However, it remains challenging for both high-throughput 3D imaging and volumetric reconstruction. Here we propose a label-free, cost-effective, and ready-to-use 3D histological imaging technique, termed microtomy-assisted autofluorescence tomography with ultraviolet excitation (MATE). With the combination of block-face imaging and serial microtome sectioning, MATE can achieve rapid and label-free imaging of paraffin-embedded whole organs at an acquisition speed of 1 cm3 per 4 h with a voxel resolution of 1.2 × 1.2 × 10 μm3. We demonstrate that MATE enables simultaneous visualization of cell nuclei, fiber tracts, and blood vessels in mouse/human brains without tissue staining or clearing. Moreover, diagnostic features, including nuclear size and packing density, can be quantitatively extracted with high accuracy. MATE is augmented to the current slide-based 2D histology, holding great promise to facilitate histopathological interpretation at the organelle level.

Collagen-Targeted Peptides for Molecular Imaging of Diffuse Cardiac Fibrosis.

BackgroundCardiac fibrosis is the excessive deposition of extracellular matrix in the heart, triggered by a cardiac insult, aging, genetics, or environmental factors. Molecular imaging of the cardiac extracellular matrix with targeted probes could improve diagnosis and treatment of heart disease. However, although this technology has been used to demonstrate focal scarring arising from myocardial infarction, its capacity to demonstrate extracellular matrix expansion and diffuse cardiac fibrosis has not been assessed.Methods and ResultsHere, we report the use of collagen‐targeted peptides labeled with near‐infrared fluorophores for the detection of diffuse cardiac fibrosis in the β2‐AR (β‐2‐adrenergic receptor) overexpressing mouse model and in ischemic human hearts. Two approaches were evaluated, the first based on a T peptide that binds matrix metalloproteinase‐2‐proteolyzed collagen IV, and the second on the cyclic peptide EP‐3533, which targets collagen I. The systemic and cardiac uptakes of both peptides (intravenously administered) were quantified ex vivo by near‐infrared imaging of whole organs, tissue sections, and heart lysates. The peptide accumulation profiles corresponded to an immunohistochemically‐validated increase in collagen types I and IV in hearts of transgenic mice versus littermate controls. The T peptide could encouragingly demonstrate both the intermediate (7 months old) and severe (11 months old) cardiomyopathic phenotypes. Co‐immunostainings of fluorescent peptides and collagens, as well as reduced collagen binding of a control peptide, confirmed the collagen specificity of the tracers. Qualitative analysis of heart samples from patients with ischemic cardiomyopathy compared with nondiseased donors supported the collagen‐enhancement capabilities of these peptides also in the clinical settings.ConclusionsTogether, these observations demonstrate the feasibility and translation potential of molecular imaging with collagen‐binding peptides for noninvasive imaging of diffuse cardiac fibrosis.

Tissue clearing and 3D imaging - putting immune cells into context.

A better understanding of cell-cell and cell-niche interactions is crucial to comprehend the complexity of inflammatory or pathophysiological scenarios such as tissue damage during viral infections, the tumour microenvironment and neuroinflammation. Optical clearing and 3D volumetric imaging of large tissue pieces or whole organs is a rapidly developing methodology that holds great promise for the in-depth study of cells in their natural surroundings. These methods have mostly been applied to image structural components such as endothelial cells and neuronal architecture. Recent work now highlights the possibility of studying immune cells in detail within their respective immune niches. This Review summarizes recent developments in tissue clearing methods and 3D imaging, with a focus on the localization and quantification of immune cells. We first provide background to the optical challenges involved and their solutions before discussing published protocols for tissue clearing, the limitations of 3D imaging of immune cells and image analysis. Furthermore, we highlight possible applications for tissue clearing and propose future developments for the analysis of immune cells within homeostatic or inflammatory immune niches.

Three-dimensional vascular and metabolic imaging using inverted autofluorescence

.Significance: Three-dimensional (3D) vascular and metabolic imaging (VMI) of whole organs in rodents provides critical and important (patho)physiological information in studying animal models of vascular network.Aim: Autofluorescence metabolic imaging has been used to evaluate mitochondrial metabolites such as nicotinamide adenine dinucleotide (NADH) and flavine adenine dinucleotide (FAD). Leveraging these autofluorescence images of whole organs of rodents, we have developed a 3D vascular segmentation technique to delineate the anatomy of the vasculature as well as mitochondrial metabolic distribution.Approach: By measuring fluorescence from naturally occurring mitochondrial metabolites combined with light-absorbing properties of hemoglobin, we detected the 3D structure of the vascular tree of rodent lungs, kidneys, hearts, and livers using VMI. For lung VMI, an exogenous fluorescent dye was injected into the trachea for inflation and to separate the airways, confirming no overlap between the segmented vessels and airways.Results: The kidney vasculature from genetically engineered rats expressing endothelial-specific red fluorescent protein TdTomato confirmed a significant overlap with VMI. This approach abided by the “minimum work” hypothesis of the vascular network fitting to Murray’s law. Finally, the vascular segmentation approach confirmed the vascular regression in rats, induced by ionizing radiation.Conclusions: Simultaneous vascular and metabolic information extracted from the VMI provides quantitative diagnostic markers without the confounding effects of vascular stains, fillers, or contrast agents.

Multidimensional Imaging of Mammary Gland Development: A Window Into Breast Form and Function.

An in-depth appreciation of organ form and function relies on the ability to image intact tissues across multiple scales. Difficulties associated with imaging deep within organs, however, can preclude high-resolution multidimensional imaging of live and fixed tissues. This is particularly challenging in the mammary gland, where the epithelium lies deeply encased within a stromal matrix. Recent advances in deep-tissue and live imaging methodologies are increasingly facilitating the visualization of complex cellular structures within their native environment. Alongside, refinements in optical tissue clearing and immunostaining methods are enabling 3D fluorescence imaging of whole organs at unprecedented resolutions. Collectively, these methods are illuminating the dynamic biological processes underlying tissue morphogenesis, homeostasis, and disease. This review provides a snapshot of the current and state-of-the-art multidimensional imaging techniques applied to the postnatal mammary gland, illustrating how these approaches have revealed important new insights into mammary gland ductal development and lactation. Continual evolution of multidimensional image acquisition and analysis methods will undoubtedly offer further insights into mammary gland biology that promises to shed new light on the perturbations leading to breast cancer.

A new pipeline for pathophysiological analysis of the mammary gland based on organoid transplantation and organ clearing.

ABSTRACTRecent developments in techniques for tissue clearing and size reduction have enabled optical imaging of whole organs and the study of rare tumorigenic events in vivo. The adult mammary gland provides a unique model for investigating physiological or pathological processes such as morphogenesis or epithelial cell dissemination. Here, we establish a new pipeline to study rare cellular events occurring in the mammary gland, by combining orthotopic transplantation of mammary organoids with the uDISCO organ size reduction and clearing method. This strategy allows us to analyze the behavior of individually labeled cells in regenerated mammary gland. As a proof of concept, we analyzed the localization of rare epithelial cells overexpressing atypical protein kinase C iota (also known as PRKCI, referred to here as aPKCι) with an N-terminal eGFP fusion (GFP-aPKCι+) in the normal mammary gland. Using this analytical pipeline, we were able to visualize epithelial aPKCι+ cells escaping from the normal mammary epithelium and disseminating into the surrounding stroma. This technical resource should benefit mammary development and tumor progression studies.

FDISCO: Advanced solvent-based clearing method for imaging whole organs.

Various optical clearing methods have emerged as powerful tools for deep biological imaging. Organic solvent-based clearing methods, such as three-dimensional imaging of solvent-cleared organs (3DISCO), present the advantages of high clearing efficiency and size reduction for panoptic imaging of large samples such as whole organs and even whole bodies. However, 3DISCO results in a rapid quenching of endogenous fluorescence, which has impeded its application. Here, we propose an advanced method named FDISCO to overcome this limitation. FDISCO can effectively preserve the fluorescence of various fluorescent probes and can achieve a long storage time of months while retaining potent clearing capability. We used FDISCO for high-resolution imaging and reconstruction of neuronal and vascular networks. Moreover, FDISCO is compatible with labeling by multiple viruses and enables fine visualization of neurons with weak fluorescence labeling in the whole brain. FDISCO represents an effective alternative to the three-dimensional mapping of whole organs and can be extensively used in biomedical studies.

Three-dimensional virtual histology enabled through cytoplasm-specific X-ray stain for microscopic and nanoscopic computed tomography

Many histological methods require staining of the cytoplasm, which provides instrumental details for diagnosis. One major limitation is the production of 2D images obtained by destructive preparation of 3D tissue samples. X-ray absorption micro- and nanocomputed tomography (microCT and nanoCT) allows for a nondestructive investigation of a 3D tissue sample, and thus aids to determine regions of interest for further histological examinations. However, application of microCT and nanoCT to biological samples (e.g., biopsies) is limited by the missing contrast within soft tissue, which is important to visualize morphological details. We describe an eosin-based preparation overcoming the challenges of contrast enhancement and selectivity for certain tissues. The eosin-based staining protocol is suitable for whole-organ staining, which then enables high-resolution microCT imaging of whole organs and nanoCT imaging of smaller tissue pieces retrieved from the original sample. Our results demonstrate suitability of the eosin-based staining method for diagnostic screening of 3D tissue samples without impeding further diagnostics through histological methods.

Label-free automated three-dimensional imaging of whole organs by microtomy-assisted photoacoustic microscopy

Three-dimensional (3D) optical imaging of whole biological organs with microscopic resolution has remained a challenge. Most versions of such imaging techniques require special preparation of the tissue specimen. Here we demonstrate microtomy-assisted photoacoustic microscopy (mPAM) of mouse brains and other organs, which automatically acquires serial distortion-free and registration-free images with endogenous absorption contrasts. Without tissue staining or clearing, mPAM generates micrometer-resolution 3D images of paraffin- or agarose-embedded whole organs with high fidelity, achieved by label-free simultaneous sensing of DNA/RNA, hemoglobins, and lipids. mPAM provides histology-like imaging of cell nuclei, blood vessels, axons, and other anatomical structures, enabling the application of histopathological interpretation at the organelle level to analyze a whole organ. Its deep tissue imaging capability leads to less sectioning, resulting in negligible sectioning artifact. mPAM offers a new way to better understand complex biological organs.

High-resolution 3D imaging of whole organ after clearing: taking a new look at the zebrafish testis

Zebrafish testis has become a powerful model for reproductive biology of teleostean fishes and other vertebrates and encompasses multiple applications in applied and basic research. Many studies have focused on 2D images, which is time consuming and implies extrapolation of results. Three-dimensional imaging of whole organs recently became an important challenge to better understand their architecture and allow cell enumeration. Several protocols have thus been developed to enhance sample transparency, a limiting step for imaging large biological samples. However, none of these methods has been applied to the zebrafish testis. We tested five clearing protocols to determine if some of them could be applied with only small modifications to the testis. We compared clearing efficiency at both macroscopic and microscopic levels. CUBIC and PACT were suitable for an efficient transparency, an optimal optical penetration, the GFP fluorescence preservation and avoiding meaningful tissue deformation. Finally, we succeeded in whole testis 3D capture at a cellular resolution with both CUBIC and PACT, which will be valuable in a standard workflow to investigate the 3D architecture of the testis and its cellular content. This paves the way for further development of high content phenotyping studies in several fields including development, genetic or toxicology.

Electrophoretic Tissue Clearing and Labeling Methods  for Volume Imaging of Whole Organs

The volume imaging of intact organs/tissues with singlecell resolution provides fundamental understanding of 3-dimensional (3D) organization of biological specimens. It will also improve our knowledge about generation and progression of diseases with chronic debilitating process such as neurodegenerative diseases or cancers, which is certainly helpful to the development of next generation diagnostic or treatment methods (Renier et al., 2014; Tainaka et al., 2014). The conventional approaches for volume imaging require physical section of biological tissues and subsequent multiple processing of images, because the technically accessible depth for acquiring tissue information (e.g., structure, protein distribution, etc.) is limited within hundreds of micrometers primarily due to the diffusion limit of the macromolecule probes (e.g., antibody) and the light scattering (Helmchen & Denk, 2005; Tang et al., 2012; Tomer et al., 2014). Therefore, the conventional approaches for volume imaging are laborintensive, time-consuming, requiring specialized apparatus, and essentially error-prone (Gong et al., 2013; Kim et al., 2014; Li et al., 2010). The recent advent of tissue clearing and labeling methods have been substantially resolved the above-mentioned obstacles through selective removal of lipid component in target tissues or organs without loss of the original protein

PEA-CLARITY: Three Dimensional (3D) Molecular Imaging of Whole Plant Organs

PEA-CLARITY: Three Dimensional (3D) Molecular Imaging of Whole Plant Organs

Vascular-mesenchymal cross-talk through Vegf and Pdgf drives organ patterning

The initiation of de novo testis cord organization in the fetal gonad is poorly understood. Endothelial cell migration into XY gonads initiates testis morphogenesis. However, neither the signals that regulate vascularization of the gonad nor the mechanisms through which vessels affect tissue morphogenesis are known. Here, we show that Vegf signaling is required for gonad vascularization and cord morphogenesis. We establish that interstitial cells express Vegfa and respond, by proliferation, to endothelial migration. In the absence of vasculature, four-dimensional imaging of whole organs revealed that interstitial proliferation is reduced and prevents formation of wedge-like structures that partition the gonad into cord-forming domains. Antagonizing vessel maturation also reduced proliferation. However, proliferation of mesenchymal cells was rescued by the addition of PDGF-BB. These results suggest a pathway that integrates initiation of vascular development and testis cord morphogenesis, and lead to a model in which undifferentiated mesenchyme recruits blood vessels, proliferates in response, and performs a primary function in the morphogenesis and patterning of the developing organ.

Imaging of molecular probe activity with Born-normalized fluorescence optical projection tomography

Optical projection tomography is a new ex vivo imaging technique that allows imaging of whole organs in three dimensions at high spatial resolutions. In this Letter we demonstrate its capability to tomographically visualize molecular activity in whole organs of mice. In particular, eosinophil activity in asthmatic lungs is resolved using a Born-normalized fluorescence optical projection tomography and employing a near-IR molecular probe. The possibility to achieve molecularly sensitive imaging contrast in optical projection tomography by means of targeted and activatable imaging reporter agents adds a new range of capabilities for investigating molecular signatures of pathophysiological processes and a wide variety of diseases and their development.