Optimized perfusion‐based CUBIC protocol for the efficient whole‐body clearing and imaging of rat organs

Whole‐organ and whole‐body optical tissue clearing methods allowing imaging in 3 dimensions are an area of profound research interest. Originally developed to study nervous tissue, they have been successfully applied to all murine organs, yet clearing and imaging of rat peripheral organs is less advanced. Here, a modification of CUBIC clearing protocol is presented. It provides a rapid and simple approach to clear the entire adult rat organism and thus all organs within as little as 4 days. Upgraded perfusion‐based rat CUBIC protocol preserves both anatomical structure of organs and signal from proteinaceous fluorophores, and furthermore is compatible with antibody staining. Finally, it enables also volumetric cells analyses and is tailored for staining of calcium deposits within unsectioned soft tissues.      

Procedures for the Quantification of Whole-Tissue Immunofluorescence Images Obtained at Single-Cell Resolution during Murine Tubular Organ Development

Whole-tissue quantification at single-cell resolution has become an inevitable approach for further quantitative understanding of morphogenesis in organ development. The feasibility of the approach has been dramatically increased by recent technological improvements in optical tissue clearing and microscopy. However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed. To provide the appropriate procedure, we here show tips for each critical step of the entire process, including fixation for immunofluorescence, optical clearing, and digital image processing, using developing murine internal organs such as epididymis, kidney, and lung as an example. Through comparison of fixative solutions and of clearing methods, we found optimal conditions to achieve clearer deep-tissue imaging of specific immunolabeled targets and explain what methods result in vivid volume imaging. In addition, we demonstrated that three-dimensional digital image processing after optical clearing produces objective quantitative data for the whole-tissue analysis, focusing on the spatial distribution of mitotic cells in the epididymal tubule. The procedure for the whole-tissue quantification shown in this article should contribute to systematic measurements of cellular processes in developing organs, accelerating the further understanding of morphogenesis at the single cell level.     

High‐throughput light sheet tomography platform for automated fast imaging of whole mouse brain

Acquiring information of the neural structures in the whole‐brain level is vital for systematically exploring mechanisms and principles of brain function and dysfunction. Most methods for whole brain imaging, while capable of capturing the complete morphology of neurons, usually involve complex sample preparation and several days of image acquisition. The whole process including optical clearing or resin embedding is time consuming for a quick survey of the distribution of specific neural circuits in the whole brain. Here, we develop a high‐throughput light‐sheet tomography platform (HLTP), which requires minimum sample preparation. This method does not require optical clearing for block face light sheet imaging. After fixation using paraformaldehyde, an aligned 3 dimensional image dataset of a whole mouse brain can be obtained within 5 hours at a voxel size of 1.30 × 1.30 × 0.92 μm. HLTP could be a very efficient tool for quick exploration and visualization of brain‐wide distribution of specific neurons or neural circuits.      

Optical clearing for multiscale biological tissues

Three‐dimensional reconstruction of tissue structures is essential for biomedical research. The development of light microscopes and various fluorescent labeling techniques provides powerful tools for this motivation. However, optical imaging depth suffers from strong light scattering due to inherent heterogeneity of biological tissues. Tissue optical clearing technology provides a distinct solution and permits us to image large volumes with high resolution. Until now, various clearing methods have been developed. In this study, from the perspective of the end users, we review in vitro tissue optical clearing techniques based on the sample features in terms of size and age, enumerate the methods suitable for immunostaining and lipophilic dyes and summarize the combinations with various imaging techniques. We hope this review will be helpful for researchers to choose the most suitable clearing method from a variety of protocols to meet their specific needs.      

光学透明技术在植物多尺度成像中的应用

光学透明技术是一种通过各种化学试剂, 将原本不透明的生物样本实现透明化, 并在光学显微镜下深度成像的技术。结合多种光学显微成像新技术, 光学透明技术可对整个组织进行成像和三维重建, 深度剖析生物体内部空间特征与形成机制。近年来, 多种植物光学透明技术和多尺度成像技术被陆续研发, 并取得了丰硕的研究成果。该文综述了生物体光学透明技术的基本原理和一些新技术, 重点介绍基于光学透明技术开发的新型成像方法及其在植物成像与细胞生物学中的应用, 为后续植物整体、组织或器官的透明、成像与三维重构及功能研究提供理论依据和技术支持。     

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.^1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light³ do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.⁴ Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.     

Evaluation of normal findings using a detailed and focused technique for transcutaneous abdominal ultrasonography in the horse

Background

Ultrasonography is an important diagnostic tool in the investigation of abdominal disease in the horse. Several factors may affect the ability to image different structures within the abdomen. The aim of the study was to describe the repeatability of identification of abdominal structures in normal horses using a detailed ultrasonographic examination technique and using a focused, limited preparation technique.

Methods

A detailed abdominal ultrasound examination was performed in five normal horses, repeated on five occasions (total of 25 examinations). The abdomen was divided into ten different imaging sites, and structures identified in each site were recorded. Five imaging sites were then selected for a single focused ultrasound examination in 20 normal horses. Limited patient preparation was performed. Structures were recorded as ‘identified’ if ultrasonographic features could be distinguished. The location of organs and their frequency of identification were recorded. Data from both phases were analysed to determine repeatability of identification of structures in each examination (irrespective of imaging site), and for each imaging site.

Results

Caecum, colon, spleen, liver and right kidney were repeatably identified using the detailed technique, and had defined locations. Large colon and right kidney were identified in 100% of examinations with both techniques. Liver, spleen, caecum, duodenum and other small intestine were identified more frequently with the detailed examination. Small intestine was most frequently identified in the ventral abdomen, its identification varied markedly within and between horses, and required repeated examinations in some horses. Left kidney could not be identified in every horse using either technique. Sacculated colon was identified in all ventral sites, and was infrequently identified in dorsal sites.

Conclusions

Caecum, sacculated large intestine, spleen, liver and right kidney were consistently identified with both techniques. There were some normal variations which should be considered when interpreting ultrasonographic findings in clinical cases: left kidney was not always identified, sacculated colon was occasionally identified in dorsal flank sites. Multiple imaging sites and repeated examinations may be required to identify small intestine. A focused examination identified most key structures, but has some limitations compared to a detailed examination.

     

In vivo monitoring blood‐brain barrier permeability using spectral imaging through optical clearing skull window

The blood‐brain barrier (BBB) plays a key role in the health of the central nervous system. Opening the BBB is very important for drug delivery to brain tissues to enhance the therapeutic effect on brain diseases. It is necessary to in vivo monitor the BBB permeability for assessing drug release with high resolution; however, an effective method is lacking. In this work, we developed a new method that combined spectral imaging with an optical clearing skull window to in vivo dynamically monitor BBB opening caused by 5‐aminolevulinic acid (5‐ALA)‐mediated photodynamic therapy (PDT), in which the Evans blue dye (EBd) acted as an indicator of the BBB permeability. Using this method, we effectively monitored the cerebrovascular EBd leakage process. Moreover, the analysis of changes in the vascular and extravascular EBd concentrations demonstrated that the PDT‐induced BBB opening exhibited spatiotemporal differences in the cortex. This spectral imaging method based on the optical clearing skull window provides a low‐cost and simply operated tool for in vivo monitoring BBB opening process. This has a high potential for the visualization of drug delivery to the central nervous system. Thus, it is of tremendous significance in brain disease therapy. Monitoring the changes in PDT‐induced BBB permeability by evaluating the EBd concentration using an optical clearing skull window. (A) Entire brains and coronal sections following treatment of PDT with/without an optical clearing skull window after injection of EBd. (B) Typical EBd distribution maps before and after laser irradiation captured by the spectral imaging method. (Colorbar represents the EBd concentration).      

A biopsy‐needle compatible varifocal multiphoton rigid probe for depth‐resolved optical biopsy

In this work, we report a biopsy‐needle compatible rigid probe, capable of performing three‐dimensional (3D) two‐photon optical biopsy. The probe has a small outer diameter of 1.75 mm and fits inside a gauge‐14 biopsy needle to reach internal organs. A carefully designed focus scanning mechanism has been implemented in the rigid probe, which, along with a rapid two‐dimensional MEMS scanner, enables 3D imaging. Fast image acquisition up to 10 frames per second is possible, dramatically reducing motion artifacts during in vivo imaging. Equipped with a high‐numerical aperture micro‐objective, the miniature rigid probe offers a high two‐photon resolution (0.833 × 6.11 μm, lateral × axial), a lateral field of view of 120 μm, and an axial focus tuning range of 200 μm. In addition to imaging of mouse internal organs and subcutaneous tumor in vivo, first‐of‐its‐kind depth‐resolved two‐photon optical biopsy of an internal organ has been successfully demonstrated on mouse kidney in vivo and in situ.      

A simple approach for non‐invasive transcranial optical vascular imaging (nTOVI)

In vivo imaging of cerebral vasculature is highly vital for clinicians and medical researchers alike. For a number of years non‐invasive optical‐based imaging of brain vascular network by using standard fluorescence probes has been considered as impossible. In the current paper controverting this paradigm, we present a robust non‐invasive optical‐based imaging approach that allows visualize major cerebral vessels at the high temporal and spatial resolution. The developed technique is simple to use, utilizes standard fluorescent dyes, inexpensive micro‐imaging and computation procedures. The ability to clearly visualize middle cerebral artery and other major vessels of brain vascular network, as well as the measurements of dynamics of blood flow are presented. The developed imaging approach has a great potential in neuroimaging and can significantly expand the capabilities of preclinical functional studies of brain and notably contribute for analysis of cerebral blood circulation in disorder models.

An example of 1 × 1.5 cm color‐coded image of brain blood vessels of mouse obtained in vivo by transcranial optical vascular imaging (TOVI) approach through the intact cranium.     

Imaging of subchondral bone by optical coherence tomography upon optical clearing of articular cartilage

Optical clearing is an effective method to reduce light scattering of biological tissues that provides significant enhancement of light penetration into the biological tissues making non‐invasive diagnosis more feasible. In current report Optical Coherence Tomography (OCT) in conjunction with optical clearing is applied for assessment of deep cartilage layers and cartilage‐bone interface. The solution of Iohexol in water has been used as an optical clearing agent. The cartilage‐bone boundary becomes visible after 15 min of optical clearing that enabling non‐invasive estimation of its roughness: S_a = 10 ± 1 µm. The results show that for 0.9 mm thick cartilage optical clearing is stopped after 50 min with an increase of refractive index from 1.386 ± 0.008 to 1.510 ± 0.009. Current approach enables more reliable detection of arthroscopically inaccessible regions, including cartilage‐bone boundary and subchondral bone, and potentially improves accuracy of the osteoarthritis diagnosis.

     

High‐resolution imaging of fluorescent whole mouse brains using stabilised organic media (sDISCO)

Optical tissue clearing using dibenzyl ether (DBE) or BABB (1 part benzyl alcohol and 2 parts benzyl benzoate) is easy in application and allows deep‐tissue imaging of a wide range of specimens. However, in both substances, optical clearing and storage times of enhanced green fluorescent protein (EGFP)‐expressing specimens are limited due to the continuous formation of peroxides and aldehydes, which severely quench fluorescence. Stabilisation of purified DBE or BABB by addition of the antioxidant propyl gallate efficiently preserves fluorescence signals in EGFP‐expressing samples for more than a year. This enables longer clearing times and improved tissue transparency with higher fluorescence signal intensity. The here introduced clearing protocol termed stabilised DISCO allows to image spines in a whole mouse brain and to detect faint changes in the activity‐dependent expression pattern of tdTomato.      

Variations in the kinetic response of several different phosphate-dependent glutaminase isozymes during acute metabolic acidosis

Summary We describe the kinetic modifications to mitochondrial-membrane-bound phosphate-dependent glutaminase in various types of rat tissue brought about by acute metabolic acidosis. The activity response of phosphate-dependent glutaminase to glutamine was sigmoidal, showing positive co-operativity, the Hill coefficients always being higher than 2. The enzyme from acidotic rats showed increased activity at subsaturating concentrations of glutamine in kidney tubules, as might be expected, but not in brain, intestine or liver tissues. Nevertheless, when brain and intestine from control rats were incubated in plasma from acutely acidotic rats enzyme activity increased at 1 mM glutamine in the same way as in kidney cortex. The enzyme from liver tissue remained unaltered. S_0.5 and n_H values decreased significantly in kidney tubules, enterocytes and brain slices preincubated in plasma from acidotic rats. The sigmoidal curves of phosphate-dependent glutaminase shifted to the left without any significant changes in V_max. The similar response of phosphate-dependent glutaminase to acute acidosis in the kidney, brain and intestine confirms the fact that enzymes from these tissues are kinetically identical and reaffirms the presence of an ammoniagenic factor in plasma, either produced or concentrated in the kidneys of rats with acute acidosis.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - EDTA NN-1,2-Ethane-diylbis [N-(carboxymethyl)glycyne] - Tris 2-amino-2-hydroxymethyl-1,3-propanediol - PDG phosphate dependent glutaminase Publication No. 145 from Drogas, Tóxicos Ambientales y Metabolismo Celular Research Group. Department of Biochemistry and Molecular Biology, University of Granada, Spain     

Label‐free optical projection tomography for quantitative three‐dimensional anatomy of mouse embryo

Recent progress in three‐dimensional optical imaging techniques allows visualization of many comprehensive biological specimens. Optical clearing methods provide volumetric and quantitative information by overcoming the limited depth of light due to scattering. However, current imaging technologies mostly rely on the synthetic or genetic fluorescent labels, thus limits its application to whole‐body visualization of generic mouse models. Here, we report a label‐free optical projection tomography (LF‐OPT) technique for quantitative whole mouse embryo imaging. LF‐OPT is based on the attenuation contrast of light rather than fluorescence, and it utilizes projection imaging technique similar to computed tomography for visualizing the volumetric structure. We demonstrate this with a collection of mouse embryo morphologies in different stages using LF‐OPT. Additionally, we extract quantitative organ information applicable toward high‐throughput phenotype screening. Our results indicate that LF‐OPT can provide multi‐scale morphological information in various tissues including bone, which can be difficult in conventional optical imaging technique.     

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps^1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks⁴. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to ''program'' neuronal subsets at will^5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates^7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters⁹. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods¹⁰, or 2-photon laser scanning microscopy¹¹. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).For preparing brain slices we implemented a relatively new piece of instrumentation called the ''Compresstome'' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the ''Compresstome'' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging. Download video file.^{(28M, mov)}     

Molecular modeling of immersion optical clearing of biological tissues

The interaction of six low-molecular tissue-clearing agents (1,2 and 1,3-propanediol, ethylene glycol, glycerol, xylitol, sorbitol) with the collagen mimetic peptide (GPH)₃ was studied by applying the methods of classical molecular dynamics (GROMACS), molecular docking (AutoDock Vina) and quantum chemistry (PM6 and B3LYP). The spatial configurations of intermolecular complexes were determined and interaction energies calculated. The dependence of the volume occupied by the collagen peptide on the clearing agent concentration in an aqueous solution was calculated. This dependence is not linear, and has a maximum for almost all the agents in the study. The correlations between the optical clearing potential and intermolecular interactions parameters, such as the time of an agent being in a hydrogen-bonded state, and the relative probability of formation of double hydrogen bonds and interaction energies, were determined. Using the correlations determined, we predicted the numeric value of the optical clearing potential of dextrose molecules in rat skin, which correlates with experimental data. A molecular mechanism of tissue optical clearing within the post-diffusion stage is suggested.

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Graphical abstract The molecular modeling of the interaction between clearing agents and collagen

     

Inside Cover: Optimized perfusion‐based CUBIC protocol for the efficient whole‐body clearing and imaging of rat organs (J. Biophotonics 5/2018)

Optical tissue clearing is a method allowing post‐mortem deep imaging of organs in three dimensions. By optimizing the CUBIC clearing protocol, the authors provide rapid and simple approach to clear the entire adult rat organism within as little as four days, which is accompanied by the variety of its staining and imaging techniques. The image was captured with polarizers and demonstrates transparent rodent heart with thread‐like crystals of clearing reagent. Further details can be found in the article by Pawe? Matryba et al. ( e201700248 ).

     

3D Printer Generated Tissue iMolds for Cleared Tissue Using Single- and Multi-Photon Microscopy for Deep Tissue Evaluation

Background

Pathological analyses and methodology has recently undergone a dramatic revolution. With the creation of tissue clearing methods such as CLARITY and CUBIC, groups can now achieve complete transparency in tissue samples in nano-porous hydrogels. Cleared tissue is then imagined in a semi-aqueous medium that matches the refractive index of the objective being used. However, one major challenge is the ability to control tissue movement during imaging and to relocate precise locations post sequential clearing and re-staining.

Methods

Using 3D printers, we designed tissue molds that fit precisely around the specimen being imaged. First, images are taken of the specimen, followed by importing and design of a structural mold, then printed with affordable plastics by a 3D printer.

Results

With our novel design, we have innovated tissue molds called innovative molds (iMolds) that can be generated in any laboratory and are customized for any organ, tissue, or bone matter being imaged. Furthermore, the inexpensive and reusable tissue molds are made compatible for any microscope such as single and multi-photon confocal with varying stage dimensions. Excitingly, iMolds can also be generated to hold multiple organs in one mold, making reconstruction and imaging much easier.

Conclusions

Taken together, with iMolds it is now possible to image cleared tissue in clearing medium while limiting movement and being able to relocate precise anatomical and cellular locations on sequential imaging events in any basic laboratory. This system provides great potential for screening widespread effects of therapeutics and disease across entire organ systems.

     

Indigogenic methods for glycosidases

Summary 4-Cl-5-Br-3-indolyl--D-fucoside (IbF) was tested in histochemical and bio-chemical experiments. Sections from differently treated parts of the small intestine of suckling and adult rats, of jejunum of adult hamsters, guinea pigs, cooks, monkeys, of human jejunal biopsies, of kidney of suckling and adult rats, adult monkeys, guinea pigs and hamsters, and of some other rat organs were used in histochemical experiments. Neutral and acid -D-galactosidases prepared from homogenates of the small intestine of suckling rats by chromatography on Sephadex G 200 were used in biochemical experiments.The recommended medium for histochemical studies consists of 0.1 M citrate phosphate buffer pH 6 (brush border enzyme) and pH 4 (lysosomal enzyme), 3.6·10^–4M IbF and 3.1·10^–3 M potassium ferri- and ferrocyanide.IbF is split quite efficiently by the brush border -D-galactosidase (=lactase) and the recommended histochemical method with IbF at pH 6 is the method of choice in studies concerned with the localization of intestinal lactase. In unfixed cold microtome sections the brush border activity can be easily detected within 15–240 minutes, depending on the lactase activity of the studied sample. In this study this activity was shown to be present in the brush border of enterocytes in the upper part of crypts reaching its maximum in differentiated enterocytes covering the sides and tops of villi in the small intestine of suckling (the highest activity) and adult rats (3–4x lower activity according to the time of appearance of the staining), and of human, monkey and hamster jejunum. In the cock jejunum traces of brush border staining were seen only after 24 hours of incubation. In enterocytes of patients with celiac sprue the brush border activity was very much reduced in dependence on the stage of the disease. Brush border staining along with a diffuse cytoplasmic reaction was found in the proximal convoluted tubules of the monkey kidney. The question of localization of enzyme activity splitting IbF in the monkey kidney deserves further investigation.IbF is also split by isolated intestinal acid -D-galactosidase and histochemically a positive reaction was found in lysosomes of many cells displaying a high activity of -D-galactosidase when IbF was used in the recommended medium of pH 4. The use of aldehyde fixation (glutaraldehyde is to be preferred) is a prerequisite for the assessment of this lysosomal localization. The lysosomal activity splitting IbF is not firmly bound structurally and escapes from cold microtome sections prepared from unfixed tissue samples into the incubation solution.The recommended method is also very suitable for processing zymograms and immunoprecipitation lines of lactase with antisera obtained by Ouchterlony's technic and by immunoelectrophoresis.