A fast, aqueous, reversible three-day tissue clearing method for adult and embryonic mouse brain and whole body.
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ABSTRACT:
SUBMITTER: Kosmidis S
PROVIDER: S-EPMC8713566 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.
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Project description:Investigation of the internal tissues and organs of a macroscopic organism usually requires destructive processes, such as dissection or sectioning. These processes are inevitably associated with the loss of some spatial information. Recently, aqueous-based tissue clearing techniques, which allow whole-organ or even whole-body clearing of small rodents, have been developed and opened a new method of three-dimensional histology. It is expected that these techniques will be useful tools in the field of zoology, in which organisms with highly diverse morphology are investigated and compared. However, most of these new methods are optimized for soft, non-pigmented organs in small rodents, especially the brain, and their applicability to non-model organisms with hard exoskeletons and stronger pigmentation has not been tested.We explored the possible application of an aqueous-based tissue clearing technique, advanced CUBIC, on small crustaceans. The original CUBIC procedure did not clear the terrestrial isopod, Armadillidium vulgare. Therefore, to apply the whole-mount clearing method to isopods with strong pigmentation and calcified exoskeletons, we introduced several pretreatment steps, including decalcification and bleaching. Thereafter, the clearing capacity of the procedure was dramatically improved, and A. vulgare became transparent. The internal organs, such as the digestive tract and male reproductive organs, were visible through sclerites using an ordinary stereomicroscope. We also found that fluorescent nuclear staining using propidium iodide (PI) helped to visualize the internal organs of cleared specimens. Our procedure was also effective on the marine crab, Philyra sp.In this study, we developed a method to clear whole tissues of crustaceans. To the best of our knowledge, this is the first report of whole-mount clearing applied to crustaceans using an aqueous-based technique. This technique could facilitate morphological studies of crustaceans and other organisms with calcified exoskeletons and pigmentation.
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Project description:BackgroundWe need to improve our understanding of the complex interactions between family carers' emotional relationships with care-recipients and carers use of support services. This study assessed carer's expectations and perceptions of adult day respite services and their commitment to using services.MethodsA mixed-method case study approach was used with psychological contract providing a conceptual framework. Data collection was situated within an organisational case study, and the total population of carers from the organisation's day respite service were approached. Fifty respondents provided quantitative and qualitative data through an interview survey. The conceptual framework was expanded to include Maslow's hierarchy of needs during analysis.ResultsCarers prioritised benefits for and experiences of care-recipients when making day respite decisions. Respondents had high levels of trust in the service and perceived that the major benefits for care-recipients were around social interaction and meaningful activity with resultant improved well-being. Carers wanted day respite experiences to include all levels of Maslow's hierarchy of needs from the provision of physiological care and safety through to the higher levels of belongingness, love and esteem.ConclusionThe study suggests carers need to trust that care-recipients will have quality experiences at day respite. This study was intended as a preliminary stage for further research and while not generalizable it does highlight key considerations in carers' use of day respite services.
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Project description:Fast Field-Cycling (FFC) is a well-established Nuclear Magnetic Resonance (NMR) technique that exploits varying magnetic fields to quantify molecular motion over a wide range of time scales, providing rich structural information from nanometres to micrometres, non-invasively. Previous work demonstrated great potential for FFC-NMR biomarkers in medical applications; our research group has now ported this technology to medical imaging by designing a whole-body FFC Magnetic Resonance Imaging (FFC-MRI) scanner capable of performing accurate measurements non-invasively over the entire body, using signals from water and fat protons. This is a unique tool to explore new biomarkers related to disease-induced tissue remodelling. Our approach required making radical changes in the design, construction and control of MRI hardware so that the magnetic field is switched within 12.5 ms to reach any field strength from 50 μT to 0.2 T, providing clinically useful images within minutes. Pilot studies demonstrated endogenous field-dependant contrast in biological tissues in good agreement with reference data from other imaging modalities, confirming that our system can perform multiscale structural imaging of biological tissues, from nanometres to micrometres. It is now possible to confirm ex vivo results obtained from previous clinical studies, offering applications in diagnosis, staging and monitoring treatment for cancer, stroke, osteoarthritis and oedema.
| S-EPMC6639535 | biostudies-literature
Project description:PurposeTendency is to moderate the injected activity and/or reduce acquisition time in PET examinations to minimize potential radiation hazards and increase patient comfort. This work aims to assess the performance of regular full-dose (FD) synthesis from fast/low-dose (LD) whole-body (WB) PET images using deep learning techniques.MethodsInstead of using synthetic LD scans, two separate clinical WB 18F-Fluorodeoxyglucose (18F-FDG) PET/CT studies of 100 patients were acquired: one regular FD (~ 27 min) and one fast or LD (~ 3 min) consisting of 1/8th of the standard acquisition time. A modified cycle-consistent generative adversarial network (CycleGAN) and residual neural network (ResNET) models, denoted as CGAN and RNET, respectively, were implemented to predict FD PET images. The quality of the predicted PET images was assessed by two nuclear medicine physicians. Moreover, the diagnostic quality of the predicted PET images was evaluated using a pass/fail scheme for lesion detectability task. Quantitative analysis using established metrics including standardized uptake value (SUV) bias was performed for the liver, left/right lung, brain, and 400 malignant lesions from the test and evaluation datasets.ResultsCGAN scored 4.92 and 3.88 (out of 5) (adequate to good) for brain and neck + trunk, respectively. The average SUV bias calculated over normal tissues was 3.39 ± 0.71% and - 3.83 ± 1.25% for CGAN and RNET, respectively. Bland-Altman analysis reported the lowest SUV bias (0.01%) and 95% confidence interval of - 0.36, + 0.47 for CGAN compared with the reference FD images for malignant lesions.ConclusionCycleGAN is able to synthesize clinical FD WB PET images from LD images with 1/8th of standard injected activity or acquisition time. The predicted FD images present almost similar performance in terms of lesion detectability, qualitative scores, and quantification bias and variance.
| S-EPMC8241799 | biostudies-literature
Project description:BackgroundDuchenne muscular dystrophy (DMD) is a fatal, X-linked genetic disorder. Although DMD is the most common form of muscular dystrophy, only two FDA-approved drugs were developed to delay its progression. In order to assess therapies for treating DMD, several murine models have recently been introduced. As the wide variety of murine models enlighten mechanisms underlying DMD pathology, the question on how to monitor the progression of the disease within the entire musculoskeletal system still remains to be answered. One considerable approach to monitor such progression is histological evaluation of calcium deposits within muscle biopsies. Although accurate, histology is limited to small tissue area and cannot be utilized to evaluate systemic progression of DMD. Therefore, we aimed to develop a methodology suitable for rapid and high-resolution screening of calcium deposits within the entire murine organism.MethodsProcedures were performed on adult male C57BL/10-mdx and adult male C57BL mice. Animals were sacrificed, perfused, paraformaldehyde-fixed, and subjected to whole-body clearing using optimized perfusion-based CUBIC protocol. Next, cleared organisms were stained with alizarin red S to visualize calcium deposits and subjected to imaging.ResultsStudy revealed presence of calcium deposits within degenerated muscles of the entire C57BL/10-mdx mouse organism. Calcified deposits were observed within skeletal muscles of the forelimb, diaphragm, lumbar region, pelvic region, and hindlimb. Calcified deposits found in quadriceps femoris, triceps brachii, and spinalis pars lumborum were characterized. Analysis of cumulative frequency distribution showed different distribution characteristics of calcified deposits in quadriceps femoris muscle in comparison to triceps brachii and spinalis pars lumborum muscles (p?<?0.001) and quadriceps femoris vs spinalis pars lumborum (p?<?0.001). Differences between the number of calcified deposits in selected muscles, their volume, and average volume were statistically significant.ConclusionsIn aggregate, we present new methodology to monitor calcium deposits in situ in the mouse model of Duchenne muscular dystrophy. Sample imaging with the presented setup is feasible and applicable for whole-organ/body imaging. Accompanied by the development of custom-made LSFM apparatus, it allows targeted and precise characterization of calcium deposits in cleared muscles. Hence, presented approach might be broadly utilized to monitor degree to which muscles of the entire organism are affected by the necrosis and how is it altered by the treatment or physical activity of the animal. We believe that this would be a valuable tool for studying organs alternations in a wide group of animal models of muscle dystrophy and bone-oriented diseases.
| S-EPMC6053777 | biostudies-literature
Project description:Tissue optical clearing techniques have provided important tools for large-volume imaging. Aqueous-based clearing methods are known for good fluorescence preservation and scalable size maintenance, but are limited by long incubation time, insufficient clearing performance, or requirements for specialized devices. Additionally, few clearing methods are compatible with widely used lipophilic dyes while maintaining high clearing performance. Here, to address these issues, m-xylylenediamine (MXDA) is firstly introduced into tissue clearing and used to develop a rapid, highly efficient aqueous clearing method with robust lipophilic dyes compatibility, termed MXDA-based Aqueous Clearing System (MACS). MACS can render whole adult brains highly transparent within 2.5 days and is also applicable for other intact organs. Meanwhile, MACS possesses ideal compatibility with multiple probes, especially for lipophilic dyes. MACS achieves 3D imaging of the intact neural structures labeled by various techniques. Combining MACS with DiI labeling, MACS allows reconstruction of the detailed vascular structures of various organs and generates 3D pathology of glomeruli tufts in healthy and diabetic kidneys. Therefore, MACS provides a useful method for 3D mapping of intact tissues and is expected to facilitate morphological, physiological, and pathological studies of various organs.
| S-EPMC7175264 | biostudies-literature