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"Microscopy, Medical"
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High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging
Understanding the structure–function relationships between diverse cell types in a complex organ environment requires detailed in situ reconstruction of cell-associated molecular properties in the context of 3D, macro-scale tissue architecture. We recently developed clearing-enhanced 3D (Ce3D), a simple and effective method for tissue clearing that achieves excellent transparency; preserves cell morphology, tissue architecture, and reporter molecule fluorescence; and is robustly compatible with direct immunolabeling. These characteristics permit high-quality multiplex fluorescence microscopy of large tissue volumes, as well as image analysis using advanced platforms such as volumetric histocytometry, collectively allowing quantitative characterization of cells with respect to their spatial positioning within tissues on the basis of phenotypic and functional markers. Ce3D clearing is fast, achieving robust transparency of most tissues within 24 h, albeit still necessitating additional time for staining, imaging, and analysis (1–2 weeks). Here, we provide detailed procedures for tissue clearing using Ce3D, including optimized workflows for tissue processing and staining, as well as treatment of difficult-to-clear organs such as the brain. We also describe a new procedure for RNA detection in Ce3D-treated tissues, as well as provide additional details for the volumetric histocytometry data processing steps. Finally, we discuss limitations and work-around strategies for improving antibody-based tissue immunolabeling, fluorophore multiplexing, large-volume microscopy, and computational analysis of large image datasets. Together, these detailed procedures and solutions for high-resolution volumetric microscopy with Ce3D enable quantitative visualization of cells and tissues at a high level of detail, allowing exploration of cellular spatial relationships in a variety of tissue settings.Li et al. detail a protocol for clearing-enhanced 3D (Ce3D), a method for tissue clearing, multiplexed immunofluorescence, RNA FISH microscopy of whole-mount tissues/thick tissue slices, and quantitative image analysis with volumetric histocytometry.
Journal Article
Simultaneous multiplane imaging with reverberation two-photon microscopy
Multiphoton microscopy has gained enormous popularity because of its unique capacity to provide high-resolution images from deep within scattering tissue. Here, we demonstrate video-rate multiplane imaging with two-photon microscopy by performing near-instantaneous axial scanning while maintaining three-dimensional micrometer-scale resolution. Our technique, termed reverberation microscopy, enables the monitoring of neuronal populations over large depth ranges and can be implemented as a simple add-on to a conventional design.
Reverberation two-photon microscopy enables video-rate multiplane neuroimaging by performing near-instantaneous axial scanning over large depth ranges while maintaining 3D micrometer-scale resolution.
Journal Article
Biomedical electron microscopy
This comprehensive reference illustrates optimal preparation methods in biological electron microscopy compared with common methodological problems.Not only will the basic methodologies of transmission electron microscopy like fixation, microtomy, and microscopy be presented, but the authors also endeavor to illustrate more specialized techniques.
eBook
Miniaturized head-mounted microscope for whole-cortex mesoscale imaging in freely behaving mice
The advent of genetically encoded calcium indicators, along with surgical preparations such as thinned skulls or refractive-index-matched skulls, has enabled mesoscale cortical activity imaging in head-fixed mice. However, neural activity during unrestrained behavior substantially differs from neural activity in head-fixed animals. For whole-cortex imaging in freely behaving mice, we present the ‘mini-mScope’, a widefield, miniaturized, head-mounted fluorescence microscope that is compatible with transparent polymer skull preparations. With a field of view of 8 × 10 mm2 and weighing less than 4 g, the mini-mScope can image most of the mouse dorsal cortex with resolutions ranging from 39 to 56 µm. We used the mini-mScope to record mesoscale calcium activity across the dorsal cortex during sensory-evoked stimuli, open field behaviors, social interactions and transitions from wakefulness to sleep.The mini-mScope is a miniature microscope that can image neural activity at the mesoscale in most of the dorsal cortex of freely behaving mice.
Journal Article
Advances in Portable Optical Microscopy Using Cloud Technologies and Artificial Intelligence for Medical Applications
The need for faster and more accessible alternatives to laboratory microscopy is driving many innovations throughout the image and data acquisition chain in the biomedical field. Benchtop microscopes are bulky, lack communications capabilities, and require trained personnel for analysis. New technologies, such as compact 3D-printed devices integrated with the Internet of Things (IoT) for data sharing and cloud computing, as well as automated image processing using deep learning algorithms, can address these limitations and enhance the conventional imaging workflow. This review reports on recent advancements in microscope miniaturization, with a focus on emerging technologies such as photoacoustic microscopy and more established approaches like smartphone-based microscopy. The potential applications of IoT in microscopy are examined in detail. Furthermore, this review discusses the evolution of image processing in microscopy, transitioning from traditional to deep learning methods that facilitate image enhancement and data interpretation. Despite numerous advancements in the field, there is a noticeable lack of studies that holistically address the entire microscopy acquisition chain. This review aims to highlight the potential of IoT and artificial intelligence (AI) in combination with portable microscopy, emphasizing the importance of a comprehensive approach to the microscopy acquisition chain, from portability to image analysis.
Journal Article
Direct wavefront sensing enables functional imaging of infragranular axons and spines
We advance two-photon microscopy for near-diffraction-limited imaging up to 850 µm below the pia in awake mice. Our approach combines direct wavefront sensing of light from a guidestar (formed by descanned fluorescence from Cy5.5-conjugated dextran in brain microvessels) with adaptive optics to compensate for tissue-induced aberrations in the wavefront. We achieve high signal-to-noise ratios in recordings of glutamate release from thalamocortical axons and calcium transients in spines of layer 5b basal dendrites during active tactile sensing.
Journal Article
Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded
To be able to resolve molecular-clusters it is crucial to access vital information (such as, molecule density, cluster-size, and others) that are key in understanding disease progression and the underlying mechanism. Traditional single-molecule localization microscopy (SMLM) techniques use molecules of variable sizes (as determined by its localization precision (LP)) to reconstruct a super-resolution map. This results in an image with overlapping and superimposing PSFs (due to a wide size-spectrum of single-molecules) that undermine image resolution. Ideally, it should be possible to identify the brightest molecules (also termed as the fortunate molecules) to reconstruct ultra-superresolution map, provided sufficient statistics is available from the recorded data. Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy explores this possibility by introducing a narrow probability size-distribution of single-molecules (narrow size-spectrum about a predefined mean-size). The reconstruction begins by presetting the mean and variance of the narrow distribution function (Gaussian function). Subsequently, the dataset is processed and single-molecules are filtered by the Gaussian function to remove unfortunate molecules. The fortunate molecules thus retained are then mapped to reconstruct an ultra-superresolution map. In-principle, the POSSIBLE microscopy technique is capable of infinite resolution (resolution of the order of actual single-molecule size) provided enough fortunate molecules are experimentally detected. In short, bright molecules (with large emissivity) holds the key. Here, we demonstrate the POSSIBLE microscopy technique and reconstruct single-molecule images with an average PSF sizes of [sigma] ± [DELTA][sigma] = 15 ± 10 nm, 30 ± 2 nm & 50 ± 2 nm. Results show better-resolved Dendra2-HA clusters with large cluster-density in transfected NIH3T3 fibroblast cells as compared to the traditional SMLM techniques. Cluster analysis indicates densely-packed HA molecules, HA-HA interaction, and a surge in the number of HA molecules per cluster post 24 Hrs of transfection. The study using POSSIBLE microscopy introduces new insights in influenza biology. We anticipate exciting applications in the multidisciplinary field of disease biology, oncology, and biomedical imaging.
Journal Article
Dual-view plane illumination microscopy for rapid and spatially isotropic imaging
Kumar
et al.
describe how to build a diSPIM from commercially available parts and use it to live-image cultured cells or worm embryogenesis. The inverted setup enables samples to be mounted directly on microscope slides, avoiding agarose embedding.
We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and
Caenorhabditis elegans
embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ∼6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3–8 h, depending on the size of the data.
Journal Article
Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain
Visualizing entire neuronal networks for analysis in the intact brain has been impossible up to now. Techniques like computer tomography or magnetic resonance imaging (MRI) do not yield cellular resolution, and mechanical slicing procedures are insufficient to achieve high-resolution reconstructions in three dimensions. Here we present an approach that allows imaging of whole fixed mouse brains. We modified 'ultramicroscopy' by combining it with a special procedure to clear tissue. We show that this new technique allows optical sectioning of fixed mouse brains with cellular resolution and can be used to detect single GFP-labeled neurons in excised mouse hippocampi. We obtained three-dimensional (3D) images of dendritic trees and spines of populations of CA1 neurons in isolated hippocampi. Also in fruit flies and in mouse embryos, we were able to visualize details of the anatomy by imaging autofluorescence. Our method is ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.
Journal Article