High-resolution imaging of catalytic activity of a single graphene sheet using electrochemiluminescence microscopy.
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ABSTRACT:
SUBMITTER: Zhu H
PROVIDER: S-EPMC8179586 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.
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Project description:Light-sheet microscopy has been developed as a powerful tool for live imaging in biological studies. The efficient illumination of specimens using light-sheet microscopy makes it highly amenable to high-speed imaging. We therefore applied this technology to the observation of amoeboid movements, which are too rapid to capture with conventional microscopy. To simplify the setup of the optical system, we utilized the illumination optics from a conventional confocal laser scanning microscope. Using this set-up we achieved high-speed imaging of amoeboid movements. Three-dimensional images were captured at the recording rate of 40 frames/s and clearly outlined the fine structures of fluorescent-labeled amoeboid cellular membranes. The quality of images obtained by our system was sufficient for subsequent quantitative analysis for dynamics of amoeboid movements. This study demonstrates the application of light-sheet microscopy for high-speed imaging of biological specimens.
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Project description:Purpose of Review:Light microscopy plays an essential role in clinical diagnosis and understanding the pathogenesis of cancer. Conventional bright-field microscope is used to visualize abnormality in tissue architecture and nuclear morphology, but often suffers from many limitations. This review focuses on the potential of new imaging techniques to improve basic and clinical research in pathobiology. Recent findings:Light microscopy has significantly expanded its ability in resolution, imaging volume, speed and contrast. It now allows 3D high-resolution volumetric imaging of tissue architecture from large tissue and molecular structures at nanometer resolution. Summary:Pathologists and researchers now have access to various imaging tools to study cancer pathobiology in both breadth and depth. Although clinical adoption of a new technique is slow, the new imaging tools will provide significant new insights and open new avenues for improving early cancer detection, personalized risk assessment and identifying the best treatment strategies.
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Project description:PurposeHigh resolution visualization of the ocular surface and intact eyeball is critical and essential for our understanding and treatment of eye diseases. This study is to achieve this goal using advanced tissue clearing and three-dimensional (3D) imaging technologies.MethodsWild type and fluorescently labeled transgenic mice of Prox-1-GFP (green fluorescent protein) or Thy1-YFP (yellow fluorescent protein) were used in the study. Eyeballs were harvested from normal or a disease model of corneal inflammation. Samples were infused with hydrogel monomers and heated for polymerization. Lipids were removed by electrophoresis. The transparent tissue-hydrogel hybrids of the anterior segments or intact eyeballs with immunolabeling or endogenous fluorescence were imaged by an advanced light sheet fluorescent microscope. High resolution 3D images and videos were captured for a wide array of structures and cell types.ResultsOptical transparency was achieved from intact eyeballs of both normal and diseased conditions. A variety of important structures and cell types, such as blood and lymphatic vessels, Schlemm's canal, nerves and endothelial cells, were detected with their natural morphology, location and organizational network.ConclusionsThis study provides the first comprehensive and 3D high resolution imaging of the intact eyeball using tissue clearing and advanced light sheet microscopy. Given that the eye is the window of the body, we anticipate this advanced technology will facilitate diverse applications in biomedical research inside and outside the eye.
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