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    Home > Biochemistry News > Biotechnology News > Technology! Chinese scholars bring new expansion microscopy techniques

    Technology! Chinese scholars bring new expansion microscopy techniques

    • Last Update: 2023-02-03
    • Source: Internet
    • Author: User
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    Images: e.
    (a) pre-dilated images of human kidneys imaged and processed with SOFI at 60 × versus dilated images with the same field of view (b) post-dilated images
    imaged with MAGNIFY at 40 ×.
    Magenta, DAPI; Orange Anti α-Actin 4 (ACTN4); Blue, waveform protein
    The (c-e) root mean square (RMS) length measurement error is a function
    of (c) DAPI, (d) ACTN4, and (e) Vimentin pre- and post-extended image measurements.
    solid line, channel mean; Shadow area, mean standard error (s.
    m); N = 5 technical replicates; The average coefficient of expansion was 8.
    64× (s.
    m 0.

    (f) Human pre-dilation images imaged and processed with SOFI at 60 × are compared
    with (g) post-dilated images taken with MAGNIFY at 40 ×.
    Magenta, DAPI; Green, anti-ATPASE inhibitory factor 1 (ATPIF).

    Post-extended image maximum intensity projection over 3 frames
    (h-i) RMS length measurement error is a function
    of (h) DAPI and (i) ATPIF image length measurements before and after expansion.
    solid line, channel mean; Shadow area, s.
    ; N = 4 technical replicates; The average inflation factor was 10.
    38× (s.
    m 0.

    (j-o) Validate MAGNIFY
    in multiple human tissue types.
    Human tissue FFPE samples are imaged at 40 × (top left).

    Image taken at 60×and, SOFI processed (bottom left).

    The white box represents the field of view
    of a high-magnification image.
    The samples are then MAGNIFY, imaging the same field of view
    after expanding in water at 10 × (upper right) and 40 × (bottom right).
    The extended image is projected on
    slices of 4-17 z.
    Magenta, DAPI; Green, ATPIF; Blue, cytokeratin generic I/II
    The expansion factor in water was (j) colon: 8.
    85×, (k) breast: 9×, (l) uterus: 8×, (m) placenta: 8.
    75 ×, (n) thymus: 10.
    00 ×, (o) thyroid: 10.
    59 ×
    (p-r) Example of 3D image of human tissue: (p) Kidney (expansion factor 8.

    Magenta, DAPI; Orange, ACTN4; Blue, screenwriter
    (q) Colon (dilation factor 9.

    Magenta, DAPI; Green, ATIPF; Blue, cytokeratin generic I/II
    (r) Uterus (dilation factor 8×).

    Magenta, DAPI; Green, ATIPF; Blue, cytokeratin generic I/II
    A dotted white box
    in the magnified area.
    Scale bar (yellow for extended image) :(a) 5 μm; (b) 5 μm (40.
    75 μm expansion after physical scale; Coefficient of expansion: 8.
    15×);(f) 5 μm;(g) 5 μm (expansion after physical scale: 51.
    9 μm; Expansion coefficient: 10.
    38×);(j-o) top: 10 μm; Bottom: 1 μm; (p-t) 5 μm
    Scale bars are all biological scales

    Image credit: Courtesy of Carnegie Mellon University

    Thanks to innovations in expansion microscopy (ExM), we can see the inside of cells and other nanoscale structures
    like never before.
    These advances help provide future insights
    into neuroscience, pathology, and many other biological and medical fields.

    In the Jan.
    2 paper, "Magnify is a universal molecular anchoring strategy for expansion microscopy," collaborators from Carnegie Mellon University, the University of Pittsburgh, and Brown University describe the new technique
    known as "Magnify.

    Leon Zhao, associate professor of biological sciences, said: "Magnify can be an effective and usable tool
    in biotechnology.

    Yongxin Zhao's biophotonics laboratory is a leader
    in super-resolution imaging of biological samples through physical expansion of samples, known as expansion microscopy.
    Through this process, the sample is embedded in an expandable hydrogel that expands uniformly to increase the distance between molecules, allowing them to be observed
    at higher resolution.
    This allows nanoscale biological structures previously only visible using expensive high-resolution imaging techniques to be seen
    with standard microscopy tools.

    Magnify is a variant of the expansion microscope that allows researchers to use a new hydrogel formulation invented by the team that preserves the spectrum of biomolecules, offers a wider range of applications in a variety of tissues, and increases the expansion rate linearly to 11 times or 1300 times
    the original volume.

    "We overcame some of the long-term challenges of expansion microscopy," says Yongxin Zhao, "and one of Magnify's main selling points is the general strategy
    of preserving tissue biomolecules, including proteins, nuclear fragments, and carbohydrates, within the expanded sample.
    " ”

    Zhao Yongxin said it was important to keep the different biological components intact because previous protocols required eliminating many of the various biomolecules
    that bind tissues together.
    But these molecules may contain valuable information
    for researchers.

    "In the past, in the past, in order for cells to be really expandable, you needed enzymes to digest proteins, so in the end, you get an empty gel with a label that indicates the location of the
    protein of interest," he said.
    With this new method, molecules remain intact and multiple types of biomolecules
    can be labeled in a single sample.

    "Before, exams were like multiple-choice questions
    If you want to label proteins, that's version one
    If you want to label the nucleus, it will be a different version, and if you want to do synchronous imaging, it is difficult
    Now with Magnify, you can select multiple items to label, such as proteins, lipids and carbohydrates, and image them together

    Laboratory researcher Aleksandra Klimas, a postdoctoral researcher, said: "This is a convenient method
    for imaging high-resolution specimens.
    " "Traditionally, you need expensive equipment, specific reagents, and training
    However, this method is widely applicable to many types of sample preparation and can be observed
    with standard microscopes in biological laboratories.

    Gallagher, who has a background in neuroscience, says the goal is to make these protocols as compatible as possible, so that researchers can benefit
    from adopting Magnify as part of the toolkit.

    "It works with different tissue types, fixing methods, and even tissues
    that are preserved and stored.
    It's very flexible because you don't necessarily need to redesign the experiment; It will work with
    what you already have.

    For researchers like Simon Watkins, founder and director of the University of Pittsburgh's Center for Bioimaging and the Pittsburgh Cancer Institute, the fact that the new protocol is compatible with a wide range of tissue types—including preserved tissue sections—is important
    For example, most extended microscopy methods are optimized for brain tissue
    In contrast, Magnify tested on samples from a variety of human organs and corresponding tumors, including the breast, brain, and colon

    "Let's say you have a tissue with dense and non-dense components, which bypasses tissue
    that previously didn't expand in equal amounts," Watkins said.
    Leon has been working this issue to make this solution applicable to organizations
    that have already archived.

    Part of his research includes the study of movable cilia
    that clear mucus in the body's trachea.
    At 200 nanometers in diameter and only a few microns in length, these structures are
    too small without time-consuming techniques such as electron microscopy.
    Ren's team collaborated with Zhao's lab to develop and deliver a lung organoid model with ciliary ultrastructure and function-specific defects, validating Magnify's ability to
    visualize clinically relevant cilia pathologies.

    "With the latest magnification technology, we can expand these lung tissues and even see some of the ultrastructure of motor cilia with a normal microscope, which will speed up basic and clinical research
    ," he said.

    The researchers were also able to observe defects
    in cilia in patient-specific lung cells known to have genetic mutations.

    "The lung tissue engineering community always needs a better way to describe the tissue systems we're studying," Ren said
    This work is an important first step, and he hopes that the collaboration with Zhao's laboratory will be further refined and applied to the pathological samples
    found in tissue banks.

    Finally, the hydrogel developed by Zhao Labs for Magnify is stronger than its predecessor, which is very fragile and causes breaks
    in the process.

    "We want to develop this technology to make it more accessible to people
    ," he said.
    "This goes in a different direction
    There is a lot of interest
    in using this tissue expansion technique for basic science.

    Alison Barth, a professor of life sciences at Carnegie Mellon University
    , studies synaptic connectivity during learning.
    She said the wide range of applications offered by the new method would be a boon
    for researchers.

    "The brain is a good place to take advantage of these super-resolution techniques, and microscopy methods will benefit synaptic phenotyping and analysis
    under different brain conditions.

    "One of the main advances in this paper is the method's ability to work
    on many different types of tissue specimens.

    Magnify is a universal molecular anchoring strategy for expansion microscopy

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