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Overview of Microscopy Techniques01:22

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The early pioneers of microscopy opened a window into the invisible world of microorganisms. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy that uses an ultraviolet light source and electron microscopy that uses short-wavelength electron beams. These advances significantly improved magnification, image resolution, and contrast. By comparison, the...
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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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Two-dimensional (2D) microscopy encompasses a range of optical techniques that capture images within a single focal plane, offering detailed representations of microscopic structures. These techniques are essential in biological and medical research, enabling the visualization of cellular and subcellular structures with different levels of contrast and specificity.There are several major types of 2D microscopy, each with strengths and applications.Bright-Field MicroscopyBright-field microscopy...
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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
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A scanning electron microscope (SEM) is used to study the surface features of a sample by using an electron beam that scans the sample surface in a two-dimensional manner. Typically, areas between ~1 centimeter to 5 micrometers in width can be imaged. SEM can be used to image bacteria, viruses, tissues as well as larger samples like insects. Conventional SEM gives a magnification ranging from 20X to 30,000X and spatial resolution of 50 to 100 nanometers.
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An Innovative Method for Exosome Quantification and Size Measurement
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New microscopy technique based on position localization of scattering particles.

Stefano Luigi Oscurato, Fabio Borbone, Robert Charles Devlin

    Optics Express
    |August 10, 2017
    PubMed
    Summary

    We developed Holographic - Single Scatterer Localization Microscopy (HSLM) for precise particle localization. This advanced technique offers superior resolution and miniaturization potential for scattering particle analysis.

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    Area of Science:

    • Optical Microscopy
    • Nanotechnology
    • Biophysics

    Background:

    • Accurate localization of scattering particles is crucial in various scientific fields.
    • Existing laser scanning techniques face limitations in resolution and miniaturization.

    Purpose of the Study:

    • To introduce a novel microscopy technique for precise localization of isolated scattering particles.
    • To demonstrate the advantages of Holographic - Single Scatterer Localization Microscopy (HSLM) over conventional methods.

    Main Methods:

    • Combining dynamical laser speckle illumination with centroid localization of backscattered light.
    • Utilizing a digital holography setup with a spatial light modulator for illumination control.

    Main Results:

    • Achieved highly accurate particle localization, exceeding the precision of diffraction-limited imaging.
    • Demonstrated improved resolution for distinguishing closely spaced scattering objects.
    • Showcased background light rejection capabilities comparable to confocal microscopy.

    Conclusions:

    • HSLM provides superior particle localization accuracy and resolution.
    • The digital holography approach enables versatile illumination and setup miniaturization without mechanical parts.