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Super-resolution Fluorescence Microscopy01:37

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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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Confocal Fluorescence Microscopy01:16

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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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Electron Microscope Tomography and Single-particle Reconstruction01:07

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Transmission electron microscopy (TEM) can be used to determine the 3D structure of biological samples with the help of techniques such as electron microscope tomography and single-particle reconstruction. While single-particle reconstruction can examine macromolecules and macromolecular complexes in vitro conditions only, tomography permits the study of cell components or small cells in vivo.
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Overview of Electron Microscopy01:25

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The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X.
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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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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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Updated: Feb 24, 2026

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors
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Microscopía correlativa de barrido electrónico y de iluminación estructurada de superresolución

Joseph R Hamiliton, Summer K Levis, Guy M Hagen

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    |February 23, 2026
    PubMed
    Resumen

    La microscopía correlativa de luz y electrones (CLEM) mejora el análisis de muestras biológicas. La microscopía electrónica de barrido (SEM) proporciona mayor resolución y valida los resultados de la microscopía de fluorescencia de iluminación estructurada de superresolución (SIM).

    Palabras clave:
    microscopía correlativamicroscopía electrónica de barridomicroscopía de fluorescenciailuminación estructuradasuperresoluciónbiología celularCiencias BiológicasMicroscopía

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    Área de la Ciencia:

    • Ciencias biológicas
    • Microscopía
    • Biología celular

    Sus antecedentes:

    • La microscopía correlativa integra múltiples técnicas de imagen para un análisis exhaustivo de las muestras.
    • Los métodos de preservación son cruciales para preparar muestras biológicas para la microscopía correlativa de luz y electrones (CLEM).

    Objetivo del estudio:

    • Comparar la calidad de imagen y la resolución de la microscopía de campo amplio (WF), la microscopía de fluorescencia de iluminación estructurada de superresolución (SIM) y la microscopía electrónica de barrido (SEM) en un flujo de trabajo correlativo.
    • Evaluar la efectividad del tratamiento químico con NanoSuit para preservar la integridad de la muestra para SEM después de la obtención de imágenes de fluorescencia.

    Principales métodos:

    • Se obtuvo una muestra de testículo de mamífero utilizando microscopía de fluorescencia WF y SIM.
    • La muestra se sometió a un tratamiento químico con NanoSuit.
    • Después del tratamiento, la muestra se examinó mediante SEM.

    Principales resultados:

    • La SEM logró una mayor resolución en comparación con WF y SIM.
    • La SEM proporcionó detalles estructurales que validaron los hallazgos de la SIM.
    • El tratamiento con NanoSuit permitió la obtención de imágenes SEM exitosas después de la microscopía de fluorescencia.

    Conclusiones:

    • La CLEM, especialmente la combinación de SIM y SEM, ofrece una visión mejorada de la ultraestructura de las muestras biológicas.
    • La SEM es una herramienta valiosa para validar datos de microscopía de fluorescencia de alta resolución.
    • El protocolo NanoSuit es eficaz para preparar muestras para análisis correlativo de SEM.