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Related Concept Videos

Confocal Fluorescence Microscopy01:16

Confocal Fluorescence 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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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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Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells
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Dual-axis confocal configuration for depth sensitive fluorescence spectroscopy.

Yongping Lin, Tse-Shao Chang, Jing Chen

    Optics Letters
    |July 30, 2021
    PubMed
    Summary

    Dual-axis confocal (DAC) fluorescence spectroscopy achieves high axial resolution and long working distance, enabling depth-resolved spectral analysis. This advanced technique offers superior performance for microscopic imaging and spectral collection.

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    Area of Science:

    • Biophotonics
    • Spectroscopy
    • Microscopy

    Background:

    • Confocal microscopy offers optical sectioning capabilities.
    • Depth-resolved fluorescence spectroscopy requires high axial resolution and dynamic range.
    • Existing single-axis confocal configurations have limitations in working distance and axial resolution.

    Purpose of the Study:

    • To present a depth-sensitive fluorescence spectroscopy system utilizing a dual-axis confocal (DAC) configuration.
    • To demonstrate the advantages of DAC configuration for fluorescence spectroscopy, including improved axial resolution and working distance.
    • To validate the DAC system's capability for depth-resolved spectral collection.

    Main Methods:

    • Implementation of a dual-axis confocal (DAC) microscopy system.
    • Utilizing Huygens-Fresnel integrals for enhanced dynamic range and out-of-focus light rejection.
    • Employing a dual-axis illumination configuration for target layer localization with white light imaging.
    • Testing with a multi-layer fluorescence phantom of Barrett's esophagus.

    Main Results:

    • Achieved an axial resolution of 3.23 µm and a working distance of 3.73 mm, surpassing single-axis configurations.
    • Demonstrated superior dynamic range and rejection of out-of-focus scattered light.
    • Successfully collected depth-resolved fluorescence spectra from microscopic regions using the DAC system.

    Conclusions:

    • The DAC configuration is highly effective for depth-resolved fluorescence spectroscopy.
    • DAC systems offer significant improvements in axial resolution and working distance compared to single-axis systems.
    • This technology enables precise spectral analysis at specific depths within microscopic samples.