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Related Concept Videos

Total Internal Reflection Fluorescence Microscopy01:05

Total Internal Reflection Fluorescence Microscopy

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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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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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A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins
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Single-shot, shadowless total internal reflection fluorescence microscopy via annular fiber bundle.

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    We developed a new method for instant, uniform total internal reflection fluorescence (TIRF) excitation using an annular fiber bundle. This versatile technique supports various light sources and illumination modes, enabling quantitative imaging.

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    Area of Science:

    • Microscopy and Imaging Technologies
    • Biophysics
    • Optical Engineering

    Background:

    • Total internal reflection fluorescence (TIRF) microscopy is crucial for high-resolution imaging of cell surfaces.
    • Achieving uniform and instantaneous TIRF excitation over large fields of view remains a challenge.
    • Existing methods often lack flexibility in light source compatibility and illumination mode switching.

    Purpose of the Study:

    • To present a novel method for generating instantaneous and uniform TIRF excitation.
    • To demonstrate the versatility and performance of the developed technique.
    • To enable quantitative shadowless TIRF imaging.

    Main Methods:

    • Utilized an annular fiber bundle with spatially incoherent light sources for TIRF excitation.
    • Integrated laser and LED light sources of different wavelengths.
    • Facilitated seamless switching between TIRF and epi illumination modes.

    Main Results:

    • Achieved instantaneous and uniform TIRF excitation over a large field of view.
    • Demonstrated successful single-molecule imaging in high-background conditions.
    • Showcased high-throughput and uniform TIRF imaging of cells.

    Conclusions:

    • The developed annular fiber bundle method provides a versatile and effective solution for TIRF excitation.
    • This technique enhances quantitative imaging capabilities, particularly for cellular studies.
    • Enables advanced applications in microscopy requiring uniform and controlled illumination.