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Related Concept Videos

Imaging Biological Samples with Optical Microscopy01:18

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Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in the 17th century to visualize blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes with enhanced magnification and resolution.
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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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Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope
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Variable immersion microscopy with a high numerical aperture.

Keita Ishida, Kanta Naruse, Yuta Mizouchi

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    |February 12, 2021
    PubMed
    Summary
    This summary is machine-generated.

    Researchers developed a variable immersion lens (VIL) to overcome optical aberrations in 3D microscopy of thick specimens. This innovation enables clearer, deeper imaging of biological samples, improving 3D microscopy resolution.

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    Area of Science:

    • Optical Microscopy
    • Biophysics
    • Imaging Science

    Background:

    • High numerical aperture (NA) 3D optical microscopy faces challenges with thick biological specimens due to aberrations from interface refractions.
    • Existing methods struggle to maintain image quality and resolution deep within scattering or refractive samples.

    Purpose of the Study:

    • To develop a novel optical system that passively minimizes aberrations in high-NA 3D microscopy.
    • To enable diffraction-limited imaging at greater depths within biological tissues.

    Main Methods:

    • Development of a variable immersion lens (VIL), a high-NA concentric meniscus lens.
    • Integration of the VIL with an aberration-corrected high-NA reflecting objective (TORA-FUJI mirror).
    • Utilizing wave-optics simulations and experimental validation with fluorescent beads.

    Main Results:

    • Wave-optics simulations demonstrated diffraction-limited 1.2-NA imaging in water at 0.3 mm depth, minimizing interface refraction aberrations.
    • The VIL system effectively corrected aberrations caused by refractive index mismatches between mounting media and samples.
    • Experimental results confirmed accurate 3D imaging of a 6 µm fluorescent bead to its true dimensions.

    Conclusions:

    • The VIL system successfully corrects two major aberrations in high-NA 3D optical microscopy.
    • This technology significantly enhances imaging capabilities for thick biological specimens, paving the way for deeper and clearer visualization.
    • The VIL offers a versatile solution for improving resolution and accuracy in advanced microscopy applications.