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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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 developed.
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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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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Related Experiment Video

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Removal of subsurface fluorescence in cryo-imaging using deconvolution.

Ganapathy Krishnamurthi1, Charlie Y Wang, Grant Steyer

  • 1School of Biomedical Engineering, 10900 Euclid Avenue, Wickenden Bldg, Cleveland, OH 44106, USA.

Optics Express
|October 14, 2010
PubMed
Summary

We evaluated image restoration methods for subsurface fluorescence removal in cryo-images. Richardson-Lucy (RL) and Wiener methods best improved image quality and cell detection, with RL recommended for preserving structural integrity.

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Area of Science:

  • Microscopy
  • Image Processing
  • Biomedical Imaging

Background:

  • Subsurface fluorescence in cryo-images presents visualization challenges.
  • Image restoration techniques are crucial for analyzing cellular structures in such data.

Purpose of the Study:

  • To compare the effectiveness of Richardson-Lucy (RL), Wiener, and Next-image algorithms for removing subsurface fluorescence.
  • To assess the impact of these methods on image quality, cell delineation, and labeled cell detection.

Main Methods:

  • Comparison of RL, Wiener, and Next-image algorithms using measured scatter point-spread-functions.
  • Application to section-and-image cryo-image volumes.
  • Evaluation using contrast-to-noise ratio, contrast-to-background ratio, and Receiver Operating Characteristic (ROC) analyses.

Main Results:

  • All tested methods reduced haze and improved cell visualization.
  • RL and Wiener demonstrated comparable contrast improvements (35% better than Next-image).
  • RL and Wiener showed superior cell detection performance compared to Next-image and no processing (ROC analysis).
  • Next-image was faster and less prone to artifacts.

Conclusions:

  • RL is recommended for optimal restoration of fluorescent structure shape and size.
  • RL and Wiener are effective for improving image quality and cell detection in subsurface fluorescence cryo-images.