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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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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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Updated: Feb 27, 2026

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions
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Stochastic Optical Reconstruction Microscopy (STORM).

Jianquan Xu1, Hongqiang Ma1, Yang Liu2

  • 1Biomedical and Optical Imaging Laboratory, Departments of Medicine and Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania.

Current Protocols in Cytometry
|July 6, 2017
PubMed
Summary
This summary is machine-generated.

Super-resolution microscopy, like Stochastic Optical Reconstruction Microscopy (STORM), offers nanoscale biological insights. Optimizing sample prep, imaging, and reconstruction is key to high-quality STORM images and avoiding artifacts.

Keywords:
single molecule localization microscopy (SMLM)stochastic optical reconstruction microscopy (STORM)super-resolution fluorescence microscopy

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Cell Biology

Background:

  • Super-resolution (SR) microscopy provides nanoscale resolution, revolutionizing biological studies.
  • Stochastic Optical Reconstruction Microscopy (STORM) is a key SR technique utilizing single-molecule localization.
  • STORM achieves 20-30 nm resolution, a significant advancement over conventional methods.

Purpose of the Study:

  • To discuss the optimization of Stochastic Optical Reconstruction Microscopy (STORM) for high-quality imaging.
  • To highlight the critical factors influencing STORM image quality: sample preparation, image acquisition, and reconstruction.
  • To address common artifacts and resolution degradation issues in STORM imaging.

Main Methods:

  • Detailed discussion on optimizing sample preparation techniques for STORM.
  • Guidelines for proper image acquisition parameters in STORM.
  • Explanation of image reconstruction algorithms and their impact on STORM resolution.

Main Results:

  • STORM provides a ten-fold resolution improvement over conventional optical microscopy.
  • Successful implementation requires careful optimization of sample preparation, image acquisition, and reconstruction.
  • Improper conditions can lead to image artifacts and reduced resolution.

Conclusions:

  • Achieving high-quality STORM images necessitates meticulous attention to all experimental stages.
  • Understanding and controlling variables in sample prep, acquisition, and reconstruction are crucial for reliable nanoscale biological imaging.
  • This unit provides a comprehensive guide to overcoming STORM limitations for advanced biological research.