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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.
Total Internal Reflection Fluorescence Microscopy01:05

Total Internal Reflection Fluorescence Microscopy

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

Updated: May 21, 2026

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy
14:23

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

Published on: March 6, 2018

Accessible STORM Imaging: An Optimized Workflow for Conventional Widefield Epifluorescence/TIRF Setups.

Jaime Fernández de Córdoba1, Ana Oña1, Gianluca D'Agostino1

  • 1Advanced Light Microscopy, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

Bio-Protocol
|May 20, 2026
PubMed
Summary

This study presents an accessible workflow for Stochastic Optical Reconstruction Microscopy (STORM), a super-resolution imaging technique. The developed protocol simplifies sample preparation and image acquisition for visualizing cellular structures with nanoscale precision.

Keywords:
ActinFWHMLysosomesMicrotubulesMitochondriaSMLMSTORMTIRFUncertainty

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High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip
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High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

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Test Samples for Optimizing STORM Super-Resolution Microscopy
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Test Samples for Optimizing STORM Super-Resolution Microscopy

Published on: September 6, 2013

Related Experiment Videos

Last Updated: May 21, 2026

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy
14:23

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

Published on: March 6, 2018

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip
14:09

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

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Test Samples for Optimizing STORM Super-Resolution Microscopy
16:52

Test Samples for Optimizing STORM Super-Resolution Microscopy

Published on: September 6, 2013

Area of Science:

  • Cell Biology
  • Microscopy
  • Biophysics

Background:

  • Stochastic Optical Reconstruction Microscopy (STORM) is a super-resolution technique enabling visualization of cellular ultrastructure beyond the diffraction limit.
  • STORM relies on sequential single-photon emission from photosensitive fluorophores for precise localization and high-resolution image reconstruction.

Purpose of the Study:

  • To describe a simple and efficient STORM workflow for sample preparation, image acquisition, and quality control.
  • To enable broader implementation of STORM by lowering technical barriers on widely available microscopy platforms.

Main Methods:

  • Developed an optimized immunofluorescence protocol including washing steps and secondary antibody fixation for single and dual-color STORM.
  • Adapted a conventional epifluorescence/TIRF microscope for STORM imaging with a 160×/1.43 NA objective and high-power laser mode.
  • Implemented a 1.6× magnification lens and 4×4 camera binning for optimal 100 nm pixel size for molecule detection.

Main Results:

  • Successfully visualized subcellular structures including mitochondria, microtubules, lysosomes, and the actin cytoskeleton.
  • Achieved localization precision of up to ~20 nm, enabling detailed ultrastructural visualization.
  • Demonstrated the protocol's effectiveness using Alexa Fluor 647 and Alexa Fluor 488-conjugated phalloidin.

Conclusions:

  • The presented STORM workflow simplifies super-resolution microscopy, making it more accessible to the research community.
  • This protocol facilitates the broader application of STORM for investigating cellular ultrastructure with nanoscale resolution.