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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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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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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: Oct 26, 2025

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
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Building a Total Internal Reflection Microscope (TIRF) with Active Stabilization (Feedback SMLM).

Simao Coelho1,2,3, Jongho Baek1,2, J Justin Gooding4,5

  • 1EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia.

Bio-Protocol
|July 30, 2021
PubMed
Summary
This summary is machine-generated.

Active stabilization using feedback loops significantly enhances high-resolution imaging quality. This protocol details building a feedback microscope for improved single-molecule localization microscopy (SMLM) precision.

Keywords:
Active stabilizationBiophysicsDrift correctionLocalization microscopySingle-molecule imagingTIRF microscopy

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Area of Science:

  • Microscopy
  • Optical Engineering
  • Biophysics

Background:

  • High-resolution imaging quality is often limited by sample drift and instability.
  • Active stabilization systems are crucial for maintaining sample position during microscopy experiments.
  • Previous methods lacked comprehensive protocols for building integrated stabilization systems.

Purpose of the Study:

  • To provide a detailed protocol for constructing a total internal reflection microscope with active feedback loops.
  • To achieve highly accurate focus lock and eliminate drift in microscopy.
  • To improve localization precision for single-molecule localization microscopy (SMLM).

Main Methods:

  • Describing a step-by-step protocol for building a total internal reflection microscope.
  • Integrating feedback loops for sample and detection stabilization.
  • Providing a list of optical components, practical tips, and an alignment strategy for system replication.

Main Results:

  • Demonstration of a replica system build for feedback-enhanced SMLM.
  • Successful implementation of active stabilization to maintain sample position.
  • Improved localization precision due to eliminated drift and accurate focus lock.

Conclusions:

  • The developed protocol enables the construction of a robust feedback-stabilized microscope.
  • This system significantly enhances data quality in high-resolution imaging, particularly for SMLM.
  • The detailed guide facilitates the replication of advanced microscopy techniques.