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Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox
Published on: July 5, 2021
Objective-type total internal reflection microscopy (excitation) for single-molecule FRET.
Cold Spring Harbor Protocols
|November 3, 2012
Summary
This study details objective-type total internal reflection (TIR) microscopy for single-molecule (sm) fluorescence detection. It enables precise distance measurements using Förster (fluorescence) resonance energy transfer (FRET) for biological studies.
Area of Science:
- Biophysics
- Optical Microscopy
- Molecular Biology
Background:
- Single-molecule (sm) fluorescence detection offers insights into biological events without averaging.
- Förster (fluorescence) resonance energy transfer (FRET) measures distances (30-80 Å) between molecules.
- Changes in FRET signal structural dynamics or molecular interactions.
Purpose of the Study:
- To describe a protocol for objective-type total internal reflection (TIR) microscopy.
- To detail the setup for laser beam and epifluorescence microscopy.
- To explain the conversion of epifluorescence microscopy into TIR mode.
Main Methods:
- Objective-type total internal reflection (TIR) microscopy setup.
- Epifluorescence microscopy adaptation for TIR.
- Laser beam configuration for smFRET experiments.
Main Results:
- Successful implementation of objective-type TIR microscopy.
- Demonstration of converting epifluorescence microscopy to TIR.
- Enabling precise single-molecule distance measurements via FRET.
Conclusions:
- Objective-type TIR microscopy is a viable method for smFRET.
- The protocol facilitates detailed study of molecular dynamics.
- This technique enhances understanding of biological processes at the molecular level.
Related Concept Videos
01:05Total 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.
01:37Super-resolution Fluorescence Microscopy
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