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Total Internal Reflection Fluorescence Microscopy

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.

Confocal Fluorescence Microscopy

Confocal Fluorescence Microscopy

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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A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

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Alignment and calibration of total internal reflection fluorescence microscopy systems.

Cold Spring Harbor Protocols

|April 5, 2012

Summary

Total internal reflection fluorescence microscopy (TIRFM) offers exquisite sensitivity for imaging cellular processes near the cell cortex. This protocol details TIRFM system alignment and calibration for enhanced biological research.

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Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

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Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

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Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Published on: October 2, 2012

Area of Science:

Cell Biology
Microscopy Techniques
Biophysics

Background:

Live cell fluorescent microscopy is crucial for understanding dynamic cellular processes.
High spatiotemporal resolution and sensitivity are key for observing transient states.
Total internal reflection fluorescence microscopy (TIRFM) provides enhanced sensitivity for near-cortex imaging.

Purpose of the Study:

To describe the procedure for alignment and calibration of TIRFM systems.
To enable accurate and sensitive imaging of cellular dynamics.
To make TIRFM more accessible and attractive to biologists.

Main Methods:

Utilizing a high numerical-aperture (NA) objective lens (>1.45).
Implementing turnkey TIRFM systems.
Describing the alignment and calibration of the TIRF illuminator with downstream optics using standard cellular samples, a 488-nm laser, and a GFP filter cube.

Main Results:

Successful alignment and calibration of TIRFM systems.
Demonstration of TIRFM's exquisite sensitivity for near-cortex cellular processes.
Increased accessibility of TIRFM for quantitative biological studies.

Conclusions:

TIRFM is a powerful technique for studying dynamic cellular events at the single-molecule or organelle level.
Proper alignment and calibration are essential for optimal TIRFM performance.
The increasing availability of TIRFM systems facilitates quantitative biological research.