Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events
Daniel S Johnson1, Jyoti K Jaiswal, Sanford Simon
1The Rockefeller University, Laboratory of Cellular Biophysics, New York, New York, USA.
Current Protocols in Cytometry
|July 4, 2012
Summary
Total internal reflection fluorescence (TIRF) microscopy enhances cell membrane imaging by improving contrast and signal-to-noise ratio. This protocol details setting up TIRF illuminators for observing cellular events with high resolution.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Total internal reflection fluorescence (TIRF) microscopy offers superior contrast for imaging events near cell membranes.
- It achieves this by confining excitation light to a thin layer, significantly improving signal-to-noise ratio compared to epi-fluorescence.
- This makes TIRF ideal for studying dynamic processes like exocytosis, endocytosis, and membrane protein behavior.
Purpose of the Study:
- To provide protocols for setting up TIRF microscopy systems.
- To introduce an automated deflection system for enhanced TIRF illumination control.
- To discuss the principles, parameters, and challenges of TIRF microscopy.
Main Methods:
- Detailed procedures for assembling through-the-objective and prism-based TIRF illuminators.
- Description of an automated deflection system for TIRF microscopy.
- Explanation of the total internal reflection phenomenon and TIRF microscope parameters.
Main Results:
- Established protocols for two common TIRF illumination setups.
- Demonstrated benefits of an automated deflection system, including aberration reduction and rapid illumination switching.
- Provided a comprehensive overview of TIRF microscopy principles and practical considerations.
Conclusions:
- TIRF microscopy is a powerful technique for high-resolution live-cell imaging at the cell membrane.
- The described protocols and automated system facilitate advanced TIRF applications.
- Understanding TIRF principles is crucial for optimizing imaging of cellular dynamics.
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
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.