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A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins
Published on: March 22, 2012
Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy.
René Schneider1, Tilman Glaser, Michael Berndt
1Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
Optics Express
|March 14, 2013
Summary
A new total internal reflection (TIR) prism enables sensitive dark-field imaging for single molecules and particles. This advanced TIR microscopy achieves high signal-to-noise ratios for observing nanoscale events like kinesin motor stepping.
Area of Science:
- Optical microscopy
- Nanotechnology
- Biophysics
Background:
- Total internal reflection (TIR) microscopy is crucial for high-performance fluorescence imaging.
- Objective-type TIR setups face challenges in dark-field imaging due to spectral overlap of illumination and detection light.
- Separating illumination and detection light spectrally is difficult in conventional TIR setups for scattering entities.
Purpose of the Study:
- To develop a novel TIR approach for improved dark-field imaging of scattering entities.
- To enable sensitive detection of single-molecule fluorescence and single-particle scattering.
- To achieve high signal-to-noise ratios in advanced microscopy applications.
Main Methods:
- Utilized a novel TIR approach employing a parabolically shaped quartz prism.
- Demonstrated homogeneous and spatially invariant illumination profiles.
- Controlled a wide range of illumination angles for versatile imaging.
Main Results:
- Achieved high signal-to-noise ratios for both single-molecule fluorescence and single-particle scattering.
- Quantitatively compared fluorescence performance against objective-type TIR.
- Demonstrated sub-nanometer localization accuracies for 40 nm gold nanoparticle (AuNP) scattering.
- Successfully reported on the 8 nm stepping of kinesin-1 motors using AuNPs.
Conclusions:
- The novel parabolic prism TIR method offers superior performance for dark-field imaging.
- This technique facilitates high-resolution observation of nanoscale dynamics, including molecular motor activity.
- The setup provides a versatile platform for single-molecule and single-particle studies in biophysics.
