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Absolute position total internal reflection microscopy with an optical tweezer.

Lulu Liu1, Alexander Woolf2, Alejandro W Rodriguez3

  • 1School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138; and capasso@seas.harvard.edu lululiu@fas.harvard.edu.

Proceedings of the National Academy of Sciences of the United States of America
|December 17, 2014
PubMed
Summary

A new optical tweezing method calibrates total internal reflection microscopy (TIRM) noninvasively. This technique precisely measures particle position over a wider range, enhancing TIRM

Keywords:
TIRFMTIRMcalibrationoptical tweezerparticle tracking

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Area of Science:

  • Microscopy and Nanotechnology
  • Optical Physics
  • Physical Chemistry

Background:

  • Total Internal Reflection Microscopy (TIRM) is a powerful technique for studying interfacial phenomena.
  • Conventional TIRM calibration methods often rely on assumptions or approximations, limiting precision and measurement range.
  • Accurate calibration is crucial for reliable quantitative measurements in TIRM.

Purpose of the Study:

  • To develop a noninvasive, in situ calibration method for TIRM using optical tweezing.
  • To significantly improve the precision and measurement range of TIRM.
  • To demonstrate the enhanced capabilities of TIRM for studying interfacial dynamics.

Main Methods:

  • Integration of optical tweezing with a standard TIRM setup.
  • In situ measurement of a probe particle's scattering intensity versus height profile.
  • Calibration based on direct experimental data rather than theoretical models or system analogs.

Main Results:

  • Achieved better than 10 nm precision in determining absolute particle position.
  • Extended the measurement range to over 1 μm from the dielectric interface.
  • Demonstrated a ~10x improvement in error and a 3x improvement in range compared to conventional TIRM.

Conclusions:

  • The optical tweezing-based calibration method substantially enhances TIRM's precision and range.
  • This technique offers a more direct and reliable approach to TIRM calibration.
  • The improved TIRM method enables more accurate studies of interfacial phenomena, such as near-wall diffusion.