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Conducting Multiple Imaging Modes with One Fluorescence Microscope
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Supercritical Angle Fluorescence Microscopy and Spectroscopy.

Martin Oheim1, Adi Salomon2, Maia Brunstein3

  • 1Saints-Pères Paris Institute for the Neurosciences, Université de Paris, CNRS, Paris, France.

Biophysical Journal
|April 30, 2020
PubMed
Summary
This summary is machine-generated.

Supercritical angle fluorescence (SAF) microscopy offers optical sectioning similar to total internal reflection fluorescence (TIRF). SAF enables precise analysis of fluorophore properties like distance and orientation near interfaces.

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Area of Science:

  • Optics and Photonics
  • Biophysics
  • Spectroscopy

Background:

  • Fluorescence detection is crucial in spectroscopy, sensing, and microscopy.
  • Total internal reflection fluorescence (TIRF) is a common technique for surface-selective assays.
  • Supercritical angle fluorescence (SAF) is an emerging technique with similar optical sectioning capabilities to TIRF.

Purpose of the Study:

  • To explore the principles and potential of SAF microscopy and spectroscopy.
  • To compare SAF with TIRF for surface analysis.
  • To identify biophysical applications and challenges of SAF.

Main Methods:

  • SAF utilizes near-field emission coupling to a higher refractive index medium.
  • Analysis of back focal plane images via Fourier filtering to decode fluorophore radiation patterns.
  • Integration of SAF functionality into existing inverted microscopes for spatially resolved measurements.

Main Results:

  • SAF allows for precise determination of emitter's refractive index, distance from interface, and orientation.
  • SAF collects both supercritical angle fluorescence and undercritical angle fluorescence components.
  • SAF can retrieve near-field information using wide-field optics.

Conclusions:

  • SAF microscopy presents a powerful tool for nanoscale analysis at interfaces.
  • SAF offers a valuable alternative or complement to TIRF in biophysical studies.
  • Further research into SAF's challenges and opportunities is warranted for broader applications.