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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.
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Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo
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Fluorescence coherence tomography.

A Bilenca1, A Ozcan, B Bouma

  • 1Harvard Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital, 50 Blossom Street, BAR 7, Boston, Massachusetts 02114, USA. abilenca@partners.org

Optics Express
|June 17, 2009
PubMed
Summary
This summary is machine-generated.

We developed spectral-domain fluorescence coherence tomography (SD-FCT), a new imaging method. This technique enables detailed cross-sectional views of fluorescent samples with high resolution and depth.

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Published on: October 20, 2017

Area of Science:

  • Biomedical Optics
  • Fluorescence Imaging
  • Optical Coherence Tomography

Background:

  • Fluorescence imaging is crucial for visualizing biological structures.
  • Existing methods face limitations in resolution, depth penetration, or field of view.
  • Coherence-gated imaging offers depth-resolved information but can be complex.

Purpose of the Study:

  • Introduce and validate a novel cross-sectional fluorescence imaging technique.
  • Demonstrate the capabilities of spectral-domain fluorescence coherence tomography (SD-FCT).
  • Assess the potential of SD-FCT for imaging semi-transparent, fluorescent samples.

Main Methods:

  • Developed a first-generation SD-FCT system using a dual-objective interferometer.
  • Employed an imaging spectrometer for spectrally detecting self-interference of fluorophore emission.
  • Utilized spontaneous emission from fluorophores along the axial dimension for depth information.

Main Results:

  • Achieved narrow axial point-spread functions (micrometers FWHM).
  • Demonstrated imaging depths of several hundred micrometers.
  • Obtained wide fields of view exceeding 1 millimeter.
  • Successfully performed cross-sectional profiling of layered fluorescence phantoms.

Conclusions:

  • SD-FCT is a novel coherence-gated fluorescence imaging modality.
  • The system provides high axial resolution, significant depth penetration, and a wide field of view.
  • SD-FCT shows promise as a valuable tool for investigating semi-transparent and fluorescently labeled samples.