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Optically sectioned wide-field fluorescence lifetime imaging microscopy enabled by structured illumination.

Taylor Hinsdale1, Cory Olsovsky1, Jose J Rico-Jimenez1

  • 1Department of Biomedical Engineering, Texas A&M University, 5045 Emerging Technologies Building, 3120 TAMU, College Station, TX 77843, USA.

Biomedical Optics Express
|July 1, 2017
PubMed
Summary
This summary is machine-generated.

Structured illumination fluorescence lifetime imaging microscopy (SI-FLIM) accurately measures fluorescence lifetimes in thick, layered samples with overlapping spectra. This advanced microscopy technique improves resolution in biological tissues like oral mucosa.

Keywords:
(110.0180) Microscopy(110.6880) Three-dimensional image acquisition(170.3880) Medical and biological imaging

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Area of Science:

  • Biophotonics and Imaging
  • Microscopy Techniques
  • Fluorescence Spectroscopy

Background:

  • Resolving fluorescence lifetimes in thick biological samples with spectrally overlapping layers is challenging.
  • Traditional fluorescence lifetime imaging (FLIM) struggles with depth resolution and spectral interference in complex tissues.
  • Need for advanced microscopy methods to accurately characterize fluorophores in heterogeneous environments.

Purpose of the Study:

  • To demonstrate structured illumination microscopy's capability to enhance fluorescence lifetime imaging (FLIM).
  • To accurately measure fluorescence lifetimes in thick samples with distinct, spectrally overlapping fluorescent layers.
  • To validate the performance of structured illumination fluorescence lifetime imaging microscopy (SI-FLIM) in biological tissues.

Main Methods:

  • Utilized structured illumination principles combined with fluorescence lifetime imaging.
  • Tested SI-FLIM on homogenous fluorophore samples (POPOP, NADH, FAD).
  • Evaluated SI-FLIM on two-layer models with spectrally overlapping fluorophores (NADH/FAD over POPOP).
  • Applied SI-FLIM ex vivo to hamster cheek pouch tissue (oral mucosa).

Main Results:

  • SI-FLIM accurately reconstructed lifetime values in homogenous fluorophore samples.
  • Accurate fluorescence lifetime measurements were achieved in two-layer models with spectral overlap.
  • SI-FLIM demonstrated improved accuracy in measuring lifetimes for distinct layers (epithelium, submucosa) of oral mucosa.

Conclusions:

  • Structured illumination fluorescence lifetime imaging microscopy (SI-FLIM) significantly enhances the resolution of fluorescence lifetimes.
  • SI-FLIM effectively resolves spectrally overlapping fluorophores in thick samples and complex biological tissues.
  • This technique offers improved accuracy for analyzing layered biological structures at the cellular and tissue level.