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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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Related Experiment Video

Updated: Sep 15, 2025

Fluorescence Lifetime Macro Imager for Biomedical Applications
06:01

Fluorescence Lifetime Macro Imager for Biomedical Applications

Published on: April 7, 2023

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Depth-Resolved Macroscopic Fluorescence Lifetime Imaging via High-Spatial-Frequency Structured Illumination.

Nanxue Yuan1, Saif Ragab1, Navid Nizam1

  • 1Center for modeling, simulation and Imaging in Medicine, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.

Biorxiv : the Preprint Server for Biology
|July 15, 2025
PubMed
Summary
This summary is machine-generated.

A new High Spatial Frequency-Fluorescence Lifetime Imaging (HSF-FLI) method accurately distinguishes deep tissue fluorescence from surface signals. This advancement improves clarity in biological interpretation and supports translational research applications.

Keywords:
FLIFRETHSFMCXMFLIStructured Light Illumination

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Area of Science:

  • Biophotonics and Imaging Science
  • Optical Physics
  • Preclinical Research

Background:

  • Macroscopic Fluorescence Lifetime Imaging (MFLI) provides robust contrast but struggles with depth localization.
  • Surface signal bias, particularly from skin, complicates biological interpretation in MFLI.
  • Accurate depth determination is crucial for advancing MFLI in translational studies.

Purpose of the Study:

  • To establish a High Spatial Frequency-Fluorescence Lifetime Imaging (HSF-FLI) framework.
  • To selectively isolate subsurface fluorescence from surface fluorescence.
  • To preserve the accuracy of lifetime estimation in depth-resolved imaging.

Main Methods:

  • Developed a modulation transfer function (MTF) relating spatial frequency to penetration depth.
  • Utilized Monte Carlo eXtreme (MCX) simulations for physics-based modeling.
  • Employed structured three-phase sinusoidal illumination and nonlinear least squares fitting.
  • Validated in vivo using mouse models and cross-validated with ex vivo measurements.

Main Results:

  • HSF-FLI successfully isolated subsurface fluorescence from surface signals.
  • Accurate lifetime estimation was maintained with depth selectivity.
  • Demonstrated in vivo utility in mouse models for drug delivery assessment.
  • Validated using MFLI-FRET in preclinical models.

Conclusions:

  • HSF-FLI effectively eliminates surface signal bias without chemical clearance.
  • Coupling structured illumination with physics-based depth modeling yields accurate, depth-selective lifetime readouts.
  • This method enables robust and fast fluorescence lifetime imaging for translational studies.