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Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy
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A readily usable two-photon fluorescence lifetime microendoscope.

Charles-Henri Hage1, Pierre Leclerc1, Marc Fabert1

  • 1Université de Limoges, XLIM - Pôle Photonique, UMR CNRS 7252, Limoges, France.

Journal of Biophotonics
|December 15, 2018
PubMed
Summary
This summary is machine-generated.

A novel two-photon fluorescence lifetime microendoscope enables sub-cellular imaging of cellular metabolism by analyzing nicotinamide adenine dinucleotide (NADH) autofluorescence. This compact system achieves high resolution and fast imaging rates for biological research.

Keywords:
FLIMbiomedical imagingfiber opticsnonlinear endoscopyultrashort pulses

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Area of Science:

  • Biomedical Optics
  • Cellular Metabolism Imaging
  • Microendoscopy

Background:

  • Intracellular energetic metabolism is crucial for cell function.
  • Accurate imaging of metabolic states requires high-resolution techniques.
  • Existing methods for in vivo metabolic imaging are often limited by resolution or invasiveness.

Purpose of the Study:

  • To demonstrate a two-photon fluorescence lifetime (2P-FLIM) microendoscope for sub-cellular resolution imaging.
  • To enable energetic metabolism imaging via intracellular nicotinamide adenine dinucleotide (NADH) autofluorescence.
  • To achieve near table-top microscope performance in a compact endoscopic probe.

Main Methods:

  • Development of a 2P-FLIM microendoscope with a ≈2 mm probe diameter and >5 m fiber length.
  • Utilized a home-designed spectro-temporal pulse shaper and custom photonic crystal fiber for high-power pulsed laser delivery (≈100 fs, 760 nm, 40 mW).
  • Incorporated a Gradient Index (GRIN) lens for 0.67 μm lateral resolution and spiral scanning for image formation.

Main Results:

  • Achieved a data acquisition rate of 16 frames per second (fps), leading to FLIM refreshing rates of 0.1–1 fps.
  • Demonstrated sub-cellular resolution imaging of intracellular NADH autofluorescence.
  • Confirmed that spiral scanning does not affect the instrument response function (IRF).

Conclusions:

  • The developed 2P-FLIM microendoscope offers a powerful tool for in vivo cellular metabolism studies.
  • The system's compact size, high resolution, and fast imaging capabilities advance biomedical research.
  • This technology facilitates detailed investigation of cellular energetic states at sub-cellular levels.