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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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 developed.
Confocal Fluorescence Microscopy01:16

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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
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Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy
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Pulsed interleaved excitation fluorescence spectroscopy with a supercontinuum source.

Linnea Olofsson1, Emmanuel Margeat

  • 1CNRS UMR5048, Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier, France.

Optics Express
|March 14, 2013
PubMed
Summary

We developed a new supercontinuum laser setup for Pulsed Interleaved Excitation (PIE) microscopy. This versatile system enhances fluorescence cross-correlation spectroscopy (FCCS) and single pair Förster Resonance Energy Transfer (spFRET) measurements.

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Published on: August 22, 2019

Area of Science:

  • Optics and Photonics
  • Biophysical Techniques
  • Laser Technology

Background:

  • Pulsed Interleaved Excitation (PIE) enhances fluorescence spectroscopy by correlating photons to excitation sources.
  • Traditional PIE methods often require multiple picosecond lasers and complex drivers.
  • Time-correlated single photon counting (TCSPC) is crucial for PIE-based measurements.

Purpose of the Study:

  • To present a novel, simplified optical configuration for generating multicolor interleaved picosecond pulses.
  • To offer a versatile alternative to conventional multi-laser setups for PIE experiments.
  • To enable advanced fluorescence measurements like FCCS and spFRET with improved efficiency.

Main Methods:

  • Utilized a commercial supercontinuum laser source.
  • Engineered an optical setup for generating arbitrary spacing and wavelengths of picosecond pulses.
  • Integrated the setup with time-correlated single photon counting (TCSPC) detection.

Main Results:

  • Successfully generated multicolor interleaved picosecond pulses across the visible spectrum.
  • Demonstrated the configuration's robustness and simplicity.
  • Showcased its applicability as a versatile source for PIE experiments.

Conclusions:

  • The presented supercontinuum laser configuration provides a practical and robust alternative for PIE.
  • This approach simplifies the generation of excitation pulses for advanced fluorescence spectroscopy.
  • It broadens the accessibility of PIE-enhanced FCCS and spFRET techniques.