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Related Concept Videos

Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

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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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.

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Related Experiment Video

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A Multimodal Wide-Field Fourier-Transform Raman Microscope
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A white light confocal microscope for spectrally resolved multidimensional imaging.

J H Frank1, A D Elder, J Swartling

  • 1Combustion Research Facility, Sandia National Laboratories, Livermore, CA 94611, USA. jhfrank@sandia.gov

Journal of Microscopy
|September 1, 2007
PubMed
Summary
This summary is machine-generated.

This study presents a novel spectrofluorometric imaging microscope. It combines a supercontinuum laser and prism spectrometer for detailed spectral analysis of fluorescence in 3D confocal images, suitable for live cell imaging.

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Last Updated: Jul 12, 2026

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Published on: December 9, 2013

Area of Science:

  • Optical microscopy
  • Spectroscopy
  • Biophotonics

Background:

  • Confocal microscopy is a powerful tool for biological imaging.
  • Characterizing fluorophores requires spectrally resolved excitation and emission data.
  • Existing systems may have limitations in spectral range or tunability.

Purpose of the Study:

  • To develop and demonstrate a spectrofluorometric imaging confocal microscope.
  • To enable spectrally resolved fluorescence excitation and detection.
  • To provide comprehensive spectral information for each point in a 3D image.

Main Methods:

  • Utilized a supercontinuum laser as a broad-spectrum excitation source.
  • Integrated a custom-built prism spectrometer for spectrally resolved detection.
  • Employed an acousto-optic tunable filter for precise excitation wavelength selection (1-nm bandwidth).

Main Results:

  • Achieved spectrally resolved confocal imaging from 450 to 700 nm.
  • Demonstrated simultaneous selection of eight excitation wavelengths.
  • Obtained fluorescence excitation and emission spectra for each location in 3D images.
  • Showcased sensitivity comparable to standard confocal systems.
  • Confirmed suitability for live-cell imaging due to scan speed.

Conclusions:

  • The developed microscope system provides extensive fluorophore characterization capabilities.
  • It offers optimal access to a wide range of fluorophores.
  • Modest chromatic aberration effects do not significantly impact spatial resolution.