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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

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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Related Experiment Video

Updated: May 9, 2026

Whole-cell Super-Resolution Imaging via DNA-PAINT on a Spinning Disk Confocal with Optical Photon Reassignment
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Light-sheet confined super-resolution using two-photon photoactivation.

Francesca Cella Zanacchi1, Zeno Lavagnino, Mario Faretta

  • 1Department of Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy. francesca.cella@iit.it

Plos One
|July 12, 2013
PubMed
Summary

This study shows two-photon photoactivation improves deep-tissue super-resolution microscopy. This technique enhances imaging depth and reduces light damage in scattering biological samples.

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Last Updated: May 9, 2026

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Published on: December 9, 2013

Area of Science:

  • Biophysics
  • Optical Microscopy
  • Cell Biology

Background:

  • Light-sheet microscopy enables biological investigations of thick samples.
  • Super-resolution imaging of thick samples using individual molecule localization is challenging due to scattering effects limiting imaging depth and reducing image quality.

Purpose of the Study:

  • To investigate the advantages of non-linear photoactivation in a selective plane illumination configuration for imaging scattering samples.
  • To improve imaging depth and reduce light-sample interactions and photo-damage in super-resolution microscopy.

Main Methods:

  • Implementing two-photon photoactivation within a light-sheet illumination setup.
  • Coupling two-photon photoactivation with individual molecule localization methods (IML-SPIM).
  • Imaging nuclear pH2AX in NB4 cells to demonstrate the technique's efficacy.

Main Results:

  • Two-photon excitation significantly improves imaging capabilities in terms of imaging depth.
  • The method is expected to reduce light-sample interactions and minimize sample photo-damage.
  • Successful super-resolution imaging of nuclear pH2AX in NB4 cells was achieved at greater depths.

Conclusions:

  • Non-linear photoactivation, specifically two-photon excitation, enhances super-resolution microscopy in scattering biological samples.
  • This approach overcomes depth limitations and reduces photodamage, enabling deeper and clearer imaging.
  • The developed IML-SPIM technique with two-photon photoactivation is a valuable tool for advanced biological imaging.