Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Dispersion, aberration and deconvolution in multi-wavelength fluorescence images

B A Scalettar1, J R Swedlow, J W Sedat

  • 1Howard Hughes Medical Institute, University of California, San Francisco 94143-0448, USA.

Journal of Microscopy
|April 1, 1996
PubMed
Summary

Wide-field microscopy images with spherical aberration due to wavelength changes can be improved. Constrained deconvolution restores resolution and intensity, enabling accurate multi-component analysis in fluorescence microscopy.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Real-space heterogeneous reconstruction, refinement, and disentanglement of CryoEM conformational states with HetSIREN.

bioRxiv : the preprint server for biology·2024
Same author

Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology·2013
Same author

Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization.

Nature cell biology·2012
Same author

Interferometer-based structured-illumination microscopy utilizing complementary phase relationship through constructive and destructive image detection by two cameras.

Journal of microscopy·2012
Same author

Hindered submicron mobility and long-term storage of presynaptic dense-core granules revealed by single-particle tracking.

Developmental neurobiology·2011
Same author

Automated data collection for electron microscopic tomography.

Methods in enzymology·2010

Area of Science:

  • Microscopy
  • Optical Physics
  • Biophysics

Background:

  • Wavelength variation in fluorescence microscopy causes significant changes in the point spread function (PSF).
  • Optical dispersion in samples and microscope optics leads to wavelength-dependent spherical aberration.
  • This aberration complicates multi-wavelength imaging by degrading resolution and intensity.

Purpose of the Study:

  • To investigate the wavelength dependence of the incoherent point spread function (PSF) in wide-field microscopy.
  • To evaluate methods for correcting spherical aberration in multi-wavelength fluorescence images.
  • To enable accurate co-localization and intensity analysis of multiple components in biological samples.

Main Methods:

  • Experimental investigation of PSF wavelength dependence.

Related Experiment Videos

  • Application of constrained, iterative deconvolution to correct for spherical aberration.
  • Creation of a library of aberrated and unaberrated PSFs for deconvolution.

    • Main Results:

    • Spherical aberration in wide-field microscopy images is dependent on wavelength.
    • Constrained deconvolution effectively restores resolution and intensity in aberrated images.
    • The deconvolution method's success is independent of the initial degree of spherical aberration.

    Conclusions:

    • Wavelength-dependent spherical aberration is a significant challenge in multi-wavelength fluorescence microscopy.
    • Constrained iterative deconvolution is a robust method for correcting these aberrations.
    • Accurate co-localization and intensity measurements are achievable with this deconvolution technique.