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Related Concept Videos

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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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...
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Related Experiment Video

Updated: Feb 22, 2026

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
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Fully Automated Targeted Confocal and Single-Molecule Localization Microscopy.

Jan Philipp Eberle1, Walter Muranyi2, Holger Erfle3

  • 1High-Content Analysis of the Cell (HiCell) and Advanced Biological Screening Facility, BioQuant, Heidelberg University, Heidelberg, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|September 20, 2017
PubMed
Summary

This study introduces an automated system for high-resolution single-molecule localization microscopy (SMLM). The new setup significantly reduces measurement times, making nanoscale imaging of biological structures statistically robust.

Keywords:
High content screening (HCS)Konstanz Information Miner (KNIME)Multiscale imagingOnline feedback image analysisSingle-molecule localization microscopy (SMLM)Super-resolution microscopy

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Single-molecule localization microscopy (SMLM) offers nanoscale imaging of biological structures.
  • SMLM typically requires long acquisition times due to the large number of images needed.

Purpose of the Study:

  • To develop an automated workflow for targeted SMLM imaging.
  • To overcome the limitations of long measurement times in SMLM.

Main Methods:

  • An automated setup combining confocal microscopy for target detection and SMLM for high-resolution imaging was constructed.
  • The Konstanz Information Miner (KNIME) platform was utilized with custom plugins to integrate microscope components.
  • A workflow was developed to automate the screening and imaging process.

Main Results:

  • The automated system successfully detected and imaged nanometre-sized biological structures.
  • The workflow enabled statistically significant data acquisition, overcoming previous time constraints.
  • Proof-of-principle imaging of HIV-1 assembly complexes in HeLa cells was achieved through automated screening.

Conclusions:

  • The developed automated SMLM system enhances efficiency and feasibility for nanoscale biological imaging.
  • This approach facilitates statistically relevant studies of biological nanostructures.
  • Automated targeted screening with SMLM is a powerful tool for biological research.