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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

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OpenSTED: open-source dynamic intensity minimum system for stimulated emission depletion microscopy.

Stephanie A Pierce1, Jordan Jacobelli2,3, Katherine S Given4

  • 1University of Colorado Anschutz Medical Campus, Department of Bioengineering, Aurora, Colorado, United States.

Neurophotonics
|June 13, 2024
PubMed
Summary

We developed an open-source Dynamic Intensity Minimum (DyMIN) system to reduce photobleaching in Stimulated Emission Depletion (STED) microscopy. This cost-effective add-on enables high-resolution, long-term live-cell imaging with improved signal-to-noise ratios.

Keywords:
dynamic minimumfluorescencemicroscopystimulated emission depletionsuper-resolution

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Area of Science:

  • Microscopy and Imaging Technologies
  • Biophysics
  • Cell Biology

Background:

  • Stimulated Emission Depletion (STED) microscopy offers super-resolution imaging but is limited by high laser power, causing photobleaching and sample damage.
  • Existing STED techniques struggle with sensitive biological samples, restricting long-term live imaging and high-resolution data acquisition.
  • A commercially available stand-alone module for Dynamic Intensity Minimum (DyMIN) to mitigate these issues is currently unavailable.

Purpose of the Study:

  • To develop and demonstrate an open-source Dynamic Intensity Minimum (DyMIN) system for reducing photobleaching in STED microscopy.
  • To enable enhanced time-lapse imaging of live biological samples with improved resolution and reduced photodamage.
  • To provide an accessible and cost-effective solution for researchers using STED microscopy.

Main Methods:

  • An open-source, three-step DyMIN system was designed and implemented on a STED microscope.
  • The system utilizes a fast multiplexer circuit and an inexpensive field-programmable gate array controlled by Labview software.
  • Software and circuit diagrams were made freely available as a stand-alone module for STED microscopes.

Main Results:

  • The custom DyMIN system demonstrated significantly reduced photobleaching in time-lapse imaging of beads, cells, and tissue samples.
  • A higher signal was recorded for bead samples using DyMIN compared to conventional STED after a 50-image sequence.
  • The system proved effective for time-lapse STED imaging of live cells and brain tissue slices, showcasing its versatility.

Conclusions:

  • The developed open-source DyMIN system is an inexpensive add-on that effectively reduces photobleaching in conventional STED microscopes.
  • This system significantly improves signal-to-noise ratios, facilitating dynamic time-lapse STED imaging of live samples.
  • The accessibility of the design empowers researchers to enhance their STED imaging capabilities without commercial limitations.