Micro-stepping extended focus reduces photobleaching and preserves structured illumination super-resolution features

Xian Hu, Salma Jalal, Michael Sheetz, Oddmund Bakke, Felix Margadant

Research output: Contribution to journalArticlepeer-review


Despite progress made in confocal microscopy, even fast systems still have insufficient temporal resolution for detailed live-cell volume imaging, such as tracking rapid movement of membrane vesicles in three-dimensional space. Depending on the shortfall, this may result in undersampling and/or motion artifacts that ultimately limit the quality of the imaging data. By sacrificing detailed information in the Z-direction, we propose a new imaging modality that involves capturing fast 'projections' from the field of depth and shortens imaging time by approximately an order of magnitude as compared to standard volumetric confocal imaging. With faster imaging, radiation exposure to the sample is reduced, resulting in less fluorophore photobleaching and potential photodamage. The implementation minimally requires two synchronized control signals that drive a piezo stage and trigger the camera exposure. The device generating the signals has been tested on spinning disk confocal and instant structured-illumination-microscopy (iSIM) microscopes. Our calibration images show that the approach provides highly repeatable and stable imaging conditions that enable photometric measurements of the acquired data, in both standard live imaging and super-resolution modes.

Original languageEnglish (US)
Article number240796
JournalJournal of Cell Science
Issue number10
StatePublished - May 2020


  • Extended focus
  • Long-term live-cell imaging
  • Microscopy
  • Photobleaching
  • Rapid projections
  • Super-resolution
  • Well-defined sectioning

ASJC Scopus subject areas

  • Cell Biology


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