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Three-dimensional photoactivated localization microscopy with genetically expressed probes.

Kelsey Temprine1, Andrew G York, Hari Shroff

  • 1Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, 13 South Drive, Bethesda, MD, 20892, USA.

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Photoactivated localization microscopy (PALM) advances to 3D imaging of whole cells. New methods optimize instrumentation for dim photoactivatable fluorescent proteins (PA-FPs), enhancing super-resolution microscopy capabilities.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Single-molecule imaging techniques like Photoactivated Localization Microscopy (PALM) achieve high resolution (~20 nm lateral, ~50-100 nm axial).
  • Original PALM applications were limited to 2D imaging near surfaces, restricting analysis of whole cells.

Purpose of the Study:

  • To describe methods for converting 2D PALM into a 3D imaging system for whole fixed cells.
  • To emphasize instrumentation compatible with dim, genetically expressed photoactivatable fluorescent proteins (PA-FPs).

Main Methods:

  • Review of 2D PALM principles.
  • Detailed explanation of astigmatic and multiphoton imaging approaches for PA-FP compatibility.
  • Discussion of open-source software for 3D PALM data analysis.

Main Results:

  • Development of methods enabling 3D PALM imaging of entire fixed cells.
  • Demonstration of compatibility with dim photoactivatable fluorescent proteins (PA-FPs).
  • Guidance on selecting appropriate instrumentation and software for 3D PALM.

Conclusions:

  • 3D PALM imaging of whole cells is now feasible with optimized instrumentation.
  • The described methods facilitate super-resolution imaging using genetically encoded PA-FPs.
  • This advancement expands the application of PALM in biological research.