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High speed structured illumination microscopy in optically thick samples.

Michael Shaw1, Lydia Zajiczek1, Kevin O'Holleran2

  • 1Analytical Science Division, National Physical Laboratory, Hampton Road, Teddington, Middlesex TW11 0LW, UK.

Methods (San Diego, Calif.)
|April 4, 2015
PubMed
Summary
This summary is machine-generated.

Structured illumination microscopy (SIM) achieves higher resolution but requires many images. New techniques enable fast super-resolution imaging from a single focal plane, even in thick specimens.

Keywords:
Fluorescence microscopyOptical sectioningStructured illumination microscopySuper-resolution microscopy

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Super-resolution Imaging

Background:

  • Structured illumination microscopy (SIM) doubles optical resolution but necessitates extensive image capture, increasing light exposure and limiting dynamic process observation.
  • Current SIM methods struggle with optically thick specimens and capturing rapid biological events due to lengthy acquisition times.

Purpose of the Study:

  • To develop and validate novel image acquisition and reconstruction techniques for fast super-resolution imaging.
  • To enable high-speed SIM imaging within optically thick biological samples.
  • To reduce light exposure and improve the ability to image dynamic processes using SIM.

Main Methods:

  • Exploiting overlaps between SIM information passbands to reconstruct images from a single focal plane.
  • Implementing 2D and 3D SIM illumination patterns for single-plane reconstruction.
  • Combining single-plane reconstruction algorithms with high-speed hardware for illumination pattern switching and fluorescence image acquisition.

Main Results:

  • Demonstrated optically sectioned, super-resolution images from a single focal plane acquisition.
  • Successfully imaged dynamic processes within biological organisms at high speeds.
  • Compared reconstruction methods using 2D and 3D SIM illumination patterns.

Conclusions:

  • Developed a technique for fast super-resolution imaging in optically thick specimens.
  • Enabled high-speed SIM imaging of dynamic biological processes by reducing image acquisition requirements.
  • The new method significantly enhances the applicability of SIM for in vivo imaging.