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A Step-by-Step Guide to Instant Structured Illumination Microscopy (iSIM).

Alexander Zhovmer1, Christian A Combs2

  • 1Laboratory of Molecular Cardiology, National Institutes of Health, Bethesda, MD, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 24, 2021
PubMed
Summary

Instant structured illumination microscopy (iSIM) provides fast, high-resolution 3D multicolor fluorescence imaging. This guide details iSIM methods, from sample preparation to post-processing, for accessible super-resolution microscopy.

Keywords:
FluorescenceInstant structured illumination microscopyReviewSuper-resolutioniSIM

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Cell Biology

Background:

  • Super-resolution microscopy techniques are crucial for visualizing cellular structures beyond the diffraction limit.
  • Instant structured illumination microscopy (iSIM) offers a rapid approach to achieving high-resolution 3D multicolor fluorescence imaging.

Purpose of the Study:

  • To provide a comprehensive guide to the theory, hardware, and practical application of iSIM.
  • To enable researchers to perform multicolor 3D iSIM imaging and subsequent data processing.
  • To offer insights into limitations, quality control, and alternative methods for iSIM.

Main Methods:

  • Description of the theoretical principles underlying iSIM.
  • Step-by-step protocols for preparing cellular fluorescent standards.
  • Guidance on acquiring multicolor 3D images using iSIM and post-processing techniques (de-striping, deconvolution) with free software.

Main Results:

  • Demonstration of iSIM's capability for rapid, multicolor 3D fluorescence imaging.
  • Achieving resolution close to twice the diffraction limit.
  • Successful application of post-processing techniques for enhanced image quality.

Conclusions:

  • iSIM is a powerful and accessible technique for high-resolution 3D multicolor fluorescence imaging.
  • The provided protocols and guidance facilitate the implementation and use of iSIM in various research settings.
  • Free software and detailed considerations enhance the accessibility and reliability of iSIM-based research.