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Construction of an instant structured illumination microscope.

Alistair Curd1, Alexa Cleasby1, Katarzyna Makowska1

  • 1School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.

Methods (San Diego, Calif.)
|July 27, 2015
PubMed
Summary

We provide instructions for building an instant structured illumination microscope (iSIM) for high-speed, super-resolution live-cell imaging. This system achieves over 100 frames per second with twofold resolution improvement.

Keywords:
ConstructionFluorescence microscopyInstant structured illumination microscopeSuper-resolution

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Area of Science:

  • Optical microscopy
  • Biophysics
  • Live-cell imaging

Background:

  • Capturing high-resolution images at fast frame rates in live cells presents a significant challenge in biological imaging.
  • Standard structured illumination microscopy (SIM) offers improved resolution but can be limited in acquisition speed.
  • The instant structured illumination microscope (iSIM) aims to overcome these limitations.

Purpose of the Study:

  • To provide detailed instructions for constructing an iSIM system.
  • To report modifications to existing iSIM hardware and software for improved performance.
  • To enable rapid, super-resolution imaging of live biological samples.

Main Methods:

  • Assembly of an iSIM involving precise alignment of micro-optics arrays and scanning mirrors.
  • Integration and control of multiple electronic components for system operation.
  • Implementation of hardware and software modifications to a previous iSIM design.

Main Results:

  • Successful construction and operation of a functional iSIM.
  • Achieved fluorescence image acquisition rates exceeding 100 frames per second.
  • Demonstrated a twofold improvement in resolution (lateral: 145 nm, axial: 320 nm) using a 1.49 NA objective and 488 nm excitation.

Conclusions:

  • The developed iSIM facilitates rapid, super-resolution live-cell imaging.
  • The provided instructions and modifications lower the barrier for iSIM construction.
  • This system is highly desirable for dynamic biological processes requiring high spatial and temporal resolution.