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Structured illumination microscopy artefacts caused by illumination scattering.

Yanquan Mo1, Fan Feng2, Heng Mao3

  • 1State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, School of Future Technology, Peking University, Beijing 100871, People's Republic of China.

Philosophical Transactions. Series A, Mathematical, Physical, and Engineering Sciences
|April 26, 2021
PubMed
Summary

Structured illumination microscopy (SIM) for live-cell imaging is hindered by light scattering in thick samples. This study shows increased sample thickness worsens aberrations, degrading super-resolution image quality and guiding future artifact reduction algorithms.

Keywords:
aberrationillumination pathoptical scatteringstructured illumination microscopy

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Super-resolution Imaging

Background:

  • Structured illumination microscopy (SIM) is vital for live-cell super-resolution imaging.
  • Aberrations in SIM imaging are often caused by light scattering within samples, a factor rarely investigated.
  • Existing methods effectively minimize reconstruction parameter and noise-related artifacts.

Purpose of the Study:

  • To investigate how sample thickness and refractive index variations affect light scattering in SIM.
  • To understand the impact of scattered light on the quality of 2D-SIM super-resolution images.
  • To provide insights for developing algorithms to mitigate SIM artifacts in thick biological samples.

Main Methods:

  • Simulated subcellular structures with varying thicknesses and refractive indices.
  • Analyzed the scattering of excitation light along the optical path in SIM.
  • Evaluated the degradation of 2D-SIM reconstructed images as a function of axial focus changes.

Main Results:

  • Increased sample thickness exacerbates aberrant interference light.
  • Aberrations significantly degrade the quality of 2D-SIM super-resolution images.
  • Image reconstruction quality diminishes with axial focus shifts due to scattering.

Conclusions:

  • Sample thickness is a critical factor influencing SIM performance due to light scattering.
  • Scattering-induced aberrations pose a significant challenge for high-resolution imaging in thick specimens.
  • This research provides a foundation for developing advanced algorithms to correct SIM artifacts caused by light scattering in biological samples.