Incoherent structured illumination improves optical sectioning and contrast in multiphoton super-resolution microscopy

Opt Express. 2015 Feb 23;23(4):5327-34. doi: 10.1364/OE.23.005327.

Abstract

Three-dimensional super-resolution imaging in thick, semi-transparent biological specimens is hindered by light scattering, which increases background and degrades both contrast and optical sectioning. We describe a simple method that mitigates these issues, improving image quality in our recently developed two-photon instant structured illumination microscope without requiring any hardware modifications to the instrument. By exciting the specimen with three laterally-structured, phase-shifted illumination patterns and post-processing the resulting images, we digitally remove both scattered and out-of-focus emissions that would otherwise contaminate our raw data. We demonstrate the improved performance of our approach in biological samples, including pollen grains, primary mouse aortic endothelial cells cultured in a three-dimensional collagen matrix and live tumor-like cell spheroids.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Endothelial Cells / cytology*
  • Equipment Design
  • Equipment Failure Analysis
  • Image Enhancement / instrumentation*
  • Image Enhancement / methods
  • Lighting / instrumentation*
  • Lighting / methods
  • Mice
  • Microscopy, Fluorescence, Multiphoton / instrumentation*
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Neoplasms, Experimental / pathology*
  • Reproducibility of Results
  • Sensitivity and Specificity