title: Incoherent structured illumination improves optical sectioning and contrast in multiphoton super-resolution microscopy.
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.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Winter
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Chandris
authors.forename: Robert S
authors.initials: RS
authors.lastname: Fischer
authors.forename: Yicong
authors.initials: Y
authors.lastname: Wu
authors.forename: Clare M
authors.initials: CM
authors.lastname: Waterman
authors.forename: Hari
authors.initials: H
authors.lastname: Shroff
mesh_terms: D000818:Animals; D042783:Endothelial Cells / Q000166:cytology*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation* / Q000379:methods; D008029:Lighting / Q000295:instrumentation* / Q000379:methods; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods*; D009374:Neoplasms, Experimental / Q000473:pathology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
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