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

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 d...

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Bibliographic Details
Published in:Optics express 2015-02, Vol.23 (4), p.5327-5334
Main Authors: Winter, Peter W, Chandris, Panagiotis, Fischer, Robert S, Wu, Yicong, Waterman, Clare M, Shroff, Hari
Format: Article
Language:English
Subjects:
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
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Description
Summary: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.
ISSN:1094-4087
1094-4087
DOI:10.1364/OE.23.005327