Open AccessTwo-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples
Author(s) -
Peter W. Winter,
Andrew G. York,
Damian Dalle Nogare,
Maria Ingaramo,
Ryan Christensen,
Ajay Chitnis,
George H. Patterson,
Hari Shroff
Publication year - 2014
Publication title -
optica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.074
H-Index - 107
ISSN - 2334-2536
DOI - 10.1364/optica.1.000181
Subject(s) - resolution (logic) , optics , microscopy , light sheet fluorescence microscopy , optical sectioning , microscope , penetration depth , materials science , image resolution , two photon excitation microscopy , biological specimen , physics , fluorescence , scanning confocal electron microscopy , computer science , artificial intelligence
Fluorescence imaging methods that achieve spatial resolution beyond the diffraction limit (super-resolution) are of great interest in biology. We describe a super-resolution method that combines two-photon excitation with structured illumination microscopy (SIM), enabling three-dimensional interrogation of live organisms with ~150 nm lateral and ~400 nm axial resolution, at frame rates of ~1 Hz. By performing optical rather than digital processing operations to improve resolution, our microscope permits super-resolution imaging with no additional cost in acquisition time or phototoxicity relative to the point-scanning two-photon microscope upon which it is based. Our method provides better depth penetration and inherent optical sectioning than all previously reported super-resolution SIM implementations, enabling super-resolution imaging at depths exceeding 100 μm from the coverslip surface. The capability of our system for interrogating thick live specimens at high resolution is demonstrated by imaging whole nematode embryos and larvae, and tissues and organs inside zebrafish embryos.