title: Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples.
abstract: 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.
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Winter
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: York
authors.affiliation: Section on Neural Developmental Dynamics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Damian Dalle
authors.initials: DD
authors.lastname: Nogare
authors.affiliation: Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Ingaramo
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Christensen
authors.affiliation: Section on Neural Developmental Dynamics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Ajay
authors.initials: A
authors.lastname: Chitnis
authors.affiliation: Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: George H
authors.initials: GH
authors.lastname: Patterson
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Hari
authors.initials: H
authors.lastname: Shroff