Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution

Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluo...

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Published inNature methods Vol. 18; no. 6; pp. 688 - 693
Main Authors Hao, Xiang, Allgeyer, Edward S., Lee, Dong-Ryoung, Antonello, Jacopo, Watters, Katherine, Gerdes, Julianne A., Schroeder, Lena K., Bottanelli, Francesca, Zhao, Jiaxi, Kidd, Phylicia, Lessard, Mark D., Rothman, James E., Cooley, Lynn, Biederer, Thomas, Booth, Martin J., Bewersdorf, Joerg
Format Journal Article
LanguageEnglish
Published New York Nature Publishing Group US 01.06.2021
Nature Publishing Group
Subjects
631/1647/245/2226
631/1647/328/2238
631/80/2373
Adaptive optics
Bioinformatics
Biological Microscopy
Biological Techniques
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Depletion
Diffraction
Fluorescence
Fluorescence microscopy
Imaging
Imaging, Three-Dimensional
Innovations
Life Sciences
Microscopy, Fluorescence - methods
Nanotechnology
Nanotechnology - methods
Optics
Optics and Photonics - methods
Optimization
Proteomics
Signal-To-Noise Ratio
Spatial discrimination
Spatial resolution
Stimulated emission
Synapses
United States
United Kingdom
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Abstract Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens. The combination of adaptive optics with an improved isoSTED nanoscope allows imaging of cells and tissues with sub-50-nm isotropic resolution.
AbstractList Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens. The combination of adaptive optics with an improved isoSTED nanoscope allows imaging of cells and tissues with sub-50-nm isotropic resolution.
Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens. The combination of adaptive optics with an improved isoSTED nanoscope allows imaging of cells and tissues with sub-50-nm isotropic resolution.
Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.
Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.
Audience Academic
Author Gerdes, Julianne A.
Bewersdorf, Joerg
Bottanelli, Francesca
Kidd, Phylicia
Hao, Xiang
Biederer, Thomas
Rothman, James E.
Antonello, Jacopo
Lessard, Mark D.
Booth, Martin J.
Schroeder, Lena K.
Allgeyer, Edward S.
Lee, Dong-Ryoung
Zhao, Jiaxi
Cooley, Lynn
Watters, Katherine
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  organization: Department of Cell Biology, Yale School of Medicine, Nanobiology Institute, Yale University, Department of Biomedical Engineering, Yale University
BackLink https://www.ncbi.nlm.nih.gov/pubmed/34059828$$D View this record in MEDLINE/PubMed
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ContentType Journal Article
Copyright The Author(s), under exclusive licence to Springer Nature America, Inc. 2021
COPYRIGHT 2021 Nature Publishing Group
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Snippet Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi...
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pubmed
crossref
springer
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Enrichment Source
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SubjectTerms 631/1647/245/2226
631/1647/328/2238
631/80/2373
Adaptive optics
Bioinformatics
Biological Microscopy
Biological Techniques
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Depletion
Diffraction
Fluorescence
Fluorescence microscopy
Imaging
Imaging, Three-Dimensional
Innovations
Life Sciences
Microscopy, Fluorescence - methods
Nanotechnology
Nanotechnology - methods
Optics
Optics and Photonics - methods
Optimization
Proteomics
Signal-To-Noise Ratio
Spatial discrimination
Spatial resolution
Stimulated emission
Synapses
Title Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution
URI https://link.springer.com/article/10.1038/s41592-021-01149-9
https://www.ncbi.nlm.nih.gov/pubmed/34059828
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https://www.proquest.com/docview/2535833572
Volume 18
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