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Remote focusing multifocal plane microscopy for the imaging of 3D single molecule dynamics with cellular context

Remote focusing multifocal plane microscopy for the imaging of 3D single molecule dynamics with cellular context
Remote focusing multifocal plane microscopy for the imaging of 3D single molecule dynamics with cellular context

Three-dimensional (3D) single molecule fluorescence microscopy affords the ability to investigate subcellular trafficking at the level of individual molecules. An imaged single molecule trajectory, however, often reveals only limited information about the underlying biological process when insufficient information is available about the organelles and other cellular structures with which the molecule interacts. A new 3D fluorescence microscopy imaging modality is described here that enables the simultaneous imaging of the trajectories of fast-moving molecules and the associated cellular context. The new modality is called remote focusing multifocal plane microscopy (rMUM), as it extends multifocal plane microscopy (MUM) with a remote focusing module. MUM is a modality that uses multiple detectors to image distinct focal planes within the specimen at the same time, and it has been demonstrated to allow the determination of 3D single molecule trajectories with high accuracy. Remote focusing is a method that makes use of two additional objective lenses to enable the acquisition of a z-stack of the specimen without having to move the microscope's objective lens or sample stage, components which are required by MUM to be fixed in place. rMUM's remote focusing module thus allows the cellular context to be imaged in the form of z-stacks as the trajectories of molecules or other objects of interest are imaged by MUM. In addition to a description of the modality, a discussion of rMUM data analysis and an example of data acquired using an rMUM setup are provided in this paper.

Cellular context, fluorescence microscopy, multifocal plane microscopy, remote focusing, single molecule localization, single molecule microscopy, single molecule tracking, three-dimensional microscopy
SPIE
Chao, Jerry
550e20b0-8365-42e3-a6fc-1048eb8c2e47
Velmurugan, Ramraj
4f2d41cd-90eb-4f61-b5e0-df6308726d28
You, Sungyong
06e1ba5a-2d3a-4267-9bfd-ac5abeff555b
Kim, Dongyoung
43a73b5c-3f2e-4ba9-8ffa-a7b454e2fd9f
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36
Chao, Jerry
550e20b0-8365-42e3-a6fc-1048eb8c2e47
Velmurugan, Ramraj
4f2d41cd-90eb-4f61-b5e0-df6308726d28
You, Sungyong
06e1ba5a-2d3a-4267-9bfd-ac5abeff555b
Kim, Dongyoung
43a73b5c-3f2e-4ba9-8ffa-a7b454e2fd9f
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36

Chao, Jerry, Velmurugan, Ramraj, You, Sungyong, Kim, Dongyoung, Ward, E. Sally and Ober, Raimund J. (2017) Remote focusing multifocal plane microscopy for the imaging of 3D single molecule dynamics with cellular context. In Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIV. vol. 10070, SPIE.. (doi:10.1117/12.2251218).

Record type: Conference or Workshop Item (Paper)

Abstract

Three-dimensional (3D) single molecule fluorescence microscopy affords the ability to investigate subcellular trafficking at the level of individual molecules. An imaged single molecule trajectory, however, often reveals only limited information about the underlying biological process when insufficient information is available about the organelles and other cellular structures with which the molecule interacts. A new 3D fluorescence microscopy imaging modality is described here that enables the simultaneous imaging of the trajectories of fast-moving molecules and the associated cellular context. The new modality is called remote focusing multifocal plane microscopy (rMUM), as it extends multifocal plane microscopy (MUM) with a remote focusing module. MUM is a modality that uses multiple detectors to image distinct focal planes within the specimen at the same time, and it has been demonstrated to allow the determination of 3D single molecule trajectories with high accuracy. Remote focusing is a method that makes use of two additional objective lenses to enable the acquisition of a z-stack of the specimen without having to move the microscope's objective lens or sample stage, components which are required by MUM to be fixed in place. rMUM's remote focusing module thus allows the cellular context to be imaged in the form of z-stacks as the trajectories of molecules or other objects of interest are imaged by MUM. In addition to a description of the modality, a discussion of rMUM data analysis and an example of data acquired using an rMUM setup are provided in this paper.

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More information

Published date: 17 February 2017
Venue - Dates: Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIV 2017, San Francisco, United States, 2017-01-30 - 2017-02-01
Keywords: Cellular context, fluorescence microscopy, multifocal plane microscopy, remote focusing, single molecule localization, single molecule microscopy, single molecule tracking, three-dimensional microscopy

Identifiers

Local EPrints ID: 423674
URI: https://eprints.soton.ac.uk/id/eprint/423674
PURE UUID: 12066ea7-5f9f-4674-9e25-eebcbb7d6080
ORCID for E. Sally Ward: ORCID iD orcid.org/0000-0003-3232-7238
ORCID for Raimund J. Ober: ORCID iD orcid.org/0000-0002-1290-7430

Catalogue record

Date deposited: 27 Sep 2018 16:30
Last modified: 14 Mar 2019 01:21

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Contributors

Author: Jerry Chao
Author: Ramraj Velmurugan
Author: Sungyong You
Author: Dongyoung Kim
Author: E. Sally Ward ORCID iD
Author: Raimund J. Ober ORCID iD

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