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Overcoming the depth discrimination barrier in widefield microscopes: 3D single molecule tracking with high axial accuracy

Overcoming the depth discrimination barrier in widefield microscopes: 3D single molecule tracking with high axial accuracy
Overcoming the depth discrimination barrier in widefield microscopes: 3D single molecule tracking with high axial accuracy

Current widefield microscopy techniques are well suited for imaging fast moving single molecules in two dimensions even within cells. However, the 3D imaging of single molecules poses several technical challenges. Foremost being that in the current microscope design only one focal plane can be imaged at any given point in time. Hence single molecule tracking in a 3D environment such as a cell is problematic since the molecule can easily move out of the focal plane that is currently being imaged. Focusing devices such as piezo nano-positioners could be used to overcome this shortcoming by sequentially scanning the sample at different planes. However, these devices are typically slow and therefore may not be suitable for 3D tracking of fast moving single molecules. Aside from this, widefield microscopes suffer from poor depth discrimination capability. Therefore, there exists significant uncertainty in determining the axial location of the single molecule, especially when the molecule is close to the plane of focus. To overcome the above limitations, we have developed a new microscopy technique called multifocal plane microscopy (MUM) that can simultaneously image distinct planes within the specimen. In contrast to standard microscopes, a MUM setup exhibits significantly improved depth discrimination capability, especially close to focus, which markedly improves the accuracy with which the axial position of the single molecule can be determined. Results are presented to illustrate the applicability of MUM for 3D single molecule tracking.

3D single molecule tracking, Fisher information matrix, Localization accuracy, Multifocal plane microscopy
SPIE
Ram, Sripad
559bd560-3817-4e53-8c7a-2f08e4518412
Chao, Jerry
550e20b0-8365-42e3-a6fc-1048eb8c2e47
Prabhat, Prashant
e79cffdb-4de8-42cc-b0f7-6d28f6d3c82e
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36
Ram, Sripad
559bd560-3817-4e53-8c7a-2f08e4518412
Chao, Jerry
550e20b0-8365-42e3-a6fc-1048eb8c2e47
Prabhat, Prashant
e79cffdb-4de8-42cc-b0f7-6d28f6d3c82e
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36

Ram, Sripad, Chao, Jerry, Prabhat, Prashant, Ward, E. Sally and Ober, Raimund J. (2008) Overcoming the depth discrimination barrier in widefield microscopes: 3D single molecule tracking with high axial accuracy. In Single Molecule Spectroscopy and Imaging. vol. 6862, SPIE.. (doi:10.1117/12.763097).

Record type: Conference or Workshop Item (Paper)

Abstract

Current widefield microscopy techniques are well suited for imaging fast moving single molecules in two dimensions even within cells. However, the 3D imaging of single molecules poses several technical challenges. Foremost being that in the current microscope design only one focal plane can be imaged at any given point in time. Hence single molecule tracking in a 3D environment such as a cell is problematic since the molecule can easily move out of the focal plane that is currently being imaged. Focusing devices such as piezo nano-positioners could be used to overcome this shortcoming by sequentially scanning the sample at different planes. However, these devices are typically slow and therefore may not be suitable for 3D tracking of fast moving single molecules. Aside from this, widefield microscopes suffer from poor depth discrimination capability. Therefore, there exists significant uncertainty in determining the axial location of the single molecule, especially when the molecule is close to the plane of focus. To overcome the above limitations, we have developed a new microscopy technique called multifocal plane microscopy (MUM) that can simultaneously image distinct planes within the specimen. In contrast to standard microscopes, a MUM setup exhibits significantly improved depth discrimination capability, especially close to focus, which markedly improves the accuracy with which the axial position of the single molecule can be determined. Results are presented to illustrate the applicability of MUM for 3D single molecule tracking.

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More information

e-pub ahead of print date: 21 February 2008
Published date: 2008
Venue - Dates: Single Molecule Spectroscopy and Imaging, , San Jose, CA, United States, 2008-01-19 - 2008-01-21
Keywords: 3D single molecule tracking, Fisher information matrix, Localization accuracy, Multifocal plane microscopy

Identifiers

Local EPrints ID: 423600
URI: http://eprints.soton.ac.uk/id/eprint/423600
PURE UUID: df0eb28a-8eff-4346-9b43-06d5a072b250
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: 16 Mar 2024 04:37

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Contributors

Author: Sripad Ram
Author: Jerry Chao
Author: Prashant Prabhat
Author: E. Sally Ward ORCID iD
Author: Raimund J. Ober ORCID iD

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