Improved single particle localization accuracy with dual objective multifocal plane microscopy
Improved single particle localization accuracy with dual objective multifocal plane microscopy
In single particle imaging applications, the number of photons detected from the fluorescent label plays a crucial role in the quantitative analysis of the acquired data. For example, in tracking experiments the localization accuracy of the labeled entity can be improved by collecting more photons from the labeled entity. Here, we report the development of dual objective multifocal plane microscopy (dMUM) for single particle studies. The new microscope configuration uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position. We show that dMUM has a higher photon collection efficiency when compared to standard microscopes. We demonstrate that fluorescent labels can be localized with better accuracy in 2D and 3D when imaged through dMUM than when imaged through a standard microscope. Analytical tools are introduced to estimate the nanoprobe location from dMUM images and to characterize the accuracy with which they can be determined.
6881-6898
Ram, Pad Sri
3d8ad969-58f2-4e76-9490-9aa4443105f9
Prabhat, Prashant
e79cffdb-4de8-42cc-b0f7-6d28f6d3c82e
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36
13 April 2009
Ram, Pad Sri
3d8ad969-58f2-4e76-9490-9aa4443105f9
Prabhat, Prashant
e79cffdb-4de8-42cc-b0f7-6d28f6d3c82e
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36
Ram, Pad Sri, Prabhat, Prashant, Ward, E. Sally and Ober, Raimund J.
(2009)
Improved single particle localization accuracy with dual objective multifocal plane microscopy.
Optics Express, 17 (8), .
(doi:10.1364/OE.17.006881).
Abstract
In single particle imaging applications, the number of photons detected from the fluorescent label plays a crucial role in the quantitative analysis of the acquired data. For example, in tracking experiments the localization accuracy of the labeled entity can be improved by collecting more photons from the labeled entity. Here, we report the development of dual objective multifocal plane microscopy (dMUM) for single particle studies. The new microscope configuration uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position. We show that dMUM has a higher photon collection efficiency when compared to standard microscopes. We demonstrate that fluorescent labels can be localized with better accuracy in 2D and 3D when imaged through dMUM than when imaged through a standard microscope. Analytical tools are introduced to estimate the nanoprobe location from dMUM images and to characterize the accuracy with which they can be determined.
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Accepted/In Press date: 3 April 2009
e-pub ahead of print date: 10 April 2009
Published date: 13 April 2009
Identifiers
Local EPrints ID: 423603
URI: http://eprints.soton.ac.uk/id/eprint/423603
ISSN: 1094-4087
PURE UUID: 42218893-da2d-4d80-88e0-c7739d3ed7a0
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Date deposited: 27 Sep 2018 16:30
Last modified: 16 Mar 2024 04:37
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Author:
Pad Sri Ram
Author:
Prashant Prabhat
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