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Investigating the usage of point spread functions in point source and microsphere localization

Investigating the usage of point spread functions in point source and microsphere localization
Investigating the usage of point spread functions in point source and microsphere localization

Using a point spread function (PSF) to localize a point-like object, such as a fluorescent molecule or microsphere, represents a common task in single molecule microscopy image data analysis. The localization may differ in purpose depending on the application or experiment, but a unifying theme is the importance of being able to closely recover the true location of the point-like object with high accuracy. We present two simulation studies, both relating to the performance of object localization via the maximum likelihood fitting of a PSF to the object's image. In the first study, we investigate the integration of the PSF over an image pixel, which represents a critical part of the localization algorithm. Specifically, we explore how the fineness of the integration affects how well a point source can be localized, and find the use of too coarse a step size to produce location estimates that are far from the true location, especially when the images are acquired at relatively low magnifications. We also propose a method for selecting an appropriate step size. In the second study, we investigate the suitability of the common practice of using a PSF to localize a microsphere, despite the mismatch between the microsphere's image and the fitted PSF. Using criteria based on the standard errors of the mean and variance, we find the method suitable for microspheres up to 1 μm and 100 nm in diameter, when the localization is performed, respectively, with and without the simultaneous estimation of the width of the PSF.

Cramér-Rao lower bound, Fisher information, localization accuracy, maximum likelihood estimation, microsphere, pixel integration, point spread function, single molecule microscopy
SPIE
Chao, Jerry
550e20b0-8365-42e3-a6fc-1048eb8c2e47
Ram, Sripad
559bd560-3817-4e53-8c7a-2f08e4518412
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36
Chao, Jerry
550e20b0-8365-42e3-a6fc-1048eb8c2e47
Ram, Sripad
559bd560-3817-4e53-8c7a-2f08e4518412
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36

Chao, Jerry, Ram, Sripad, Ward, E. Sally and Ober, Raimund J. (2016) Investigating the usage of point spread functions in point source and microsphere localization. In Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIII. vol. 9713, SPIE.. (doi:10.1117/12.2208631).

Record type: Conference or Workshop Item (Paper)

Abstract

Using a point spread function (PSF) to localize a point-like object, such as a fluorescent molecule or microsphere, represents a common task in single molecule microscopy image data analysis. The localization may differ in purpose depending on the application or experiment, but a unifying theme is the importance of being able to closely recover the true location of the point-like object with high accuracy. We present two simulation studies, both relating to the performance of object localization via the maximum likelihood fitting of a PSF to the object's image. In the first study, we investigate the integration of the PSF over an image pixel, which represents a critical part of the localization algorithm. Specifically, we explore how the fineness of the integration affects how well a point source can be localized, and find the use of too coarse a step size to produce location estimates that are far from the true location, especially when the images are acquired at relatively low magnifications. We also propose a method for selecting an appropriate step size. In the second study, we investigate the suitability of the common practice of using a PSF to localize a microsphere, despite the mismatch between the microsphere's image and the fitted PSF. Using criteria based on the standard errors of the mean and variance, we find the method suitable for microspheres up to 1 μm and 100 nm in diameter, when the localization is performed, respectively, with and without the simultaneous estimation of the width of the PSF.

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More information

e-pub ahead of print date: 9 March 2016
Published date: 2016
Venue - Dates: Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIII, San Francisco, United States, 2016-02-15 - 2016-02-17
Keywords: Cramér-Rao lower bound, Fisher information, localization accuracy, maximum likelihood estimation, microsphere, pixel integration, point spread function, single molecule microscopy

Identifiers

Local EPrints ID: 423673
URI: https://eprints.soton.ac.uk/id/eprint/423673
PURE UUID: 274700da-ca40-4c75-a3d1-64a282ced84d
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: Sripad Ram
Author: E. Sally Ward ORCID iD
Author: Raimund J. Ober ORCID iD

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