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High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells

High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells
High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells

Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in three dimensions in live cells. Here, we introduce an algorithm, the MUM localization algorithm (MUMLA), to determine the 3D position of a point source that is imaged using MUM. We validate MUMLA through simulated and experimental data and show that the 3D position of quantum dots can be determined over a wide spatial range. We demonstrate that MUMLA indeed provides the best possible accuracy with which the 3D position can be determined. Our analysis shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. Here, using MUM and MUMLA we report for the first time the full 3D trajectories of QD-labeled antibody molecules undergoing endocytosis in live cells from the plasma membrane to the sorting endosome deep inside the cell.

0006-3495
6025-6043
Ram, Sripad
559bd560-3817-4e53-8c7a-2f08e4518412
Prabhat, Prashant
e79cffdb-4de8-42cc-b0f7-6d28f6d3c82e
Chao, Jerry
550e20b0-8365-42e3-a6fc-1048eb8c2e47
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36
Ram, Sripad
559bd560-3817-4e53-8c7a-2f08e4518412
Prabhat, Prashant
e79cffdb-4de8-42cc-b0f7-6d28f6d3c82e
Chao, Jerry
550e20b0-8365-42e3-a6fc-1048eb8c2e47
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36

Ram, Sripad, Prabhat, Prashant, Chao, Jerry, Ward, E. Sally and Ober, Raimund J. (2008) High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells. Biophysical Journal, 95 (12), 6025-6043. (doi:10.1529/biophysj.108.140392).

Record type: Article

Abstract

Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in three dimensions in live cells. Here, we introduce an algorithm, the MUM localization algorithm (MUMLA), to determine the 3D position of a point source that is imaged using MUM. We validate MUMLA through simulated and experimental data and show that the 3D position of quantum dots can be determined over a wide spatial range. We demonstrate that MUMLA indeed provides the best possible accuracy with which the 3D position can be determined. Our analysis shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. Here, using MUM and MUMLA we report for the first time the full 3D trajectories of QD-labeled antibody molecules undergoing endocytosis in live cells from the plasma membrane to the sorting endosome deep inside the cell.

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More information

Accepted/In Press date: 5 September 2008
Published date: 15 December 2008

Identifiers

Local EPrints ID: 423595
URI: http://eprints.soton.ac.uk/id/eprint/423595
ISSN: 0006-3495
PURE UUID: ec26b15d-d8f5-4b15-8e03-0affbc4e7a75
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: Prashant Prabhat
Author: Jerry Chao
Author: E. Sally Ward ORCID iD
Author: Raimund J. Ober ORCID iD

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