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Simultaneous imaging of several focal planes in fluorescence microscopy for the study of cellular dynamics in 3D

Simultaneous imaging of several focal planes in fluorescence microscopy for the study of cellular dynamics in 3D
Simultaneous imaging of several focal planes in fluorescence microscopy for the study of cellular dynamics in 3D

Fluorescence microscopy of live cells is an important tool to investigate cellular trafficking pathways. The existing microscope design is very well suited to image fast moving vesicles, tubules and organelles in one focal plane. More problematic is the imaging of cellular components that move between different focal planes. This is due to the fact that tracking of such cellular components requires that the focal plane of the microscope be changed. This has to be done with a focusing device, which is relatively slow. More importantly, only one focal plane can be imaged at a time. Therefore, while the cell is imaged at one focal plane, important events could be missed at other focal planes. To overcome these shortcomings, we present a modification of the classical microscope design with which two or more focal planes can be imaged simultaneously. In this design, the emission light collected by a single stationary objective lens is split into multiple channels. Light in each channel is focused on a CCD camera by a tube lens. By ensuring that the camera position with respect to the tube lens focal plane position is not the same in any two channels, distinct planes within the specimen can be simultaneously imaged. Here we discuss the implementation of a configuration with which four focal planes can be imaged simultaneously.

Fluorescence microscopy, Multi-focal plane imaging, Three-dimensional (3-D) tracking
SPIE
Prabhat, Prashant
e79cffdb-4de8-42cc-b0f7-6d28f6d3c82e
Ram, Sripad
559bd560-3817-4e53-8c7a-2f08e4518412
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36
Prabhat, Prashant
e79cffdb-4de8-42cc-b0f7-6d28f6d3c82e
Ram, Sripad
559bd560-3817-4e53-8c7a-2f08e4518412
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ober, Raimund J.
31f4d47f-fb49-44f5-8ff6-87fc4aff3d36

Prabhat, Prashant, Ram, Sripad, Ward, E. Sally and Ober, Raimund J. (2006) Simultaneous imaging of several focal planes in fluorescence microscopy for the study of cellular dynamics in 3D. In Proceedings of SPIE - The International Society for Optical Engineering. vol. 6090, SPIE.. (doi:10.1117/12.644343).

Record type: Conference or Workshop Item (Paper)

Abstract

Fluorescence microscopy of live cells is an important tool to investigate cellular trafficking pathways. The existing microscope design is very well suited to image fast moving vesicles, tubules and organelles in one focal plane. More problematic is the imaging of cellular components that move between different focal planes. This is due to the fact that tracking of such cellular components requires that the focal plane of the microscope be changed. This has to be done with a focusing device, which is relatively slow. More importantly, only one focal plane can be imaged at a time. Therefore, while the cell is imaged at one focal plane, important events could be missed at other focal planes. To overcome these shortcomings, we present a modification of the classical microscope design with which two or more focal planes can be imaged simultaneously. In this design, the emission light collected by a single stationary objective lens is split into multiple channels. Light in each channel is focused on a CCD camera by a tube lens. By ensuring that the camera position with respect to the tube lens focal plane position is not the same in any two channels, distinct planes within the specimen can be simultaneously imaged. Here we discuss the implementation of a configuration with which four focal planes can be imaged simultaneously.

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More information

Published date: 2006
Venue - Dates: Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII, , San Jose, CA, United States, 2006-01-24 - 2006-01-26
Keywords: Fluorescence microscopy, Multi-focal plane imaging, Three-dimensional (3-D) tracking

Identifiers

Local EPrints ID: 424098
URI: http://eprints.soton.ac.uk/id/eprint/424098
PURE UUID: 0059d3ba-b9b2-4c96-bfaf-dd32123721c7
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: 04 Oct 2018 16:30
Last modified: 16 Mar 2024 04:37

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Contributors

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

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