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Proceedings Paper

Overcoming the depth discrimination barrier in widefield microscopes: 3D single molecule tracking with high axial accuracy
Author(s): Sripad Ram; Jerry Chao; Prashant Prabhat; E. Sally Ward; Raimund J. Ober
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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.

Paper Details

Date Published: 21 February 2008
PDF: 7 pages
Proc. SPIE 6862, Single Molecule Spectroscopy and Imaging, 68620O (21 February 2008); doi: 10.1117/12.763097
Show Author Affiliations
Sripad Ram, Univ. of Texas Southwestern Medical Ctr. (United States)
Jerry Chao, Univ. of Texas Southwestern Medical Ctr. (United States)
Univ. of Texas at Dallas (United States)
Prashant Prabhat, Univ. of Texas Southwestern Medical Ctr. (United States)
Univ. of Texas at Dallas (United States)
E. Sally Ward, Univ. of Texas Southwestern Medical Ctr. (United States)
Raimund J. Ober, Univ. of Texas Southwestern Medical Ctr. (United States)
Univ. of Texas at Dallas (United States)

Published in SPIE Proceedings Vol. 6862:
Single Molecule Spectroscopy and Imaging
Jörg Enderlein; Zygmunt K. Gryczynski; Rainer Erdmann, Editor(s)

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