Overcoming the depth discrimination barrier in widefield microscopes: 3D single molecule tracking with high axial accuracy

Sripad Ram; Jerry Chao; Prashant Prabhat; E. Sally Ward; Raimund J. Ober

doi:10.1117/12.763097

21 February 2008 Overcoming the depth discrimination barrier in widefield microscopes: 3D single molecule tracking with high axial accuracy

Sripad Ram, Jerry Chao, Prashant Prabhat, E. Sally Ward, Raimund J. Ober

Author Affiliations +

Proceedings Volume 6862, Single Molecule Spectroscopy and Imaging; 68620O (2008) https://doi.org/10.1117/12.763097
Event: SPIE BiOS, 2008, San Jose, California, United States

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.

Citation Download Citation

Sripad Ram, Jerry Chao, Prashant Prabhat, E. Sally Ward, and Raimund J. Ober "Overcoming the depth discrimination barrier in widefield microscopes: 3D single molecule tracking with high axial accuracy", Proc. SPIE 6862, Single Molecule Spectroscopy and Imaging, 68620O (21 February 2008); https://doi.org/10.1117/12.763097

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available