Resolution in 3D in multifocal plane microscopy

Chao, Jerry, Ram, Sripad, Abraham, Anish V., Ward, E. Sally and Ober, Raimund J. (2008) Resolution in 3D in multifocal plane microscopy. In Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XV. vol. 6861, SPIE.. (doi:10.1117/12.763084).

Abstract

Using single molecule microscopy, biological interactions can be imaged and studied at the level of individual biomolecules. When characterizing an imaged biological interaction, the distance separating the two participating biomolecules can provide valuable information. Therefore, the resolvability of an imaging setup is of practical significance in the analysis of the acquired image data. Importantly, the resolvability of the imaging setup needs evaluation in the 3D context, since in general biomolecules reside in 3D space within the cellular environment. We recently introduced an information-theoretic 2D resolution measure which shows that the resolution limit due to Rayleigh's criterion can be overcome. This new result predicts that the resolution of optical microscopes is not limited, but rather can be improved with increased photon counts detected from the single molecules. The 2D result was subsequently extended to the 3D context, and the proposed information-theoretic 3D resolution measure can readily be used to determine the resolvability of a conventional single focal plane imaging setup. Here, we consider the 3D resolution measure for a multifocal plane microscope setup, an imaging system which allows the concurrent imaging of multiple focal planes within a specimen. The technique is useful in applications such as the tracking of subcellular objects in 3D. By comparing their 3D resolution measures, we find a two-plane setup to outperform a comparable conventional single-plane setup in resolvability over a range of axial locations for the single molecule pair. Moreover, we investigate and compare the impact of noise on the resolvability of the two setups.

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Contributors

Author: E. Sally Ward

Author: Raimund J. Ober

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