Dual objective fluorescence microscopy for single molecule imaging applications.

Abstract

Fluorescence microscopy is an invaluable tool for studying biological processes in cells. In the recent past there has been significant interest in imaging cellular processes at the single molecule level. Single molecule experiments remove ensemble averaging effects and provide information that is typically not accessible through bulk experiments. One of the major requirements in single molecule imaging applications is that a sufficient number of photons be detected from the single molecule. This is not only important for the visual identification of single molecules, but also plays a crucial role in the quantitative analysis of the acquired data. Here, we demonstrate the use of a dual objective imaging configuration for single molecule studies. The configuration uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position. The use of opposing objective lenses has been previously demonstrated in 4pi confocal microscopy and I5M to achieve high axial resolution when compared to confocal/widefield microscopes. Here we demonstrate that the dual objective imaging configuration provides higher photon collection efficiency when compared to a regular microscope for a given illumination condition. As a result, single molecules can be localized with better accuracy when imaged through opposing objective lenses than when imaged through a regular optical microscope. Analytical tools are introduced to estimate the 2D location of single molecules and to characterize the accuracy with which they can be determined.