Abstract
The ability to track single fluorescent particles in three-dimensions with sub-diffraction limit precision as well as sub-millisecond temporal resolution has enabled the understanding of many biophysical phenomena at the nanometer scale. While there are several techniques for achieving this, most require complicated experimental setups that are expensive to implement. These methods can offer superb performance but their complexity may be overwhelming to the end-user whose aim is only to understand the feature being imaged. In this work, we describe a method for tracking a single fluorescent particle using a standard confocal or multi-photon microscope configuration. It relies only on the assumption that the relative position of the measurement point and the particle can be actuated and that the point spread function has a global maximum that coincides with the particle's position. The method uses intensity feedback to calculate real-time position commands that "seek" the extremum of the point spread function as the particle moves through its environment. We demonstrate the method by tracking a diffusing quantum dot in a hydrogel on a standard epifluorescent confocal microscope.
Highlights
Due to the invention of specialized instruments with nanometer-scale resolution, such as the atomic force microscope and the scanning electron microscope, our knowledge of biological features at the nanometer scale has greatly improved in recent times
Because of the manner by which they acquire their images their temporal resolutions are far slower than inherently parallel instruments, such as widefield light microscopes
By labelling an individual leg of a myosin V motor with a fluorophore and observing the fluorophore’s emission as the motor traversed an actin filament, both qualitative and quantitative information regarding its walking behavior were derived [2]. The effectiveness of this method was demonstrated in the context of tracking phospholipids in rat kidney fibroblasts [3] and of the influenza virus during infection [4] while its utility and impact is illustrated by several recent review articles [5,6,7]
Summary
Due to the invention of specialized instruments with nanometer-scale resolution, such as the atomic force microscope and the scanning electron microscope, our knowledge of biological features at the nanometer scale has greatly improved in recent times. Most active methods localize the particle in real-time and use the resulting estimated position in a feedback algorithm that reduces the tracking error These methods are typically implemented using confocal or two-photon imaging modalities and can be broadly categorized as either orbital, in which the excitation path is modified to allow for rapid scanning of the excitation volume along a given a given closed path (typically circular), or multi-point, in which the detection path is modified to incorporate multiple detectors at different points and different planes.
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