Abstract
While fluorescence microscopes and atomic force microscopes are widely used to visualize, track, and manipulate single biomolecules, the resolution of these methods is limited by sample drift. To minimize drift, active feedback methods have recently been used to stabilize single molecule microscopes on the sub-nanometer scale. However, these methods require high intensity lasers which limits their application in single molecule fluorescence measurements. Furthermore, these feedback methods do not track user-defined regions of the sample, but rather monitor the relative displacement of an unknown point on a fiducial marker, which limits their use in biological force measurements. To overcome these limitations, we have developed a novel method to image, track and stabilize a sample using low laser intensities. We demonstrate the capabilities of our approach by tracking a user-chosen point on a fiducial marker at 8.6 kHz and stabilizing it with sub-nanometer resolution. We further showcase the application of our method in single molecule fluorescence microscopy by imaging and stabilizing individual fluorescently-tagged streptavidin proteins under biologically relevant conditions. We anticipate that our method can be easily used to improve the resolution of a wide range of single molecule fluorescence microscopy and integrated force-fluorescence applications.
Highlights
Imaging, tracking and manipulation of single biological molecules are widely used to obtain key mechanistic insights into biological processes
With the detector signal correlated to fiducial displacement, sample movement is monitored in real-time and the sample is stabilized by moving the microscope stage
While back-scatter based active stabilization achieves impressive angstrom-level stability over a significant bandwidth (0.1–10 Hz), it requires high laser intensities which quickly and irreversibly bleach fluorescent dyes. This precludes the use of this active-feedback method in single molecule fluorescence microscopy, which requires significantly lower laser intensities
Summary
Imaging, tracking and manipulation of single biological molecules are widely used to obtain key mechanistic insights into biological processes. Since cryogenic temperature measurements, which have traditionally been used to combat undesired drift[16,17], are not compatible with biological experiments, several active stabilization methods for microscopes have recently been developed[18,19,20,21,22,23] Of these techniques, back-scatter based active stabilization provides the high spatial and temporal resolution required to track molecular dynamics. While the mapping technique used in back-scatter based active stabilization provides high displacement resolution, it does not lend itself to pinpointing a specific feature on the fiducial When this method has been used to stabilize an AFM18, the AFM tip is not directly imaged, and as such the precise location of the tip apex is unknown. To showcase its application in single molecule fluorescence microscopy, we demonstrate this method by stabilizing individual fluorescently-tagged streptavidin proteins under biologically relevant conditions
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