Abstract
We report a simple single-molecule fluorescence imaging method that increases the temporal resolution of any type of array detector by >5-fold with full field-of-view. We spread single-molecule spots to adjacent pixels by rotating a mirror in the detection path during the exposure time of a single frame, which encodes temporal information into the spatial domain. Our approach allowed us to monitor fast blinking of an organic dye, the dissociation kinetics of very short DNA and conformational changes of biomolecules with much improved temporal resolution than the conventional method. Our technique is useful when a large field-of-view is required, for example, in the case of weakly interacting biomolecules or cellular imaging.
Highlights
A camera-based detector has been widely used to study many biological systems with single-molecule fluorescence techniques because of its high sensitivity and high-throughput[1, 2]
Assuming that the binding rate is 106 M−1 s−1 and the accessible concentration of fluorophore-labeled biomolecules is 10 nM in TIRF microscopy, the rate of the complex formation amounts to ~10−2 s−1
During the exposure period (∆t = 20 ms), we swept the galvo mirror in N steps, which resulted in N spaced single-molecule spots produced on the image plane with higher temporal resolution (∆t/N = 4 ms) (Fig. 1b,c, Supplementary Fig. 2)
Summary
A camera-based detector has been widely used to study many biological systems with single-molecule fluorescence techniques because of its high sensitivity and high-throughput[1, 2]. Cropping the field-of-view (FOV) is the simplest way to improve temporal resolution, it decreases the number of molecules that can be observed simultaneously[3]. This is especially disadvantageous for studying weakly interacting biomolecules because the complex formation will occur very rarely[4]. During the exposure period (∆t = 20 ms), we swept the galvo mirror in N steps (for instance, N = 5), which resulted in N spaced single-molecule spots produced on the image plane with higher temporal resolution (∆t/N = 4 ms) (Fig. 1b,c, Supplementary Fig. 2). The beam splitter was used to obtain unmodified images either for comparison or for determination of an initial point of the event
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