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Single-molecule binding experiments on long time scales
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/content/aip/journal/rsi/81/8/10.1063/1.3473936
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Figures

Image of FIG. 1.

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FIG. 1.

Single-molecule binding. The top row shows a single-molecule image from a RNA aptamer experiment (scale bar equals 500 nm), below which is a schematic illustrating the binding and unbinding of the fluorescently labeled ligand. At the bottom is the intensity trajectory for the single-molecule image from which the on- and off-event durations can be determined.

Image of FIG. 2.

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FIG. 2.

Imaging setup. (a) Both the laser used for single-molecule imaging and the lamp used for bead imaging during autofocusing are shuttered. The shutters, piezoelectric objective drive, and camera are controlled by software. (b) Spectra are shown for the separate laser and lamp excitation filters, the dichroic mirror, and the dual band emission filter, as well as for a labeled ligand used during the RNA aptamer experiments (GTP-TMR: guanosine triphosphate labeled with tetramethylrhodamine), and the reference beads.

Image of FIG. 3.

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FIG. 3.

Autofocusing. (left) Autofocusing is performed by scanning through the -axis while illuminating the beads alone. (center) Convolution with a “Mexican hat” edge/contrast detection kernel is used to compute a focus metric for scoring the images. (right) The resulting curve is fit with a Gaussian which yields the focus.

Image of FIG. 4.

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FIG. 4.

Autofocusing metric vs bead number. The amplitudes of the Gaussians that were fitted to the focus scores during autofocus -scans (25 steps of ) are plotted with respect to the number of beads being used for focusing. Three to six beads are typically used for experiments.

Image of FIG. 5.

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FIG. 5.

Long run position mean square displacement (MSD). The MSD is calculated from the focused positions (inset graph) for an autofocusing run with a 15 sec interval and no pumping. Data have been fit to an equation modeling a diffusion-with-drift process: , where is the time, is the linear monotonic drift coefficient, is the diffusion coefficient, and the square root of the intercept yields the intrinsic focal resolution.

Image of FIG. 6.

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FIG. 6.

Lateral drift. The distance moved is based on the change in bead positions between images. Data shown are taken from a long run (the imaging interval is 15 s) without pumping to refresh solution.

Image of FIG. 7.

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FIG. 7.

Stretching of fluorophore lifetime. A fluorescently labeled DNA oligonucleotide (-biotin-GCG TAG ACT GAC TG-Cy3-) was immobilized at the surface and imaged to measure photobleaching. The rate of bleaching (a) is stretched out to (b) by imaging at intervals (2 min) using a shutter. The intensities shown have had the camera’s dark count subtracted (a) and (b), and been normalized to the laser power (b) only, as discussed in Sec. II F, to correct for long time scale laser fluctuation.

Image of FIG. 8.

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FIG. 8.

Loss of binding activity. The intensity trajectory for a RNA aptamer stops exhibiting binding despite the continued periodic addition of fresh solution. Such loss appears to be due to photodamage of the RNA.

Image of FIG. 9.

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FIG. 9.

Pumping solution from a chilled reservoir. A flow cell with a temperature probe incorporated into the channel near the input port was used to monitor temperature recovery when flowing in solution from an ice-cold reservoir. Tubing length was and the volume flowed in was 0.55 ml.

Image of FIG. 10.

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FIG. 10.

Nonspecific interaction with the surface. Data from 30 h of a run in which fresh labeled ligand (GTP-TMR) was pumped in every 3 h in the absence of the binding molecule (RNA aptamer). Data were smoothed with a moving median (window of 11 frames) to reduce noise.

Image of FIG. 11.

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FIG. 11.

Image intensity correlates with laser power. Laser power (lower trace, left axis) was monitored during a run. The mean intensity of a corner region of the images outside the illumination area (with the camera dark count subtracted) was calculated (upper trace, right axis). Over a ∼33 hour period, the correlation coefficient of the laser power and the image intensity was 0.98.

Image of FIG. 12.

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FIG. 12.

Long intensity trajectory from a RNA aptamer (class V M02) binding run. On and off events have been identified.

Image of FIG. 13.

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FIG. 13.

Event distributions and single-molecule kinetic space from a RNA aptamer (Class V M02) binding run. (a) On and off event distributions can be fit by single exponentials, corresponding to a single-rate binding process. (b) On and off event durations for each individual molecule can be averaged, allowing for a single-molecule plot of the kinetic space of the molecules in an experiment.

Image of FIG. 14.

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FIG. 14.

Event correlations. 2D histograms are shown for an aptamer (class V M02) binding run. On events (top row) and off events (bottom row) are binned to correlate the durations of every event with event (left column) or with event (right column).

Image of FIG. 15.

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FIG. 15.

Accessible kinetic space for the RNA aptamer experiments. (a) To achieve a desired threshold number of events (20 events) given a viable total run time (48 h) and empirically representative degradation factor (0.35), the total possible binding cycle time (based on the on and off event durations) of the molecules is limited (green bins indicate combinations which exceed the threshold). (b) In this limited “kinetic space,” only a small percentage of the possible values are therefore accessible.

Image of FIG. 16.

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FIG. 16.

Buildup of nonspecifically bound particles during load-wash cycles. Labeled ligand (30 nM GTP-TMR) is added in the absence of RNA aptamers to quantify interaction with the surface. This run was performed with ATP as one of a series of attempts to improve blocking.

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/content/aip/journal/rsi/81/8/10.1063/1.3473936
2010-08-27
2014-04-20

Abstract

We describe an approach for performing single-molecule binding experiments on time scales from hours to days, allowing for the observation of slower kinetics than have been previously investigated by single-molecule techniques. Total internal reflection fluorescence microscopy is used to image the binding of labeled ligand to molecules specifically coupled to the surface of an optically transparent flow cell. Long-duration experiments are enabled by ensuring sufficient positional, chemical, thermal, and imagestability. Principal components of this experimental stability include illumination timing, solution replacement, and chemical treatment of solution to reduce photodamage and photobleaching; and autofocusing to correct for spatial drift.

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Scitation: Single-molecule binding experiments on long time scales
http://aip.metastore.ingenta.com/content/aip/journal/rsi/81/8/10.1063/1.3473936
10.1063/1.3473936
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