Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time

Abstract

Here we present a technique called single-molecule high-resolution colocalization (SHREC) of fluorescent dyes that allows the measurement of interfluorophore distances in macromolecules and macromolecular complexes with better than 10-nm resolution. By using two chromatically differing fluorescent molecules as probes, we are able to circumvent the Rayleigh criterion and measure distances much smaller than 250 nm. The probes are imaged separately and localized individually with high precision. The registration between the two imaging channels is measured by using fiduciary markers, and the centers of the two probes are mapped onto the same space. Multiple measurements can be made before the fluorophores photobleach, allowing intramolecular and intermolecular distances to be tracked through time. This technique's lower resolution limit lies at the upper resolution limit of single molecule FRET (smFRET) microscopy. The instrumentation and fluorophores used for SHREC can also be used for smFRET, allowing the two types of measurements to be made interchangeably, covering a wide range of interfluorophore distances. A dual-labeled duplex DNA molecule (30 bp) was used as a 10-nm molecular ruler to confirm the validity of the method. We also used SHREC to study the motion of myosin V. We directly observed myosin V's alternating heads while it walked hand-over-hand along an actin filament.

Fig. 1.

Precise alignment of the two imaging channels to within 3.3 nm. Fiducial grids with 0.5-μm spacing were made by translating a fluorescent bead detectable in both channels by a piezo stage. At each point in the grid, an image of the bead was taken. (a) The resulting stack of images was projected through z to observe the positions that the calibration series visited. (b) The center locations of the beads were found by means of a fit to a 2D Gaussian function. (c) The subsequent pairs of locations were used to calculate a transformation mapping of the Cy5 channel onto the Cy3 channel.

Fig. 4.

Time trace of a differentially labeled myosin V molecule walking along an actin filament. The labels (Cy3 and Cy5) are covalently attached to calmodulins that were exchanged onto the myosin V molecule. In this trace, both of the fluorescent probe's locations are taking 72-nm steps, indicating that the calmodulins were exchanged close to the motor domain. The alternating positions of the probes provide a direct observation of myosin V's hand-over-hand walking mechanism.

Fig. 2.

Determining the accurate end-to-end distance from the skewed distribution of distance measurements. (a) The distance probability distribution was calculated by means of Monte Carlo simulations. (b) The skew in the histograms toward large values can be understood from a geometric argument. If one end of the DNA molecule is measured to reside at the origin, then only points lying on the circumference of a circle with radius s and origin (0, 0) will yield the true end-to-end distance. It is more likely that a point will lie outside of the dashed-line semicircle than inside it, which gives the distance distribution a long tail. (c) Despite the non-Gaussian nature of the distance distribution, the end-to-end separation can be calculated using the geometric mean, variance, and localization errors.

Fig. 3.

End-to-end distances of 482 duplex DNA molecules (30 bp) as measured by SHREC.