Simultaneous Multicolor Single-Molecule Tracking with Single-Laser Excitation via Spectral Imaging

Abstract

Single-molecule tracking (SMT) offers rich information on the dynamics of underlying biological processes, but multicolor SMT has been challenging due to spectral cross talk and a need for multiple laser excitations. Here, we describe a single-molecule spectral imaging approach for live-cell tracking of multiple fluorescent species at once using a single-laser excitation. Fluorescence signals from all the molecules in the field of view are collected using a single objective and split between positional and spectral channels. Images of the same molecule in the two channels are then combined to determine both the location and the identity of the molecule. The single-objective configuration of our approach allows for flexible sample geometry and the use of a live-cell incubation chamber required for live-cell SMT. Despite a lower photon yield, we achieve excellent spatial (20-40 nm) and spectral (10-15 nm) resolutions comparable to those obtained with dual-objective, spectrally resolved Stochastic Optical Reconstruction Microscopy. Furthermore, motions of the fluorescent molecules did not cause loss of spectral resolution owing to the dual-channel spectral calibration. We demonstrate SMT in three (and potentially more) colors using spectrally proximal fluorophores and single-laser excitation, and show that trajectories of each species can be reliably extracted with minimal cross talk.

Figure 1

The single-molecule spectral imaging setup. (A) Optical scheme of the single-molecule spectral imaging system, constructed based on a standard, single-objective, single-molecule localization microscope is shown. To achieve simultaneous recording of the positions and emission spectra of all fluorescent molecules in the field of view, signals from the sample were split into two channels at ∼30:70 for the positional and spectral channels, respectively. The signals were then projected to the left and right sides of the detector after passing through the lenses L3S (spectral) and L3P (positional), respectively, and combining on a knife-edge mirror. In the spectral channel, a prism assembly consisting of an equilateral prism and a pair of mirrors was inserted in the infinity space between lenses L2 and L3S to disperse the signal. The prism assembly was mounted on a translational stage so that it could be moved in and out of the beam path, and the pair of mirrors after the prism was used to align the light path so that the overall direction of light propagation did not change in this channel. (B) A picture of the prism assembly in the microscope setup. The prism was mounted on its side so that the signals came in at the minimum dispersion angle and induced dispersion in the direction perpendicular to the closing direction (horizontal in this case) of the slit. To see this figure in color, go online.

Figure 2

Calibration of the single-molecule spectral imaging system. (A) Principles of spectral measurement on the single-molecule spectral imaging system are shown. The positional (empty circle) and spectral (filled circle) images were first aligned with high precision before the prism was inserted (top panel). With the prism inserted, the spectral images of all fluorescent objects became elongated, and the intensity profile of each elongated image represented the emission spectrum of the corresponding object (bottom panel). For each object in the overlaid image, its ssd was defined as the distance between the centroid of its positional image and the (sub-) pixel position of maximum emission intensity in the spectral image. In this case, objects two and three are the same type of fluorophore and they exhibit the same ssd (i.e., ssd2 = ssd3) values, whereas object one is in a different color exhibiting a different ssd value (ssd1); (B) Overlaid positional (pos) (red) and spectral (spec) (green) images of fluorescent beads (broad emission between 500 and 800 nm when excited at 488 or 561 nm) after passing through a series of narrow bandpass filters as indicated. The data point at ∼572 nm was taken with 488 nm excitation and the remaining data were taken with 561 nm excitation. The positional and spectral images were intentionally overlapped with subpixel precision at 661 ± 5.5 nm (i.e., ssd = 0.0). Thus, images acquired at shorter wavelengths had negative ssd with the spectral image to the left of the positional image, and the opposite in images taken at longer wavelengths (left). Shown on the right, histograms of ssd at each wavelength with the means and SD indicated. (C) The calibration curve showing the relationship between the center wavelength and the measured ssd values is shown. Inset is the SD of ssd at each wavelength. To see this figure in color, go online.

Figure 3

Multicolor single-molecule tracking with the single-molecule spectral imaging system in living cells. (A) The lower panel shows the average single-molecule spectra of CF633, CellMask DR, and CF680R molecules measured in live U2OS cells. The upper panel shows the mean (vertical line) and spread (horizontal line) of emission maxima for each fluorophore. (B) Series of single-molecule positional (pos) (red) and spectral (spec) (green) images of CF680R recorded at 20 ms per frame and shown in 40 ms (every two frames) intervals are shown. Here, the spectra have not been corrected by image registration; (C) Left: a representative raw image frame taken on a live U2OS cell simultaneously labeled with WGA-CF633, CellMask DR, and HT-CF680R, showing three distinct populations of molecules based on the separation between the positional (red) and spectral (green) images of the single molecules. Markers 1, 2, and 3 indicate CF633, CellMask DR, and CF680R molecules, respectively. Frame acquisition time was 20 ms. All three dyes were excited with the same 637 nm laser. Right: a histogram of ssd values from a three-color SMT experiment and the results of fitting with three Gaussian distributions, where the green curves represent individual fittings and the red line is the sum of all three fittings. Areas with orange shades indicate ranges of ssd values of which the associated localization events were kept and assigned to specific fluorophores. Other localization events were discarded. (D) Example diffusion trajectories of the three molecular species in a live U2OS cell, obtained simultaneously on the single-molecule spectral imaging system but separated into the three channels during sample processing are shown. Also shown on the left is an overview of a part of a cell, and on the right is a zoom-in view of the boxed region in the left plot. The scale bars represent 2 μm (C, left and D, left) and 500 nm (D, right), respectively. MSSMI, multispectral single-molecule imaging.

Figure 4

Single-molecule diffusion properties measured with single-molecule spectral imaging and analyzed with vbSPT (5). The diffusion state graphs obtained from single-color (top) and three-color (bottom) experiments are shown for WGA-CF633 (left), CellMask DR (middle), and HT-CF680R (right) as connected circles. Size of a circle indicates probabilities of the molecules residing in that specific state. Diffusion constants of state i are labeled as D_i (units: μm²/s), probability of molecules in state i is P_i, and the probability of transitioning from state i to state j within one frame is labeled as p_ij.