Optically resolving individual microtubules in live axons

Abstract

Microtubules are essential cytoskeletal tracks for cargo transportation in axons and also serve as the primary structural scaffold of neurons. Structural assembly, stability, and dynamics of axonal microtubules are of great interest for understanding neuronal functions and pathologies. However, microtubules are so densely packed in axons that their separations are well below the diffraction limit of light, which precludes using optical microscopy for live-cell studies. Here, we present a single-molecule imaging method capable of resolving individual microtubules in live axons. In our method, unlabeled microtubules are revealed by following individual axonal cargos that travel along them. We resolved more than six microtubules in a 1 microm diameter axon by real-time tracking of endosomes containing quantum dots. Our live-cell study also provided direct evidence that endosomes switch between microtubules while traveling along axons, which has been proposed to be the primary means for axonal cargos to effectively navigate through the crowded axoplasmic environment.

Figure 1. Schematic Representations of Imaging Strategy and Platform

(A) An illustration of imaging scheme. Fluorescently labeled organelles that are traveling along microtubules in an axon appear as large diffraction-limited spheres. At each time point, the central positions of those organelles (containing a single fluorescent molecule) are determined to high accuracy by fitting with a 2D Gaussian function. As organelles travel along the axon, their time-lapse central positions trace out the microtubule tracks that they are moving along. Cumulative trajectories depict densely packed microtubules that are otherwise unresolvable using conventional optical imaging. (B) Platform for single molecule imaging of axonal transport. DRG neurons are cultured inside the microfluidic chamber that separates the chemical environment of the distal axons from that of the cell bodies. Fluorescent QD-NGF is applied only to the distal axons where it binds its membrane receptor and is subsequently endocytosed and retrogradely transported toward the cell body. Imaging is restricted to the microchannels or to the cell body chamber, so only internalized and transported QD-NGF molecules are observed using a pseudo-TIRF imaging setup. The angle of the incident laser beam is adjusted to be slightly less than the critical angle so the refracted beam penetrates ∼1 μm into the solution.

Figure 2. Localization Accuracy of Immobilized Quantum Dots

(A) The point-spread-function of a single QD-NGF molecule overlaid with the fitted 2D Gaussian function. The central position is determined to high accuracy after 2D Gaussian fitting. (B) Spread of center positions of an immobilized quantum dot over 321 frames. (C) Dependence of localization accuracy versus the brightness of the quantum dot. By excluding points below 5000 a.u., a centroid standard deviation of 11.5 nm was achieved. (D) The distribution of the standard deviations of 30 QD-NGF molecules imaged over 800 frames (80 s), with the mean at 11.5 nm.

Figure 3. Resolving Individual Axonal Microtubules

A and B show the x-y positional trajectories and the corresponding x-t time traces of 9 endosomes traveling in the same axon. These trajectories include dim points that have ∼70 nm FMHM resolution. Even though the trajectories are overlapped spatially, they are clearly separated in the time regime, allowing one to use the localization-based method to achieve superresolution. In (A) the red, blue, and magenta trajectories initially overlap and follow the same MT but are well separated temporally in (B). The red endosome makes a rapid lateral movement indicated by the black arrow and then proceeds along the same MT as the green endosome. Another switching event is marked for the blue trace. (C) High-resolution (∼27 nm) long-distance traces constructed using a 5000 a.u. brightness cutoff. Unlike EM studies that restrict their field-of-view to several hundred nanometers, our wide-field recording method traces out microtubule structure over several tens of microns. The insets c1, c2, and c3 show zoomed-in fine structures of the microtubules. In some parts of the axon, they are overlapped (the black and purple traces in c2), but in other areas they are clearly separated.

Figure 4. Capturing Microtubule Track-Switching Event During Axonal Transport

A and B display the x-y trajectory and the corresponding x-t time trace for an endosome that clearly exhibits microtubule track switching during axonal transport. The plots are colored by segments to denote a series of detailed events: initial pausing (black); directional retrograde transport (colored red); large lateral movement with continued retrograde transport (green); dissociation from the microtubule and Brownian diffusion in the cytosol (orange); association with a new microtubule and short anterograde transport (blue); a long pause (yellow) followed by retrograde transport along the new microtubule (gray) (see Movie S3).