Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots

Abstract

Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery.

Inhibitory Receptor Diffusion Dynamics

“…Whilst there have been huge technological advancements over a relatively short period of time, there remains inherent limitations in the currently used techniques for analyzing lateral membrane diffusion. QDs are a popular choice due to their photostability, bright fluorescence, long trajectories and ability to multiplex (Cutler et al, 2013;Kakizuka et al, 2016;Renner et al, 2017). However, their large size complexed with antibodies can sterically hinder lateral mobility (Abraham et al, 2017) and low-density labeling strategies mean only a fraction of the molecules are probed.…”

Biological nanoscale fluorescent probes: From structure and performance to bioimaging

“…Generally speaking, directly prepared QDs have poor biocompatibility, have specific toxicity, and are prone to non-specific adsorption and aggregation during use in the biological environment. Therefore, they need to be modified in biomedicine (usually using natural coupling [64] or encapsulation [65]). Then they can possess good water solubility, biocompatibility, and low cytotoxicity during use [14,66,67].…”

Symmetrically dispersed spectroscopic single-molecule localization microscopy

“…As a result, sSMLM has shown great potential in understanding fundamental biomolecular processes in cell biology and material science [1][2][3][4][5][6][7] . It also enables the characterization of nanoparticle properties based on the emission spectrum at the single-particle level [8][9][10] . Similar to other localizationbased super-resolution techniques, such as stochastic optical reconstruction microscopy (STORM) and point accumulation for imaging in nanoscale topography (PAINT), the localization precision of sSMLM is fundamentally limited by the number of collected photons per emitter 11 .…”

“…Similar to other localizationbased super-resolution techniques, such as stochastic optical reconstruction microscopy (STORM) and point accumulation for imaging in nanoscale topography (PAINT), the localization precision of sSMLM is fundamentally limited by the number of collected photons per emitter 11 . However, sSMLM suffers from further photon budget constraints since the collected photons of each molecule need to be divided into two separate channels to simultaneously capture the spatial and spectral information [5][6][7][8][9][10] . Thus, the spatial localization precision of sSMLM also depends on the splitting ratio between the spatial and spectral channels and is typically limited to 15-30 nm in cell imaging 2,3,5,6 .…”