High-density mapping of single-molecule trajectories with photoactivated localization microscopy

Abstract

We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.

Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics

“…Although we used similar frame-capture rates for the live-cell experiments here, we assembled the position data into several equal time interval subgroups, which then formed the individual PALM frames of a time-lapse movie. Thus, although individual single-molecule frames were captured fast enough to visualize molecular dynamics at the 20-Hz level, as in previous PALM-based diffusion studies 13 , here we concentrate instead on the nanoscale dynamics of the AC morphology, which occur over the time scale of minutes and require collating the molecular position information from hundreds or thousands of single-molecule frames. For example, we captured 7,500 singlemolecule frames lasting 40 ms each without pause (Fig.…”

Fluorescence nanoscopy. Methods and applications

“…Photoinduced activation may not be necessary: PALM with independently running acquisition (PALMIRA) exploits spontaneous activation and deactivation and asynchronous detection, which results in faster acquisitions [40]. When the interest is restricted to imaging the cell membrane, single particle tracking of photoswitchable fluorophores (sptPALM) and transiently labelling the surface with synthetic dyes that become fluorescent upon binding (point accumulation for imaging in nanoscale topography, PAINT) are two variations of stochastic LM that achieve higher number of localisations: sptPALM allows hundreds of individual molecules to be visualised and localised at the same time with tens of nanometres precision, mapping the trajectories of single molecules at high molecular density [41]; PAINT involves transiently labelling the surface of the cell such that a fluorescent signal appears as a diffraction-limited spot when the fluorophore binds to the membrane and disappears when it dissociates or bleaches [42][43][44]. PAINT and sptPALM are advantageous over conventional single particle tracking because many overlapping trajectories can be followed as long as the distance between fluorescent molecules at any time is greater than several times the width of their PSF.…”

“…HIV Viral assembly is widely studied using HIV-Gag, the main structural protein of HIV. This protein has received much attention since the early proof-of-concept reports of superresolution, studying the assembly and dynamics of tdEosGag [34,41]. Gag clusters have been imaged at subdiffraction resolution to quantitatively characterise their size and morphological characteristics in different stages of their formation [209].…”

Comparing video‐rate STED nanoscopy and confocal microscopy of living neurons

“…While the possibility of overcoming the diffraction limit in living cells was demonstrated a decade ago [19], it was not before 2007 that dynamic imaging with nanoscale resolution in the focal plane was demonstrated at 80 frames per second [20] and in 2008 that video-rate imaging was achieved in living cells [21]. Another approach for investigating dynamic processes with subdiffraction resolution is based on stochastically switching and tracking single molecules [22], which was demonstrated in 2007 [23] and 2008 [24]. So-called structured illumination microscopy, with diffraction-limited resolution of 100 nm, was recently used for dynamic imaging with 11 frames per second in combination with TIRF illumination [25].…”