In this study, we have implemented PALM microscopy for long-term super-resolution dynamic imaging. The necessary tools and techniques are widely available, which makes this a relatively low-cost approach that may be easily set up in many laboratories using fluorescence microscopy.
We initially validated our experimental strategy by studying the organization of the actin cytoskeleton in dendritic spines in fixed neurons. From the high-resolution PALM images of mature dendrites it was possible to quantify morphological parameters of spines that are comparable with the results obtained by electron microscopy 
. We did not observe any correlations between the size of the spine head and the dimensions of the neck, in line with previous findings 
. Upon closer inspection, the sub-spine distribution of actin is not homogeneous. The actin density within the spine head was generally higher in the center compared to the outer regions. These low-density regions appeared to consist of a filamentous mesh. The thickness of these structures is below the pointing accuracy of our detection and could thus not be determined accurately. However, it would be tempting to speculate that these represent actin filaments that have so far only been observed by electron microscopic analysis of the spine cytoskeleton 
Subsequently, we performed live PALM experiments to observe the baseline dynamics of the spine morphology over tens of minutes. The quantification of the morphological spine parameters in living neurons is in very good agreement with the values obtained in fixed neurons, suggesting that the chemical fixation with paraformaldehyde did not affect the cytoskeletal structure noticeably. Furthermore, we observed that the temporal rearrangement of the spine cytoskeleton was more pronounced in the spine head and the length of the neck, while the width of the neck remained remarkably constant. This could reflect the fact that the actin filaments in the spine head are organized in a complex manner in contrast to the neck where the F-actin appears to have a linear organization 
. Changes of the spine morphology may thus be expected to occur mainly in the head width and the length of the spine rather than the width of the neck structure. Since the neck represents a bottleneck to the diffusion of synaptic proteins and ions between the head and the shaft, the precise determination of its dimensions in live systems is important to understand its involvement in regulatory mechanisms 
. We found that the neck opening (width of around 150 nm) occupies as little as 1% of the surface area of the spine head (mean diameter of 600 nm).
In this context it should be noted that our measurements refer to the dimension of the cytoskeleton as determined by the detection of the ABP-tdEosFP expression construct. In order to relate our findings to the localization of the plasma membrane, we combined PALM imaging with QD tracking of a diffusing membrane construct (GFP-GPI). This technique allowed us to visualize both, the actin cytoskeleton and the plasma membrane simultaneously at super-resolution. Our measurements showed a close correspondence between the cytoskeleton and the membrane, typically within a range of 50 nm. This distance includes the width of the plasma membrane (5–10 nm) in addition to the size of the antibodies (~5 nm) and the streptavidin-coupled QDs (~25 nm) used for labeling. The measured distances in the spine heads had a broader distribution compared to the neck. The likely cause of this difference is that F-actin filaments are distributed very unequally within the spine head and that consequently low density regions of the cytoskeleton at the periphery of the spine are sampled less efficiently than the core of the spine.
To provoke morphological changes of the spine cytoskeleton, we induced a sustained synaptic depolarization by bath application of AMPA and measured the concomitant alterations of the spine shape. We found that this treatment reproducibly led to the shrinkage of synaptic spines, followed by a reduction of the actin levels in spines. This is in agreement with the data by Halpain and colleagues, who found that glutamate receptor activation triggered a loss of F-actin and the collapse of the spines 
. A different report has suggested that AMPA application causes a rounding of the spine heads and the loss of spine dynamics 
. This difference may be explained by a concentration-dependence of the glutamatergic activation in the two studies (i.e. 10 µM versus 1–2 µM AMPA). We have therefore reassessed the effect of AMPA application at 2 µM and 10 µM concentration. While we occasionally observed the rounding of the spine head in response to glutamate receptor activation with 2 µM AMPA, the most consistent effect was the reduction of the size of the spines at either concentration, with or without the rounding effect. Our findings are thus suggestive of a depolymerization of the F-actin cytoskeleton during depolarization. In order to explore how the depolymerization of the actin cytoskeleton translates into changes of the position of the plasma membrane, we also performed dual-color PALM/QD tracking. We found that the correspondence between the two structures was equally close before and after the 2 µM application of AMPA. In other words, the position of the plasma membrane adapts dynamically to the rearrangement of the actin cytoskeleton.
In addition to the morphological changes, the levels of actin in the spine head decreased during AMPA application. This redistribution of actin could also be observed in the diffraction-limited images, where the fluorescence intensity of diffusing fluorophores contributes to the overall fluorescence. In contrast, in the PALM image reconstruction only fluorophores that are bound to F-actin are likely to be detected as well focused spots (with 2D-Gaussian shape) and thus represented in the image. Therefore, the decrease in the signal intensity of the spine head relative to the shaft is due not only to depolymerization of actin but also to an actual redistribution of actin through the spine neck. Since the observed redistribution of depolymerized actin from the spine head into the dendritic shaft occurred gradually and on a somewhat slower time scale, our findings also imply that the spine neck represents a barrier that limits the diffusion of actin monomers to the dendrite, and underlines the importance of the spine neck for the compartmentalization of the spine head as the site of synaptic neurotransmission.
The use of a low-affinity probe (ABP-tdEosFP) for live PALM imaging confers several beneficial features: 1) bleached fluorophores are replenished, in contrast to probes that are fused to the protein of interest, thereby enabling long-term recording of a given cellular structure; 2) the probe is not directly incorporated into the actin filaments, reducing the risk of altering the cytoskeletal organization; 3) the cell may tolerate higher levels of expression, meaning that there is a larger pool of photoactivatable probes. This also implies an effective increase of the sampling of the structure since a large number of binding sites are available for ABP-tdEosFP. Our experimental strategy also represents a step forward in comparison to the indirect measurements achieved by SPT techniques 
, since we reconstruct the entire spine cytoskeleton itself at a relatively high temporal resolution. Our approach can therefore be used to study the long-term morphological rearrangement of actin during synaptic plasticity. Sub-diffraction dynamic imaging (20 s per frame) of dendritic spines in organotypic slices at less than 100 nm resolution was previously shown by Nägerl et al.
using stimulated emission depletion microscopy (STED) 
. However, in this study the authors made use of a diffusive cytosolic fluorophore (YFP) to visualize the shape of the spine, without resolving the internal organization of the cytoskeletal structure. Furthermore, in comparison to this approach we managed to reduce the high density of photons irradiating the sample by five orders of magnitude (from ~400 MW/cm2
at 598 nm to ~4 kW/cm2
at 561 nm) thus reducing the risk of phototoxicity. Indeed, we did not observe any consistent changes of any of the measured spine parameters or their dynamics over time (see , movie S1
In conclusion, our work brings together several innovative approaches to study the dynamics of cellular structures at super-resolution, namely long-term dynamic PALM imaging and the use of a low-affinity probe. Furthermore, we have combined live PALM imaging with QD-based single particle tracking in order to visualize two cellular structures (membrane and cytoskeleton) simultaneously at super-resolution. We believe that this novel approach is a powerful combination of techniques that can be applied to correlate single molecule dynamics with cellular structures at the nanometer scale, such as the movement of neurotransmitter receptors in relation to the postsynaptic density