The development of new methods for imaging living cells with nanometric resolution is one of the most addresses topics in modern biology. Atomic force microscopy (AFM) provides an unpaired resolution in the out of plane z coordinate, - often around 1nm and even better -, and in the xy plane about 10nm and less. AFM was successfully applied to robust living cells [1] however, since this technique is based on the occurrence of a physical contact with the sample which can be too invasive for soft samples, application to fragile living cells such as neurons, can be problematic [2-5].

Here we propose a simple but effective approach enabling the investigation of the dynamics of neuronal growth cone (GC) with nanometric resolution, combining AFM with time lapse Differential Interference Contrast (DIC) imaging. We applied this novel method to investigate the morphology of the GCs at the neurite ends in embryonic stem (ES) cell derived neurons [6-7]. During early development of the central nervous system (CNS), there is an exuberant outgrowth of projections which later need to be refined to achieve precise connectivity [8]. Exuberant and/or erroneous neuronal connections need to be pruned to achieve precise connectivity. GC pruning occurs through retraction, degeneration, or a combination of both [9]. Analyzing GCs topography with AFM and movements with time lapse DIC imaging we verified a new pruning mechanism, not previously reported in literature, in which GC are sequentially dissolved, with an initial flattening, followed by the formation of hole and a final fragmentation on the whole structure.

Results

Cell Culture and AFM Imaging

ES-derived neuronal precursors were obtained using the protocol described previously in Ban et al. (2007) [10]. Cells were plated on coverslips and induced to differentiate for 24 hours. The structure of the differentiating living GCs was analyzed by AFM (Nanowizard II instrument; JPK, Berlin, Germany) in tapping mode at 0.6 scan line per second and with a maximum of 256 scan lines, using soft cantilevers (up to 0.03 N/m). Cells were plated at a density of 3x104 cells/cm2 in order to obtain isolated GCs to avoid overlapping structures and coverslips with living cells were mounted into a stage (BiocellTM, JPK) maintaining temperature at 37°C, covered with 1.5 ml of differentiation medium.

AFM Live Imaging

We investigated GC dynamics of living neurons derived from ES-cells 24 hours after differentiation.

GC cycles of protrusion and retraction were observed. An example of a growing GC is displayed in figure 1a-c. The external surfaces were measured with AFM imaging and GC edges (see red arrow of the fig. 1c) could advance by 1-3 microns within 20 minutes. The height of their central domain increased from 150 to more than 300 nm and subsequently decreased to 200 nm (height profile fig. 1d). The observed growth of imaged GC indicates that the physiological dynamics was poorly affected by AFM scans and that the cantilever tip did not damage the GC. In this way, we were able to follow more than 10 GCs for hours and all of them had compact and smooth surfaces.

Growth is be followed by retraction [11], indeed an example of a retracting GC is displayed in figure 1 e-g. Here AFM images were collected repetitively over 2-4 hours (160 min between fig. 1e and 1g) to follow complete cycles of growth and retraction. During retraction, the GC's height varied between 100 and 200 nm progressively thinning (fig. 3h). After 120 minutes the GC portion indicated by the red arrow (fig. 1f) retracted leaving holes and isolated fragments. The two filaments, possibly cytoskeleton components, initially detected (fig. 1e) completely disappeared (fig. 1h). After 160 minutes the neurite retracted leaving behind it a large fragment isolated from the retracted neurite (indicated by the pink arrow in fig. 1g): no structure with a height larger than 10 nm connecting the fragment to the retracted neurite was detected. We observed the retraction of 7 GCs and in all cases GCs left behind them fragments.
The acquisition of AFM images of living GCs requires several minutes and therefore due to the movement of GCs and to fluidity of the membrane, a high resolution AFM image cannot be achieved. When the same GC is fixed, protein crosslinking increases the stability/rigidity of the membrane and a higher AFM resolution can be obtained.

Effect of Fixation on GC Morphology

With fixed cells, we operated the AFM in contact mode with very low force settings (100 pN-1 nN). In these conditions image resolution is limited only by the radius of curvature of the tip and by the nature of the tip-sample interactions. Therefore by scanning fixed GCs it is possible to resolve details with dimension less than 50 nm. GCs were fixed in situ either with glutaraldehyde and paraformaldehyde after AFM live imaging (fig. 2). In both cases fixation did not modify significantly GCs shape. The height profiles (fig. 2c and 2f) obtained along the blue and the red lines do not superimpose perfectly but GCs volume changed by less than 12%. We compared the structures of 8 GCs before and after fixation and in all GCs the volume was not significantly altered.