microscopy. Both isolated axons and cell bodies were imaged for more than 24 hr after injury. Using the platform we describe, we characterized the trajectories and mobility of injured cells and correlated them with the initiation of a regenerative response. As described by Wu et. al. [30], we also observed the secretion of chemoattractant molecules at the injury site and we found that RGC-5 cells responded to this signal, moving the somas toward the injury site. We also found that this chemoattractant signal is a function of the damage level of the cells. Finally, we demonstrate how this platform can be used in high- content screening, cutting tens of cells per dish and automating the image acquisition and analysis of approximately 300 images per cell. By allowing calculation of kinematic parameters that describe the axonal and soma response to injury, this platform can be used for mechanistic studies in axonal injury and development of new axoprotective therapies. Results and Discussion We coupled a picosecond laser, an inverted microscope with a high-resolution motorized stage, an environmental chamber, and custom-written image analysis tools to create a semi-automated platform for high-content study of the response of individual axons to transection. In a typical experiment, a culture dish of differentiated RGC-5 cells was placed on the microscope stage inside a compact incubator. The sample was then manually scanned to preselect axons to be injured and areas to be imaged. Subsequent steps were performed automatically under the control of custom software. Cell damage was sequentially induced by exposing axons to the focused laser beam at each selected position (Fig. 1). Cells were then sequentially imaged over up to 26 hr by positioning the motorized stage at each selected location, automatically adjusting the focus, normalizing the illumination intensity, and acquiring a transmission bright-field microscopy image. The stage was then moved to the following cell to repeat this sequence. The resultant time-lapse series allowed observation in parallel of the morphological evolution of RGC-5 axons and somas after axotomy under a variety of conditions. Parameters for laser axotomy were optimized in preliminary experiments to induce a focal axonal transection, without concomitant injury to other areas of the axon or soma. Specifically, the power of the laser beam was adjusted so that there was no immediate visible damage at the transection site but yet transection could be observed within several minutes. Typically this was achieved with exposure duration of 5 s and the pulse energy at the sample set at approximately 7 nJ (500 mW mean power). At these energy levels, RGC-5 axons were transected and yet the somas were able to re-extend their axons, i.e. axon transection was achieved while preserving the integrity of the proximal axon and soma (Fig. 2). Fig. 2A shows the whole cell and a magnified image of the axon to be injured. Immediately after laser injury (Fig. 2B), there was swelling of the distal axon adjacent to the injury site without visible transection. Within 20 min an approximately 1 mm separation became apparent between the ends of the axon proximal and distal to the injury site (Fig. 2C). The injury subsequently induced a degenerative process in the distal axon and a contraction in both ends of the isolated axon (Fig. 2C, D). The loss of membrane integrity was evidenced by bubbles and distal axon fragmentation (Fig. 2D). The entire image sequence is shown in Supplemental Video S1 including depiction of the soma trajectory as detected by our algorithm. The injury site is indicated trough the video by a blinking white dot. The biological response to axonal injury differs among cells, presumably due to differences in the level and duration of induced Cell Mobility after Laser Axotomy Figure 1. Axon transected with a focused picosecond laser beam. The pulse energy was 7.5 nJ at 76 MHz, with an exposure of 5 sec. doi:10.1371/journal.pone.0026832.g001 damage, baseline susceptibility, environmental conditions, and interactions with adjacent cells. As an example of the power of this platform to study the neurobiology of axonal injury, we correlated two different aspects of the cellular response to injury. First, we distinguished three groups of RGC-5 cells based on morphological responses to axonal injury, that is whether they initially regenerated their transected axon. In group A (65% of total), cells either re-sealed the injured axonal membrane and hence reversed axon transection, reconnected the transected sections (Supplemental Video S2), or grew new processes at the damage site (Supplemental Video S3). In Supplemental Video S2 after a process was cut, the cell body began migrating toward the injury site, while at the same time the cut process started to degenerate, as evidenced by the retraction of its most distal part. This cell was then able to reconnect the transected process and degeneration stops. In Supplemental Video S3 there was a rapid axon degeneration after laser transection. The cell body moved toward the injury site and new processes grew at the same location as the laser transection site. We grouped these two kind of responses- reconnection of transected processes or regrowth of new processes at the injury site-together characterized by recovery or regener- ation of RGC-5 cells after axonal injury. In group B (35% of total), we include cells that neither repaired nor replaced their injured axons. This response is illustrated in the Supplemental Video S4 ,where after injury the transected process degenerated in less than 1 hour, but the cell body remained in the same position for approximately 500 min. After this latency the cell moved away from the injury site. We defined this behavior as a non-regenerative RGC-5 response to axonal injury. Finally in group C (5% of total), injured cells died within one day of injury. This response is illustrated in Supplemental Video S5. Using this classification of RGC-5 cells response to injury, we measured cell motility after axotomy and found that it depended on the regenerative response and the initial axon length. PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e26832