Cell Mobility after Laser Axotomy Figure 6. Cell motility after injury depends on whether an axon regeneration program is initiated. Somas that regenerated or repaired injured axons were more likely to migrate towards the site of axon transection (A), while somas that did not maintain their axons migrated away from the transection site (B). doi:10.1371/journal.pone.0026832.g006 Newton, NJ) was used for both laser axotomy and time-lapse imaging. To keep the sample at constant temperature (37uC), a PDMI-2 micro-incubator (Harvard Apparatus, Holliston, MA) connected to a TC-202A temperature controller (Harvard Apparatus, Holliston, MA) was mounted onto the motorized stage. The microscope was equipped with a stepper motor (Prior Scientific A500-H249, Rockland, MA) for automating image focusing. A CCD camera, (QImaging Retiga EXi 1394, Surrey, BC, Canada) was used for imaging. Software for automating laser injury and imaging was programmed using LabVIEW (National Instrument, Austin, TX). An autofocus system based on the intensity of the back-reflection of a secondary laser, was used to compensate for mechanical and thermal drifts during experiments lasting 24 hr or more. Axonal transection and imaging Axonal transection and imaging were performed using the same microscope without removal of the sample. Dishes containing differentiated RGC-5 cells were placed in the microscope incubator and approximately 20–30 locations on the dish were manually selected by the operator for subsequent injury and imaging. Cellular injury was induced by exposing the axon to the focused laser beam (pico-TRAIN High Q Laser, Rankweil, Austria). Fig. 1 shows an example of how cells were inured. The focus of the laser beam is inidcated. The pulse energy was varied between 5 nJ and 7.5 nJ while the exposure duration was set to 5 sec. The wavelength of the laser was 1.064 mm and the pulse width was 6ps. Under these exposure conditions, cell damage was not always immediately visible after exposure, but axon transection and degeneration became evident with time (see Fig. 2). Details of the microscopy set-up and alignment protocols are further described in the Supplemental Materials File S1 and also in Fig. S1, where a schematic of the microscope is presented. After injury, the morphological evolution of axons and somas was imaged by time-lapse transmission bright field microscopy over up to 26 hours, by positioning the motorized stage to each previously selected location. Once in position, the program adjusted the focus and illumination intensity, acquired an image, moved the stage to the following cell and repeated the sequence. The time between sequential images was 10–20 sec, depending on the distance between cells, and each cell was imaged approxi- mately 300 times with a duty cycle of 5 to 10 minutes, depending on the total number of cells imaged and the distance between them. The cell density was kept low to avoid overlap of axons. Therefore in order to image enough cells, a relatively large area (approximately 15 mm2) had to be scanned. Figure 7. Intermediate steps in the image analysis algorithm. A. Standard deviation of the image intensity over a 90-pixel matrix. B. Thresholding. C. Opening and filling morphological operations. D. Erosion yielding the final mask. doi:10.1371/journal.pone.0026832.g007 PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e26832