Motion and Morphometry of Fibroblasts

Fibroblasts were plated on the collagen-coated insert of a Petri dish as described above. 2D images at 4x or 10x magnification were acquired through an Olympus CK2 microscope housed in an incubator at 37°C and 5% CO₂. Illumination and acquisition were synchronized using Fire-I software (www.unibrain.com/products/fire-i-software/). A series of JPEG images were acquired every 45 seconds with a XCD-V50 camera (Sony, San Diego, CA). The images were imported into J3D-DIAS4.2 [13 (link), 14 (link), 22 (link)–24 ] and compiled into JDIAS movie format for motion and shape analysis. Cells were outlined at 7.5 minute intervals for 6 hours. Stacked perimeter plots were generated and length, width, perimeter, area and instantaneous velocity calculated as described previously [25 ]. J3D-DIAS4.2 calculates persistence as the ratio of net to total path length at 5 frame intervals and averaged over the total path length.