Metamorph image analysis software

Cells were transiently transfected with TGN38-mCherry with cytoYF_x5 and cytoCR_x5. Cells were first induced to form iPOLYMER for 1 h, then imaged while maintained at 37°C and 5% CO₂. The images were taken every 5 sec. The velocity of the TGN38-posibive vesicles was estimated by dividing the distance that the vesicles traveled within the xy plane between two consecutive frames by the 5 sec interval. The images were analyzed using the “track points” function in a Metamorph image analysis software (Molecular Devices).

The stained and mounted grafts were imaged on a Nikon A1 inverted confocal microscope using a 10× objective lens and a 488-nm argon laser. The photomultiplier tube detector was set at 500-530 nm filter. Although some adjustment was made depending on the positioning of the graft, most were imaged with an 8 × 8 × 20 XYZ array with 256 × 256 pixels resolution and optical sectioning of 78.5 μm and a z-step of 45 μm. The images were analyzed using MetaMorph image analysis software (Molecular Device Inc., Downingtown, PA) with a protocol previously demonstrated by Wolle et al.^{19 (link)} This protocol was found in that study to be comparable to Fiji enhanced image analysis software (http://Fiji.sc). Briefly, the maximum projection image was opened up and an analysis area was manually drawn around the tissue. The threshold tool was then used to set the defaults to a 12-bit data set and inclusive threshold. The threshold intensity was adjusted until all dead cells were included within the range of threshold while excluding all live cells. Calculated estimates of cell loss were exported to Microsoft Excel as the percent threshold drop signal were then exported to Microsoft Excel.
At the same time, tissues were evaluated subjectively for the maintenance of the staining using all or none rating.