The good phase contrast results from the correct alignment of the imaging areas with the objective grids, so it is a matter of moving the plate to the right spot and control the phase-contrast quality visually. In terms of the scratch assay, I would suggest staining the live cells with Calcein AM and image the samples with 488 nm fluorescence to have perfect contrast in each sample. Calcein AM is also fixable so you can image the samples at the later time point. However, Calcein AM does not fair well when permeabilized so you can't combine it with antibody staining. I also felt sometimes dissatisfied with the quality of the phase contrast imaging for the scratch assay, eventually switching to fluorescence.

If you must use phase contrast, try larger wells (6 well plates). with the scratch in the center of a 35 mm well, you can avoid strange optical effects from the edge of the well. Still adjust the condenser and phase rings for imaging each well to optimize captured image quality. Also make sure to have enough medium, and the same amoint of medium in each well to avoid meniscus effects.

Hi Ishita Virmani! I use p10 pipette to make a scratch and Olympus IX50 to take a picture

Hi Anton Lennikov! Thank you your suggestion!