Low magnification image tiles (10x) were acquired with a Phasics SID4BIO camera on a Zeiss microscope. iPSC cultures were maintained using a custom stage incubator on an automated stage. We implement an image acquisition strategy that compensates for non-flat illumination wavefronts to enable imaging of an entire well plate, including the meniscus region normally obscured in Zernike phase contrast imaging. Polynomial fitting and background mode correction was implemented to enable comparability and stitching between multiple tiles. LFOV imaging of reference materials indicated that image acquisition and processing strategies did not affect quantitative phase measurements across the LFOV. Analysis of iPSC colony images demonstrated mass doubling time was significantly different than area doubling time.
These measurements were benchmarked with prototype microsphere beads and etched-glass gratings with specified spatial dimensions designed to be QPI reference materials with optical pathlength shifts suitable for cell microscopy.
This QPI workflow and the use of reference materials can provide non-destructive traceable imaging method for novel iPSC heterogeneity characterization.