Induced pluripotent stem cells (iPSCs) are reprogrammed cells that can have heterogeneous biological potential. Quality assurance metrics of reprogrammed iPSCs will be critical to ensure reliable use in cell therapies and personalized diagnostic tests. We present a quantitative phase imaging (QPI) workflow which includes acquisition, processing, and stitching multiple adjacent image tiles across a large field of view (LFOV) of a culture vessel. These LFOV images enable quantitative characterization of colony heterogeneity and growth in an iPSC population. 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. Low magnification image tiles (10x) were acquired with a Phasics SID4BIO camera on a Zeiss 200M microscope. iPSC cultures were maintained using a custom stage incubator on a Ludl automated stage. We implement a novel 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. Background correction was implemented to zero the background phase shifts within each tile, enabling comparability and stitching between multiple tiles. LFOV imaging of reference materials indicated that image acquisition and processing strategies did not bias quantitative phase measurements across the LFOV. Analysis of iPSC colony images demonstrated mass doubling time was significantly different than area doubling time. This QPI workflow and the use of reference materials can provide non- destructive traceable imaging method for novel iPSC heterogeneity characterization.
Proceedings Title: Proceedings of SPIE
Conference Dates: January 27-February 1, 2018
Conference Location: San Francisco, CA
Conference Title: SPIE Photonics West BIOS: Quantitative Phase Imaging IV
Pub Type: Conferences
stem cells, quantitative phase imaging, reference materials