The Importance Of Digital Holographic Microscopy For Automated, Real-Time Monitoring Of Human Adult Stem Cell Confluence In Large-Scale Cultures

To guarantee robust cell expansion on a large-scale, an automated control method becomes essential for obtaining sustainable and useful stem-cell-based products. Given that both stem cell behavior and the differentiation mechanisms are sensitive to cell density, monitoring of cell confluence is mandatory. Actual observation protocols of traditional polystyrene T-flasks, or multitray stacks, are ineffective for large scale manufacturing.

Integrity™ Xpansion™ multiplate bioreactors have been developed to enable noninvasive, real-time observation of stem cell growth at large scales. The specific design of the bioreactor, combined with the iLine™ – a differential digital holographic microscope (DDHM) of the newest generation – enables automatic multiplate cell monitoring. The DDHM technology captures 3-D information, enabling label-free image processing and automatic cell confluence counting.

iLine Microscope
Ovizio’s quantitative imaging technology is based on Differential Digital Holographic Microscopy (DDHM) and can be used for label-free multilayer monitoring. In digital holography, the scattered light beam from an illuminated object interferes with a reference beam on a CCD camera. This interference pattern is used for a 3-D numerical  reconstruction of the object. Differential digital holography is where the scattered light beam from an object, by means of a small lateral shift, interferes with itself on a CCD camera. Advanced processing is used for the 3-D reconstruction of the object. In DDHM the light source can be distant from the objective and can thus be used to monitor large objects. Holograms contain information of the volume observed and are used to construct intensity images, quantitative phase images and 3-D images covering the shape and density of an object. The phase image is equivalent to the optical thickness of the sample. One of the key features of holography is the capability to refocus images post acquisition.