Label free cell tracking in 3D tissue engineering constructs with high resolution imaging

Abstract

Within the field of tissue engineering there is an emphasis on studying 3-D live tissue structures. Consequently, to investigate and identify cellular activities and phenotypes in a 3-D environment for all in vitro experiments, including shape, migration/proliferation and axon projection, it is necessary to adopt an optical imaging system that enables monitoring 3-D cellular activities and morphology through the thickness of the construct for an extended culture period without cell labeling. This paper describes a new 3-D tracking algorithm developed for Cell-IQ®, an automated cell imaging platform, which has been equipped with an environmental chamber optimized to enable capturing time-lapse sequences of live cell images over a long-term period without cell labeling. As an integral part of the algorithm, a novel auto-focusing procedure was developed for phase contrast microscopy equipped with 20x and 40x objectives, to provide a more accurate estimation of cell growth/trajectories by allowing 3-D voxels to be computed at high spatiotemporal resolution and cell density. A pilot study was carried out in a phantom system consisting of horizontally aligned nanofiber layers (with precise spacing between them), to mimic features well exemplified in cellular activities of neuronal growth in a 3-D environment. This was followed by detailed investigations concerning axonal projections and dendritic circuitry formation in a 3-D tissue engineering construct. Preliminary work on primary animal neuronal cells in response to chemoattractant and topographic cue within the scaffolds has produced encouraging results.

4-D Hippocampus Measurements of Neurite Growth in vitro

“…To facilitate detailed examination of the neuronal cell/ECM interactions in which complex topographical and biochemical cues of the environment can be present, a scalable quantitative procedure for measuring neurite extension and characterising axonal formation within the 3-D continuous culture must be developed [17]. This was achieved by taking advantage of the limited depth of field of the phase-based microscopy systems, where images are retrieved with discernable quality differences given the relatively narrow depth of field or in-focus limits; that is, cell object/features captured at different depths from the camera may appear out of focus if they are away from the focus plane.…”

“…Using the widely documented computer vision technique of shape-fromfocus (SFF), several measures of quality of image focus (or focus depth measures) have been studied to best compute the in-focus image frame for a particular point in the image space [12][13]. An important step in applying the SFF technique in this study thus entails the focus depth computation of each scene point for every image frame from the z-stack of the focusing sequence; that is, to generate the so-called all-infocus image [4,17].…”