Diametrical optical coherence tomography for the analysis of 3D cell cultures

RWTH Publications (RWTH Aachen)

Abstract

Due to their ability to realistically mimic in vivo conditions, three-dimensional (3D) cellcultures play an increasingly important role in biomedical research. However, theirstructural and cell biological complexity – one of their greatest advantages – also presentschallenges, particularly in imaging. Established imaging technologies for conventionalcell cultures are often unsuitable for 3D cultures due to their lack of depthsectioningand insufficient penetration depth. While techniques capable of depth-sectioningcan visualize entire 3D cultures, they remain constrained by penetration depthlimitations and are often invasive or destructive. Optical Coherence Tomography (OCT)offers a noninvasive alternative for high-resolution volumetric imaging of 3D cell cultures.While OCT is capable of reaching a penetration depth of approximately 600 μmin 3D cell cultures, which is superior to other imaging technologies, even this depth isoften insufficient for existing and emerging research applications.The primary objective of this thesis therefore is to noninvasively visualize entire 3D cellcultures with diameters greater than one millimeter using OCT. To achieve this, two diametricalOCT systems operating at center wavelengths of 850 nm and 1300 nm weredesigned based on quantifiable and parameterizable 3D cell culture characteristics andrealized. To ensure the diametrical positioning of the two scanning systems of a diametricalOCT setup an alignment method based on measuring the coupling efficiency wasimplemented. The application of this method resulted in a mean alignment uncertaintyof 57.9 μm (±32.7 μm).With the goal of further improving this alignment uncertainty, a marker-based and amarkerless registration method for OCT volume scans were conceptualized, implemented,and compared. The marker-based method computes a transformation matrix,enabling stitching of the diametrical scans, using volume scans of a registration target.In contrast, the markerless method determines the transformation matrix by detectingkeypoints within the sample using the scale-invariant feature transform (SIFT). Themarker-based approach achieved a mean registration uncertainty of 5.0 μm (±3.5 μm),representing an 83.3% improvement compared to the initial alignment uncertainty. Themarkerless method’s registration uncertainty and repeatability were quantified; however,direct comparison with the marker-based approach is not possible due to differencesin data and methodology.Validation measurements of four biomedical and two technical samples were performedto validate the combined functionality of the diametrical OCT systems, the alignmentmethod, and the marker-based registration method. The results confirm the capabilityof the combined methods and systems to, depending on their optical properties,noninvasively visualize 3D cell cultures with diameters ranging from 1.0 mm to 2.5 mm.Additionally, the findings demonstrate potential applications in technical imaging.

Authors 1

1. Enno Hachgenei corresponding Aachen

   RWTH Aachen University

   Affiliation as printed

   RWTH Aachen