Label-free higher harmonic generation microscopy for real time visualization of Osteogenesis Imperfecta 3D fibroblasts culture

Abstract

The inherited bone disease Osteogenesis Imperfecta (OI) caused by genetic mutation can lead to brittle bones that fracture easily and serious skeletal deformities due to collagen defects. To understand this disease, in vitro models of 2D monolayer fibroblast cultures obtained from subdermal skin biopsies were developed by worldwide research groups, and nowadays were extended to 3D models to further mimic this disease. The models allow for studying of the collagen formation and possible abnormalities in extracellular matrix (ECM) and will enable studies of the mechanisms underlying defects in the quality and quantity of collagen fibres in OI skin fibroblasts when compared to the healthy cells. The most common methods for measuring the components in fibroblasts cultures are collagen immunostaining, which is time consuming and prohibits time-lapse measurements, or second harmonic generation microscopy (SHG) which only enables visualization of collagen fibres but not fibroblasts. Here by our label-free higher harmonic generation microscopy with 1050nm / 1070nm laser, we have extended SHG to with third harmonic generation microscopy (THG) to visualize fibroblasts cell morphology and their nuclei, also with multiphoton autofluorescence (MAF) signal from the cytoplasm of the fibroblasts. Our techniques allow for the visualisation of the 3D cultures in real-time, resulting in the possibility of making time-lapse videos. For both OI patient and control fibroblast cultures, fibroblasts and their nuclei were clearly visualised by forward THG signal; the collagen fibres by the forward SHG channel, and the cytoplasm signal with the backward autofluorescence channel. First results on the comparison between cultures of OI patients and controls reveal differences in fibroblasts orientation, collagen orientation and density.