Abstract
Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) are non-linear techniques that allow label-free, non-destructive and non-invasive imaging for cellular and tissue analysis. Although live-imaging studies have been performed previously, concerns that they do not cause any changes at the molecular level in sensitive biological samples have not been addressed. This is important especially for stem cell differentiation and tissue engineering, if CARS/SHG microscopy is to be used as a non-invasive, label-free tool for assessment of the developing neo-tissue. In this work, we monitored the differentiation of human fetal-femur derived skeletal cells into cartilage in three-dimensional cultures using CARS and SHG microscopy and demonstrate the live-imaging of the same developing neo-tissue over time. Our work conclusively establishes that non-linear label-free imaging does not alter the phenotype or the gene expression at the different stages of differentiation and has no adverse effect on human skeletal cell growth and behaviour. Additionally, we show that CARS microscopy allows imaging of different molecules of interest, including lipids, proteins and glycosaminoglycans, in the bioengineered neo-cartilage. These studies demonstrate the label-free and truly non-invasive nature of live CARS and SHG imaging and their value and translation potential in skeletal research, regeneration medicine and tissue engineering.
Highlights
Coherent anti-Stokes Raman scattering (CARS) combined with microscopy is a powerful chemical imaging technique that maps the distribution of molecules in biological systems in their native state, without the need for an external label[8]
The current study examines these issues with analysis of the development of human fetal femur-derived skeletal cells into cartilage, and sets out to conclusively establish, through gene expression analysis and concomitant imaging, that the non-linear imaging process itself does not have any observed effect during cell differentiation
Human fetal femur-derived skeletal cells were differentiated into cartilage and analysed using live-CARS and second harmonic generation (SHG) microscopy
Summary
Coherent anti-Stokes Raman scattering (CARS) combined with microscopy is a powerful chemical imaging technique that maps the distribution of molecules in biological systems in their native state, without the need for an external label (such as stains or fluorophores)[8]. Simultaneously with CARS, second harmonic generation (SHG), a well-established technique that allows imaging of collagen fibres in tissues, can be carried out with appropriate laser sources[16] For both non-linear techniques, CARS and SHG, given that relatively high peak powers are used, it is necessary to establish that no subtle changes are induced that are detrimental to the biological system under study (even if the laser powers are within damage thresholds). There has been no investigation, to date, detailing the potential biological effects on using non-linear imaging techniques such as CARS and SHG on live tissue when the excitation powers are within damage thresholds. Our work establishes the translation potential of label-free multimodal non-linear imaging approaches for biomedicine and paves the way for their application to cell-based therapies, human skeletal regeneration research and tissue engineering
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