Label free optical imaging of engineered neural tissue formation by second harmonic signals from collagen type I

Sanen, K.; Paesen, R.; Martens, W.; Lambrichts, I.; Phillips, J. B. and Ameloot, M. (2014). Label free optical imaging of engineered neural tissue formation by second harmonic signals from collagen type I. In: 18th International Microscopy Congress Proceedings.



A variety of optical microscopy techniques can visualise individual cells in their extracellular matrix (ECM), most of them requiring exogenous dyes. Many labels have been subject of discussion because of phototoxic effects and perturbation of native cellular behavior. Interestingly, some biological molecules and structures can generate intrinsic optical signals, thereby making the use of exogenous dyes redundant. Cellular autofluorescence can be observed by one- or two-photon excitation (TPE) of for example NADH and flavins. Another intrinsic optical effect is Second Harmonic Generation (SHG), where laser light interacting with non-centrosymmetric molecules such as collagen type I generates frequency-doubled light. The resulting images with high contrast and submicron resolution can be further analyzed to obtain specific quantitative information.

Despite the advantages of these nonlinear optical microscopy methods, their use in the biomedical field is not widespread. In areas of tissue engineering, these techniques could be of great value for non-invasive characterization of biomaterials. Collagen type I hydrogels have been proposed for many regenerative applications due to their native-like ECM properties, inherent biocompatibility and suitability as carriers for different cell types. When a collagen type I hydrogel solution seeded with dental pulp stem cells (DPSCs) is casted into a mould with tethering bars positioned at each end, the contractile forces generated by DPSCs create a uniaxial tension along the tethered hydrogel (Fig 1a), resulting in longitudinal cell alignment within this 3D matrix (Fig 1b).[1] Although this engineered neural tissue (EngNT) containing DPSCs represents the desired end result for neuroregenerative applications [1], time-lapse experiments monitoring changes of hydrogel architecture are lacking.

In order to truly understand ECM remodeling by enclosed cells, it is essential to monitor the interaction of these cells with the 3D construct in time without the use of fluorescent labels. To this end, we performed TPE and second harmonic imaging of live EngNT, which revealed a marked change in collagen type I organization before and after cell alignment (Fig 2). Furthermore, we applied our in house developed image correlation spectroscopy approach [2] to characterize the spatial organization and structural characteristics of collagen type I fibers in time. This research demonstrates the application of nonlinear label free optical techniques for high resolution biomedical imaging.

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