Collagen matrices as an improved model for in vitro study of live cells using Raman microspectroscopy

摘要

Due to its high lateral resolution, Raman microspectrsocopy is rapidly becoming an accepted technique for the subcellular imaging of single cells. Although the potential of the technique has frequently been demonstrated, many improvements have still to be realised to enhance the relevancy of the data collected. Although often employed, chemical fixation of cells can cause modifications to the molecular composition and therefore influence the observations made. However, the weak contribution of water to Raman spectra offers the potential to study live cells cultured in vitro using an immersion lens, giving the possibility to record highly specific spectra from cells in their original state. Unfortunately, in common 2-D culture models, the contribution of the substrates to the spectra recorded requires significant data pre-processing causing difficulties in developing automated methods for the correction of the spectra. Moreover, the 2-D in vitro cell model is not ideal and dissimilarities between different optical substrates within in vitro cell cultures results in morphological and functional changes to the cells. The interaction between the cells and their microenvironment is crucial to their behavior but also their response to different external agents such as radiation or anticancer drugs. In order to create an experimental model closer to the real conditions encountered by the cell in vivo, 3-D collagen gels have been evaluated as a substrate for the spectroscopic study of live cells. It is demonstrated that neither the medium used for cell culture nor the collagen gels themselves contribute to the spectra collected. Thus the background contributions are reduced to that of the water. Spectral measurements can be made in full cell culture medium, allowing prolonged measurement times. Optimizations made in the use of collagen gels for live cells analysis by Raman spectroscopy are encouraging and studying live cells within a collagenous microenvironment seems perfectly accessible.