Imaging intra-cellular wear debris with coherent anti-Stokes Raman scattering spectroscopy

Abstract

Aseptic loosening of artificial joints is caused by an osteolytic reaction to wear debris mediated by macrophages and other cells. Imaging these wear particles within cells can be a key process in understanding particle-cell interactions. However, the compounds used in surgical implants are not easily visualised as no tagging molecule can be added without altering the properties of the material. We were interested in using a label free optical technique known as coherent anti-Stokes Raman scattering spectroscopy (CARS) to image these particles in cells.

In this thesis we studied how to use CARS to image physiologically relevant wear particles within cells. We characterised the responses from our CARS system and found them to be in good agreement to the Raman spectra we obtained for the same materials. We showed that the forward scattered CARS signal was consistently larger than the backwards scattered signal for the same size particles, and also generated a larger contrast, especially between sub-micron sized particles and the non-resonant background.

Wear particles of polyethylene isolated from a pin-on-plate wear simulator were shown to be in a similar size range to those retrieved from revision tissue. When incubated in our model macrophage cells we were able to image areas of CARS signal that indicated the location of these particles in the cell. Furthermore, using multiple CARS images taken at different Raman resonances we were able to distinguish between three different polymeric compounds added to cells, showing the specificity of the technique. The inherent 3D sectioning capabilities of multiphoton microscopy were used to generate projected images of the cells and contents, as well as estimating the particle loads within cells.

These results show that CARS could be an important tool in imaging intra-cellular polyethylene and characterising the interactions of wear particles with cells and the surrounding tissue.