Simple coherent anti-Stokes Raman spectroscopy system for minimally-invasive 3-D microscopy of lipid rafts in migratory cells

Recent data from several laboratories has shown that lipid rafts in the cell membrane act as platforms to allow the correct spatial clustering and interaction of proteins essential for driving directed cell migration. Among other functions, lipid raft structures are linked with intracellular signalling proteins to specific sites at the membrane, thus enabling biological activation. To date, most of the evidence for the existence and role of lipid rafts has come from indirect methods such as detergent extraction of the plasma membrane. Rafts have proven difficult to visualise in living cells due to the difficulties associated with fluorescent labelling. A non-invasive strategy is therefore sought to investigate lipid structures in migratory living cells. We propose to bridge this technology and information gap by developing a dedicated instrument for minimally invasive visualisation of lipid rafts in situ, using coherent anti-Stokes Raman scattering (CARS) microscopy. CARS microscopy is an established technique that uses two laser sources to spectroscopically determine the chemical composition of a sample. In CARS imaging, the two light sources interact in a sample to generate a signal that is used to create a contrast image. A signal can be generated from any sample provided that the lasers can deliver the wavelengths necessary to excite the molecular vibration. Also, no chemical fluorescent labelling is required in CARS imaging, which reduces the sample perturbation. Hence, CARS microscopy is ideally suited to visualising lipid raft structures in situ. The current technology employed for CARS imaging is expensive, high-maintainance and is difficult to operate. By developing a simplified and low-cost workstation for both conventional and multiplex CARS microscopy using recent developments in photonics technology, we will perform spectroscopic imaging of lipid rafts and further structures without compromising cell viability.

Lipid rafts are concentrated clusters of unknown size comprising cholesterol and sphingolipids which makes them spectroscopically distinguishable from the surrounding plasma membrane. These rafts are highly localised platforms that allow recruitment of transmembrane receptors and associated intracellular signalling proteins to specific sites at the membrane, facilitating biological activation. However, most of the evidence for the existence and role of lipid rafts has come from indirect methods such as detergent extraction of the plasma membrane. Disruption of lipid rafts using chemical agents such as methyl-b-cyclodextrin has confirmed their importance in allowing correct spatio-temporal signalling. Rafts have proven difficult to visualise in living cells and has relied largely on the binding of fluorescent conjugates such as the cholera toxin subunit B (CT-B) to cross-link the raft-associated glycosphingolipid GM1. Attempts to image lipid rafts in situ by inserting chemically tagged forms of lipid components into cells has largely proven ineffective as exogenous lipids can be toxic or cause disruption to the membranes, altering the cell phenotype. A non-invasive strategy is sought to study lipid rafts in migratory living cells. We propose to bridge this technology and information gap by developing a simple-to-use diagnostic instrument for minimally invasive visualisation of lipid rafts in situ, using coherent anti-Stokes Raman scattering (CARS) microscopy. Conventional CARS microscopy employs two electronically coupled wavelength-tunable laser sources to match the molecular vibration of the sample. This represents a very complex and difficult to operate instrument. This project brings together the developments in nonlinear optics and nonlinear laser scanning microscopy to create a simple to use single excitation source for three-dimensional multiplex CARS microscopy to visualise lipid raft structures used as signalling platforms in migratory cells.