PhD Project
Professor Halina Rubinsztein-Dunlop, Dr Tim McIntyre

Our project in SMP is to improve the resolution of widefield CARS microscopy by using structured illumination on the input laser beams. In structured illumination methods, used with great success in fluorescence microscopy, a standing wave or grating is projected onto the sample. A series of images at different phases of the standing wave are acquired and combined in post-processing to yield a single super-resolved image, i.e. one with resolution better than the diffraction limit for the system in question.

We have developed (and published) a theoretical proposal for using structured illumination to increase the lateral resolution of widefield CARS to around 120 nm (for standard illumination wavelengths). More recently, we have undertaken preliminary proof-of-concept experiments in collaboration with the CARS group at the Innsbruck Medical University. Work is ongoing to refine the superCARS processing scheme.

A key goal is also to implement a fully functioning superCARS microscope experiment at UQ, building on our existing setup. Student projects will centre around improving and enhancing this setup, including achieving greater stability of the microscope itself, refining delivery of the CARS laser beams to attain greater ease of alignment, and setting up a new laser system (to be purchased this year). While projects are focused on improving the experimental design of the microscope, there is also the potential for experiments characterising the microscope, followed by investigation of simple biological systems, to demonstrate the value of the superCARS technique to research in the life sciences.

What is CARS?

Coherent Anti-Stokes Raman Scattering (CARS) microscopy is a vibrant young field offering great promise for research in the life sciences. Unlike conventional microscopy modalities, such as fluorescence microscopy, this technique involves no need to tag the sample with (fluorescent) dyes to identify the species of interest. Instead, CARS generates endogenous contrast from a sample, by tuning its three input laser frequencies to a vibrational transition in the target molecule. As a result, there is less risk of perturbing the system under study, through the introduction of tag molecules.

Most CARS microscopy work to date has focused on the collinear configuration, in which the constituent laser beams of the CARS process are delivered to the microscopy sample via the objective lens. While providing (lateral) resolutions equivalent to that of the confocal microscope, this configuration has the drawback that signal is only collected from one point on the sample at a time. In other words, the sample must be scanned in order to build up a full image.

An alternative, widefield CARS configuration has, however, been developed at the Innsbruck Medical University. This method provides a field-of-view for each measurement of approximately 50 µm. Unfortunately, as it stands, the widefield configuration has a relatively poor lateral resolution.