Solid immersion lens interferometric scattering microscopy for improved sensitivity and mass-resolution

Interferometric scattering microscopy (iSCAT) is an optical imaging technique in which scattered and reflected light interfere at a detector to obtain an image. iSCAT can be used for single molecule label-free detection, single-molecule tracking and quantitative mass imaging of proteins in solution. Alternative optical imaging techniques such as fluorescence microscopy are not suitable for quantitative mass imaging due to labelling efficiency and dynamic range problems. Recently, a considerable improvement in the performance of fluorescence microscopy was obtained through the incorporation of a super-hemispherical solid immersion lens into the experimental set-up. In this project, the effect of incorporating a super-hemispherical solid immersion lens in an iSCAT experimental set-up will be analysed. The potential impact of this project is to improve the sensitivity and mass-resolution of current iSCAT experimental set-ups. This will improve the technique's ability to study many relevant biological and chemical problems in the academic field, ranging from protein self-assembly to drug screening. The aims and objectives of this project is to improve the performance of iSCAT through replacing the currently used oil immersion objective with a solid immersion lens. Use of a solid immersion medium with higher refractive index than immersion oil (n = 1.515) will lead to a considerably greater effective numerical aperture, enabling collection of more scattered photons and a higher spatial resolution. Furthermore, removing the microscope immersion oil and replacing it with solid components will improve the mechanical stability of the experimental set-up, reducing drift of the sample relative to the detector. This could bring about improvements in sensitivity since, while averaging images collected over time reduces the shot noise induced fluctuations, after ~100 ms sample drift (rather than number of photons) becomes the limiting factor. This work will entail testing different high refractive index materials for the solid immersion lens, including cubic zirconia, moissanite and various types of optical glass. For each material, the extent of monochromatic aberration and image quality will be analysed to determine the ideal material. As well as this, the effect of replacing an air objective with an aspheric lens will be probed. Replacing the air objective is advantageous because it will reduce the cost of the experimental set-up, making iSCAT more accessible to the wider academic community. The research methodology is novel as it aims to push the current boundaries of a ground-breaking technique beyond what is currently possible. Currently, all iSCAT experimental set-ups consist of an oil-immersion objective and therefore incorporating a solid immersion lens is a novel approach. It is expected that the performance of iSCAT will be improved through the research completed in this project.

This project falls within the EPSRC Biophysics and soft matter physics research area, which is under the Physical Sciences theme. No companies or collaborators are involved in the project.