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

干涉散射显微镜（iSCAT）是一种光学成像技术，其中散射光和反射光干涉检测器以获得图像。iSCAT可用于溶液中蛋白质的单分子无标记检测、单分子跟踪和定量质量成像。由于标记效率和动态范围问题，荧光显微镜等替代光学成像技术不适合定量质量成像。最近，通过在实验装置中加入超半球形固体浸没透镜，荧光显微镜的性能得到了相当大的改善。在这个项目中，将分析在iSCAT实验装置中加入超半球形固体浸没透镜的效果。该项目的潜在影响是提高当前iSCAT实验装置的灵敏度和质量分辨率。这将提高该技术在学术领域研究许多相关生物和化学问题的能力，从蛋白质自组装到药物筛选。该项目的目的是通过用固体浸没透镜取代目前使用的油浸物镜来提高iSCAT的性能。使用比浸没油（n = 1.515）具有更高折射率的固体浸没介质将导致更大的有效数值孔径，从而能够收集更多的散射光子和更高的空间分辨率。此外，去除显微镜浸泡油并用固体成分代替它将提高实验装置的机械稳定性，减少样品相对于检测器的漂移。这可能会提高灵敏度，因为随着时间的推移，虽然对收集的图像进行平均可以减少弹片噪声引起的波动，但在~100 ms后，样品漂移（而不是光子数）成为限制因素。这项工作将需要测试用于固体浸没透镜的不同高折射率材料，包括立方氧化锆、莫桑石和各种类型的光学玻璃。对于每种材料，将分析单色差和图像质量的程度，以确定理想的材料。除此之外，还将探讨用非球面透镜代替空中物镜的效果。替换空中物镜是有利的，因为它将降低实验装置的成本，使iSCAT更容易被更广泛的学术界使用。研究方法是新颖的，因为它旨在推动突破性技术的当前边界超越目前的可能性。目前，所有的iSCAT实验装置都是由油浸物镜组成的，因此结合固体浸没透镜是一种新颖的方法。期望通过本课题所完成的研究，能够提高iSCAT的性能。该项目属于EPSRC生物物理和软物质物理研究领域，属于物理科学主题。没有公司或合作者参与该项目。

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.