DESIGN, IMPLEMENTATION AND OPTIMIZATION OF A HIGH-SPEED LASER-SCANNING MICROSCOPE FOR SCANNING ANGLE INTERFERENCE MICROSCOPY

Abstract

Fluorescence imaging has become an invaluable tool for the investigation of subcellular structure and organization. However, there is a fundamental limit to the resolution that can be achieved using traditional imaging systems such as fluorescence microscopes. Scanning Angle Interference Microscopy (SAIM), a technique capable of nanometer-scale localization along the optical axis, overcomes this limit using surface generated interference effects. The spatial frequency of standing waves near a reflective surface is dependent on the polar angle of the incident light. SAIM exploits this phenomenon by carefully controlling the incident angle of the excitation light on a fluorescent sample. In order to achieve high precision localizations, the excitation light must be homogeneous within the sample plane. However, coherent light sources, such as lasers, typically form characteristic fringe and speckle artifacts on length scales relevant to subcellular imaging. To eliminate these artifacts and improve image quality we have designed and built a circle-scanning microscope specifically optimized for live-cell SAIM imaging. In our microscope the excitation laser is continuously rotated through the azimuth while maintaining a constant polar angle in each exposure. Furthermore, we employ high-speed shuttering triggered by a state-of-the-art scientific camera to achieve precise synchronization between sample excitation and the camera’s exposure window, minimizing photodamage to the sample. We demonstrate potential instrument-induced artifacts in SAIM imaging using a conventional acquisition scheme and how our purpose-built instrument addresses and overcomes each. Finally, we present a detailed protocol for the instrument construction, sample preparation and execution of high-speed live-cell SAIM experiments using the circle-scanning instrument and custom, open-source instrument control hardware and graphical interface.