Development of a Total Internal Reflection Fluorescence Structured Illumination Microscopy (TIRF-SIM) System for Bio-Imaging

Author: Joseph Brett, Physics

Mentor: Dr. Steve Smith, Department of Nanoscience and Nanoengineering

As bio-technology advances, new and improved imaging methods for studying microscopic systems are needed to enable further discovery of the dynamic processes which define life. Total Internal Reflection Fluorescence Structured Illumination Microscopy (TIRF-SIM) is a relatively new microscopy method which promises increased resolution and high imaging speeds. Nonlinear SIM is a super-resolution microscopy method which can theoretically achieve resolution far below the diffraction limit but is practically limited by the achievable signal to noise ratios. Linear SIM is able to achieve a resolution double the standard diffraction-limited resolution in biologically relevant timeframes. SIM uses patterned illumination to gather high-resolution data from the imaged structure, and multiple raw images are combined and analyzed to give one super-resolution image. TIRF is a standard-resolution imaging method used to reduce out of focus background light, increasing image contrast. Combining SIM and TIRF results in high contrast, super-resolution images which can be acquired at a relatively high framerate. Our system uses a spatial light modulator (SLM) to create the appropriately patterned sample illumination. The SLM is a sophisticated liquid crystal device which changes the polarization of light waves reflecting off it. An acousto-optic modulator is used to control illumination, ensuring the SLM is only illuminated when the correct patterns are displayed. Patterns from the SLM are filtered by a spatial mask to give a smooth, sinusoidal illumination at the sample. An azimuthal polarization device from ArcOptix is used to change the light polarization for different patterns, preserving pattern integrity. A high-speed EMCCD is used for image acquisition. These components are synchronized together using a combination of hardware, wiring, and software. The system is controlled using a LabView VI. Each component of the system has presented unique challenges and obstacles while developing the system. Details of the system design, construction, and image acquisition are presented.