UC Santa Cruz

3D Structured Illumination Microscopy (SIM) is a well established method of extendinglateral and axial resolution in fluorescence microscopy, up to two times that of the diffractive limit of resolution of wide-field microscopy. The operating principle of SIM is (spatial) frequency mixing between the illumination light and the specimen structure. The structured illumination pattern is generated inside the sample using three monochromatic polarized beams diffracted by a grating which interfere to form an extruded modulation field. The sinusoidal grating is shifted laterally to laterally translate the modulation field through the specimen, axial sample translation passes the specimen through the axial modulation component and scrambles axial modulation within the captured image plane. Commercial 3D SIM microscopes are available from several vendors but are expensive and typically only available in academic imaging facilities. Challenges in application of 3D SIM to study living biological specimens include the limited 3D acquisition time required to record each 3D SIM image volume. Multi-focus SIM (MF-SIM) has been previously demonstrated in a proof of concept experiment on a fixed specimen in a single color. My thesis is part of a research project to provide a live specimen compatible MF-SIM system. My work has included leveraging modern electronic and opto-mechanical devices, alternative illumination and data reconstruction strategies to reduce the complexity of the illumination path, by removing any unnecessary moving or modulated devices, and utilizing a modern phase-based Liquid Crystal on Silicon (LCoS) Spatial Light Modulator (SLM) utilizing calibrated sinusoidal phase grating patterns to avoid spurious orders generated from aliased pixel lattice patterns caused by binary gratings. This approach frees us of some mechanical components and allows us to extend the functionality of our SIM system to easily accept two or more optimized SIM patterns, which can be interleaved for multi-color acquisitions. This allows us to run samples with dual flourophores and study the dynamics and interactions of fine structures in samples such as the synaptonemal complex in the model organism C. elegans. Combining 3D SIM with Multifocus Microscopy (MFM) techniques which utilize specialized custom diffractive optics. This approach gives us the ability to simultaneously image 7, 9, or more planes while running a 3D SIM pattern. This greatly speeds up our SIM temporal resolution at the expense of the total intensity being split between the focal planes. Due to the fundamental difference between translating the sample axially through a modulation field, and collecting multiple focal planes sitting in a modulation field, a novel approach is required to recover the encoded axial data and perform a true 3D reconstruction. To evaluate MF-SIM more closely I have been creating tools to simulate 3D SIM modulation fields, synthetic data sets and synthetic MF-SIM data.