报告题目：Enhancing contrast, field of view and axialresolution of structured illumination microscopy

报告人：Henning Ortkrass 博士（Bielefeld University）

主持人：韦小明  教授

报告时间：2024年10月16日（星期三）上午10:30

报告地点：物理楼（18号楼）二楼213室学术报告厅

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                                     物理与光电学院

                                     2024年10月13日

摘要：Super-resolved structured illuminationmicroscopy (SR-SIM) is among the most flexible, fastest and least perturbingfluorescence microscopy techniques capable of surpassing the opticaldiffraction limit. Current custom-built instruments are easily able to delivertwo-fold resolution enhancement at video-rate frame rates, but the cost of theinstruments is still relatively high and the physical size of the instrumentsis still prohibitively large. We have developed compact, cost-efficient andhigh-speed 2D-, grazing-incidence- and TIRF-SIM to overcome these limitations. Afiber-based illumination path allows multi-color imaging with a field-of-view(FOV) of up to 230x230 µm². A novel method called multi-angle TIRF SIM allowsan image reconstruction with an enhanced axial resolution.

The setupconsists of a fiber-coupled laser combiner with 405 nm, 491 nm, 561 nm and 639 nmlaser sources, a fiber-switch to select the desired illumination angle withintegrated phase shifters and a hexagonal fiber collimator with a lenstelescope that projects the beams into the back-focal plane of the objectivelens. All modules of the illumination path are home-built to achieve higheststability and a compact size. The rotation of the illumination angle isperformed by two galvo-mirrors that select the corresponding pair of fibers andphase shifts of the zero order beam and one of the two first order beams areperformed by home-built, MEMS-mirror based phase-shifters that modulate theoptical path length. The phase shifters and galvo-mirrors allow transitiontimes between illumination patterns of less than 1 ms. The hexagonal collimatorallows to adjust the pattern spacing in the sample seamlessly, within atransition time of 100 ms.

The capability of the setup is demonstrated bymulticolor imaging of fixed liver endothelial cells (LSECs) and mouse braintissue slices with TIRF- and 2D-SIM with a FOV of up to 230x230 µm² and alateral resolution of up to 85 nm. Moreover, an improved version of this setupwas used with a wide-field FLIM detector for super-resolved FLIM-SIM, which wasapplied for live-cell imaging and dye-multiplexing.