High-speed TIRF and 2D super-resolution structured illumination microscopy with a large field of view based on fiber optic components

Super-resolved structured illumination microscopy (SR-SIM) is among the most flexible, fast, and least perturbing fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-built instruments are easily able to deliver two-fold resolution enhancement at video-rate frame rates, but the cost of the instruments is still relatively high, and the physical size of the instruments based on the implementation of their optics is still rather large. Here, we present our latest results towards realizing a new generation of compact, cost-efficient, and high-speed SR-SIM instruments. Tight integration of the fiber-based structured illumination microscope capable of multi-color 2D- and TIRF-SIM imaging, allows us to demonstrate SR-SIM with a field of view of up to 150 × 150 µm2 and imaging rates of up to 44 Hz while maintaining highest spatiotemporal resolution of less than 100 nm. We discuss the overall integration of optics, electronics, and software that allowed us to achieve this, and then present the fiberSIM imaging capabilities by visualizing the intracellular structure of rat liver sinusoidal endothelial cells, in particular by resolving the structure of their trans-cellular nanopores called fenestrations

Grazing incidence to total internal reflection fluorescence structured illumination microscopy enabled by a prism telescope

In super-resolution structured illumination microscopy (SR-SIM) the separation between opposing laser spots in the back focal plane of the objective lens affects the pattern periodicity, and, thus, the resulting spatial resolution. Here, we introduce a novel hexagonal prism telescope which allows us to seamlessly change the separation between parallel laser beams for 3 pairs of beams, simultaneously. Each end of the prism telescope is composed of 6 Littrow prisms, which are custom-ground so they can be grouped together in the form of a tight hexagon. By changing the distance between the hexagons, the beam separation can be adjusted. This allows us to easily control the position of opposing laser spots in the back focal plane and seamlessly adjust the spatial frequency of the resulting interference pattern. This also enables the seamless transition from 2D-SIM to total internal reflection fluorescence (TIRF) excitation using objective lenses with a high numerical aperture. In linear SR-SIM the highest spatial resolution can be achieved for extreme TIRF angles. The prism telescope allows us to investigate how the spatial resolution and contrast depend on the angle of incidence near, at, and beyond the critical angle. We demonstrate this by imaging the cytoskeleton and plasma membrane of liver sinusoidal endothelial cells, which have a characteristic morphology consisting of thousands of small, transcellular pores that can only be observed by super-resolution microscopy