Super-resolution structured illumination microscopy of cellular nanopores using near-infrared fluorescent probes

Currently, super-resolution optical microscopy is mostly conducted with fluorophores that are excited and that emit in the visible part of the electromagnetic spectrum. Extending this range to the near-infrared (NIR) and even the shortwave-infrared (SWIR) part of the spectrum is highly desirable, because light in this wavelength range is less hazardous for live imaging and can penetrate much deeper into tissue. Here, we detail our development of a novel super-resolution structured illumination microscope that is purpose-built for imaging in the NIR wavelength range. Structured illumination via laser beam interference is accomplished by splitting the excitation light along two arms of a Michelson interferometer. Arbitrary illumination angles and a wide range of pattern spacings can be obtained by controlling a two-axis galvanometric mirror. Phase shifts of the pattern are accomplished by changing the angle of an anti-reflection-coated planoparallel glass plate inserted in one arm of the interferometer. Fluorescence is detected using a NIR-enhanced 26 Megapixel scientific Complementary Metal-Oxide-Semiconductor (sCMOS) camera. We characterize this system by imaging fluorescent beads with a diameter below the diffraction limit, and demonstrate its suitability for biological imaging by resolving cellular nanopores in the plasma membrane of liver sinusoidal endothelial cells.