Super-resolution biological microscopy using virtual imaging by a microsphere nanoscope.

A classical microscope resolves features of around half of the wavelength of illumination λ . [ 1 ] Breaking this limit is possible in near-fi eld scanning optical microscopy using a local evanescent illumination spot, [ 2 ] in integral imaging three-dimensional microscopy, [ 3 ] or by applying a material of negative refractive index, referred to as a ‘superlens’. [ 4–6 ] A far-fi eld superlens can transform scattered evanescent waves into propagating waves, [ 7,8 ] which then can be further processed by conventional optics, but its nanofabrication process is complex. Other advanced super-resolution concepts are based on reducing the focal spot size, like done in confocal fl uorescence microscopy, [ 9 ] modifying the effective pointspread function of the excitation beam using a second laser that suppresses fl uorescence emission from fl uorophores located away from the center of excitation, like in stimulated emission depletion microscopy (STED), [ 10 ] or employing photo-switchable fl uorescent probes to resolve spatial differences in dense populations of molecules, as in photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). [ 11,12 ] Compared to these high-performance and sophisticated techniques, a dielectric lens of plano-spherical-convex shape, known as a solid-immersion lens (SIL), remains an attractive and simple solution for projecting information directly into the farfi eld. [ 13,14 ] Imaging of objects beyond the diffraction limit was demonstrated with self-assembled nanoscale lenses in a SILtype implementation, [ 15–17 ] but reducing the size of a SIL to the nanoscale is challenging. Also micrometer-scale spheres could convert the near-fi eld evanescent fi eld with high frequency spatial information into a propagating mode, [ 18 ] as a ‘photonic nanojet’ exits from such microspheres with a waist smaller than the diffraction limit, [ 19–24 ] forming the basis for their superior imaging capability. [ 25–28 ] Here, we propose the use of high-refractive index ( n p = 1.92) glass microspheres for facile and affordable super-resolution fl uorescent imaging of sub-cellular organelles and biomolecules. The microspheres are simply put on a sample that is immersed in oil or water, and project the sample’s near-fi eld nano-features into the far-fi eld, generating a magnifi ed virtual image. Using a conventional microscope objective, we image control nanostructures and fl uorescent nanobeads with a minimum feature size of ∼ λ /7. We demonstrate the potential of the technique by resolving the structure of fl uorescently stained centrioles, mitochondria, chromosomes, and study the effect of doxycycline treatment on mitochondrial encoded protein expression in a mouse liver cell line. A schematic of several microsphere nanoscopes combined with an immersion objective is illustrated in Figure 1 . The transparent barium titanate glass microspheres simply self-assemble on top of an object that is immersed in liquid medium with refractive index n m (1.33 for experiments performed in water, or 1.52 for experiments performed in oil). Fluorescent or bright-fi eld images are obtained through a 40 × water immersion objective with numerical aperture (NA) = 0.8, or a 63 × oil immersion objective with NA = 1.4, respectively. For testing the microsphere nanoscope in the fl uorescent mode, a mercury vapor short arc lamp and suitable fi lter sets for different fl uorophores are mounted on Fluorescence Imaging