Super-resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI).

AIMS: The spatial resolution of light microscopy is limited by the wavelength of visible light (the 'diffraction limit', approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI. METHODS: Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled, using standard immunohistochemical markers (neurofilament-H, myelin-associated glycoprotein, glial fibrillary acidic protein, GFAP). Image sequences were processed for STORM (effective pixel size 8-32 nm) and for SOFI (effective pixel size 80 nm). RESULTS: In human, rat and mouse, subcortical white matter high-quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. CONCLUSIONS: Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach.

Preoperative opacification of acrylic intraocular lenses in storage.

The preoperative opacification of acrylic intraocular lenses (IOLs) was investigated in order to determine its cause. Opacified IOLs were examined by energy dispersive X-ray (EDX), the buffer solutions were analysed by inductively coupled plasma optical emission spectroscopy (ICP-OES) and the rubber seals used in the bottles in which the IOLs were stored were ashed and tested. The deposit covering the opacified lenses contained a significant amount of zinc, which was absent from fresh IOLs and buffer solution. The source of this was found to be the rubber seals used to seal the glass bottles in which the IOLs were stored. There were two types of rubber seals used, red and grey in colour. The buffer solutions in which opacification had occurred was also contaminated with zinc, but this was only noticeable when using the red seals. This contamination was reproduced by boiling red seals in fresh buffer solution for eighty minutes, to simulate autoclaving. It was concluded that zinc from the zinc oxide used as filler in the rubber seals was leaching into the buffer solution and causing the IOLs to become opacified. This was found to be much worse in the case of the red seals than for the grey ones. However, minute crystals were found on the IOLs stored using the grey ones, which could potentially act as nucleation points for postoperative opacification.