Resolution of Ultramicroscopy and Field of View Analysis

In a recent publication we described a microscopical technique called Ultramicroscopy, combined with a histological procedure that makes biological samples transparent. With this combination we can gather three-dimensional image data of large biological samples. Here we present the theoretical analysis of the z-resolution. By analyzing the cross-section of the illuminating sheet of light we derive resolution values according to the Rayleigh-criterion. Next we investigate the resolution adjacent to the focal point of the illumination beam, analyze throughout what extend the illumination beam is of acceptable sharpness and investigate the resolution improvements caused by the objective lens. Finally we conclude with a useful rule for the sampling rates. These findings are of practical importance for researchers working with Ultramicroscopy to decide on adequate sampling rates. They are also necessary to modify deconvolution techniques to gain further image improvements

Chemical Clearing and Dehydration of GFP Expressing Mouse Brains

Generally, chemical tissue clearing is performed by a solution consisting of two parts benzyl benzoate and one part benzyl alcohol. However, prolonged exposure to this mixture markedly reduces the fluorescence of GFP expressing specimens, so that one has to compromise between clearing quality and fluorescence preservation. This can be a severe drawback when working with specimens exhibiting low GFP expression rates. Thus, we screened for a substitute and found that dibenzyl ether (phenylmethoxymethylbenzene, CAS 103-50-4) can be applied as a more GFP-friendly clearing medium. Clearing with dibenzyl ether provides improved tissue transparency and strikingly improved fluorescence intensity in GFP expressing mouse brains and other samples as mouse spinal cords, or embryos. Chemical clearing, staining, and embedding of biological samples mostly requires careful foregoing tissue dehydration. The commonly applied tissue dehydration medium is ethanol, which also can markedly impair GFP fluorescence. Screening for a substitute also for ethanol we found that tetrahydrofuran (CAS 109-99-9) is a more GFP-friendly dehydration medium than ethanol, providing better tissue transparency obtained by successive clearing. Combined, tetrahydrofuran and dibenzyl ether allow dehydration and chemical clearing of even delicate samples for UM, confocal microscopy, and other microscopy techniques

External transpedicular spine fixation in severe spondylodiscitis – salvage procedure

Specific and non-specific infections of the spine are rare. Due to their potential for severe instabilities, deformities and the impairment of neurological structures, the treatment is often prolonged and needs an interdisciplinary management. The clinical presentation is uncharacteristic, therefore diagnosis is often delayed. There are no prospective randomized studies for therapy recommendation. The surgical concept includes eradication of the infection and the reliable stabilization of involved segments. This concept is successful in most cases of endogenous vertebral osteomyelitis. The therapy of the exogenous spine infections after macro and micro surgery is more difficult, due to the critical wound situation and the involvement of the posterior parts of the spine. In these cases, infection-associated instability of the anterior part is complicated by critical posterior wound conditions.We present three cases of severe exogenous vertebral infections, where temporary external transpedicular spine fixation was used for salvage procedure, till soft tissue conditions have permitted a definitive internal stabilization