**9. Further developments**

The next step in evaluating NLM for a diagnostic approach in humans during ongoing neurosurgery would be to utilize an adapted miniaturized confocal instrument specially designed for neurosurgery applications. The technical settings for the laser system can be directly transferred from the system used in this study. However, one important problem should be addressed concerning the reprocessing of the microscopic device. The way of reprocessing will be an essential step to use the microscope in a sterile condition within routine neurosurgical procedures. The first data in humans during ongoing neurosurgery are meanwhile available (Sanai et al., 2011 (Epub), Schlosser, H.G., Bojarski, C. (2011 (Epub)). Neurosurgery, however the problem of reprocessing is not completely fixed. The confocal laser technique for the reusable equipment has been licensed by Zeiss, Germany, for neurosurgery from its initial developer in Australia. Here the integration of NLM into a conventional microscope system is advanced including the option of navigation and matching image-guidance data. A hand-held device has been designed and used in animal research (Sankar et al., 2010) and in humans (Schlosser, H.G., Bojarski, C. 2011 (Epub)). For the reusable system used in endoscopy in the last years a setrilizablility has not been achieved. So the application in routine neurosurgical procedures is severely limited. One has to think to introduce a disposable system which is already in clinical use for different applications. An adaptation of a reusable system could be the step to provide a confocal neurolasermicroscope for routine clinical use in neurosurgery.

Furthermore, the use of contrast agents has to be adapted to the in vivo situation. We would prefer using intravenously injected fluorescein (Makale, 2007) instead of topically applied acriflavine. Fluorescein is used for decades in ophthalmology and is permitted as a medical investigational drug with a very low rate of side effects (Lipson and Yannuzzi, 1989). Moreover, fluorescein is an established contrast agent in confocal endomicroscopy in gastroenterology where it distributes the entire gastrointestinal tissue up to 250 µm in depth (Hoffman et al., 2006). When fluorescein is applied in neurosurgery one has to consider the effect of passing the blood brain barrier (BBB), presumably only a small amount of serum albumin unbound fraction of fluorescein will pass BBB and the dye mainly stay intravascular. The amount of cellular staining has to be explored in further studies. In the neoplastic tissue the vessels probably will show a different pattern compared to healthy brain. We would expect abnormal branching and looping of the vasculature as well as abrupt changes in diameter contributing to stricture-like structures. The extravascular distribution of fluorescein, however, in a disturbed BBB as in neoplastic conditions may show the pathologic vascularisation in combination with a cellular staining.

The next step after defining the pathologies in an NLM atlas and after ascertain the affiliated operative proceeding clinical studies have to proof the benefit for the patients depending on the disease.
