**2. Technical background**

We used a miniaturized confocal laser microscope (Optiscan, Australia, Fig. 1) for tissue examination as described earlier (Schlosser et al., 2009 (Epub)). This technique was first described as confocal laser endoscopy in gastrointestinal endoscopy and was used for highresolution in-vivo imaging while endoscopy of the upper or lower gastrointestinal tract was performed (Kiesslich et al., 2004). The ability to detect or exclude premalignant conditions

Confocal Neurolasermicroscopy - Modern Perspectives for Glioma Resection on a Cellular Level 389

Topically applied acriflavine hydrochloride (0.05% in saline, Sigma Aldrich, Germany) accentuates the superficial cell borders and their nuclei (Hoffman et al., 2006). Acriflavine is not approved for the use in humans due to a low but theoretical risk of inducing mutagenesis as described in cell culture systems (Ferenc et al., 1999). However, in special clinical indications there are several studies in which this substance was used safely (Leong

In our pilot study NLM was performed in an ex vivo approach on small tissue samples of patients suspicious to suffer from GBM (WHO IV, n=9) or meningeoma (grade I n=2, grade II n=1) after diagnostic radiology. Open tumor resection was performed in the neurosurgical OR in all patients. No prior histological diagnosis was available before neurosurgery. One sample of tumor tissue was used for direct comparison of NLM and histopathology of the same area. After the examination with NLM the specimen was transferred to neuropathology for conventional tissue examination. Additional fragments of the tumor center and border were analysed by NLM. Different additional fragments were sent for conventional pathological diagnosis. These were not marked as study material and therefore

Fig. 3. (Removed with approval Schlosser, Cen Eur Neurosurg, 2010, 71(1):13-9)

the infiltration zone. The margin of the tumor appears similar to a parabolic curve.

Histopathology and Neurolasermicroscopy (NLM) of a GBM (a-f same patient, a-c center; df infiltration zone, bar = 100 µm). a, d: H&E staining of tumor border (a) and infiltration zone (d). H&E staining of tumor center shows a high density of tumor cells; b, e:

immunohistochemical staining with MIB-1antibody to assess proliferation in tumor center (b) and infiltration zone (e). c, f: NLM of tumor center (c) and infiltration zone (f) with detection of a high density of tumor cells in the center and markedly reduced cell density in

et al., 2008) (Günther et al., 2010).

assessment and diagnosis was blinded.

**3. Pilot study** 

and/or other pathologies which were normally not visible created a complete new field of gastrointestinal diagnosis followed by specific treatment before classical histology was accessible (Bojarski et al., 2009). For the use in humans intravenous applied fluorescein sodium distributes throughout the mucosa, however, most of the serum albumin bound fraction of fluorescein highlights the blood vessels and the capillaries.

Fig. 1. Distal tip of the confocal endomicroscope EC-3870CIFK (Pentax, Europe) used in gastrointestinal endoscopy. As a protrusion on the end of a conventional endoscope a microscopic lens is mounted (diameter of the lens is 5 mm). All other features of the distal tip are standard in routine endoscopy.

Fig. 2. (With kind permission of Peter Delaney, Optiscan, Australia and Ralf Kiesslich, University Medicine Mainz, Germany). The use of confocal endomicroscopy in human gastrointestinal tract. When the confocal lens was hold in gentle contact with the gastrointestinal mucosa, blue laser light generated a series of images every 7µm up to an imaging depth of 250µm. Typical contrast dyes were fluorescein and acriflavine as indicated. Topically applied acriflavine hydrochloride (0.05% in saline, Sigma Aldrich, Germany) accentuates the superficial cell borders and their nuclei (Hoffman et al., 2006). Acriflavine is not approved for the use in humans due to a low but theoretical risk of inducing mutagenesis as described in cell culture systems (Ferenc et al., 1999). However, in special clinical indications there are several studies in which this substance was used safely (Leong et al., 2008) (Günther et al., 2010).
