## 2 Materials and Methods
### Observation and Biopsy Sequences  
1. _Assessment of bulk tumor fluorescence with correlative pathology:_  
The aim of fluorescence assessment and tissue biopsy in bulk tumor is to determine whether a tumor shows fluorescence and to correlate fluorescence with tumor histology. To collect this biopsy, surgeons are asked to switch to blue light as soon as bulk tumor is encountered during surgery, to determine whether any fluorescence is visible and to take a biopsy from a fluorescing part of the tumor. A neuronavigation screenshot is taken from the exact location of the biopsy (Figure 1). The entire sequence is recorded using the microscope's video system.  
2. _Interrogation of \"indeterminate\" tissues:_  
During the course of conventional white light surgery, the surgeon might encounter tissue which appears uncertain. From such \"indeterminate\" tissue the surgeon might require more information based on fluorescence. In such cases, the observation of fluorescence might lead to a change in surgical decision or surgical strategy. Such instances of indeterminate tissue may be found at the dural tail, in scar tissue with cases of recurrent meningioma, in the bone flap or at bony resection margins. In cases in which the surgeon requires more information based on fluorescence, the region in question is marked by the navigation pointer and a screenshot is taken. At this stage, the surgeon records his presumed diagnosis for the tissue area he or she desires more information for (\"likely\" or \"unlikely\" tumor) and his surgical decision based on his impression under white light (to reset or not to reset). The surgeon then switches to blue light, records whether he or she sees fluorescence, and states whether he or she plans to change surgical strategy based on fluorescence status. Under white light, the uncertain tissue is then biopsied for later histological evaluation. The entire process is recorded using the microscope's video camera for later assessment by the review panel (Figure 2).  
Figure 1: _Cont._  
3. _Identification of residual tumor at the end of surgery:_  
After clearing a field from tumor under white light, with the surgeon no longer being able to identify residual tumor in that field of view, the surgeon has the possibility of switching to blue light to determine whether any pathological tissue can be identified based on fluorescence. If the surgeon finds residual fluorescing tissue after switching to blue light, this area is pointed out using the neuronavigation pointer (and a navigation screenshot is taken). A biopsy is taken from the exact same region after switching back to white light if this can be performed safely. This procedure is also recorded by video from beginning to end. Such interrogations might be performed at multiple stages of surgery, provided a particular area (field of view) has been cleared of tumor according to the impression of the surgeon under white light. The interrogation of an inconspicuous bone flap also constitutes an \"end of surgery\" scenario if the surgeon wants to rule out any tumor infiltration in the bone. If any fluorescent bone is encountered, then this is biopsied prior to drilling of the bone flap (Figure 3).