5-ALA fluorescence and laser Doppler flowmetry for guidance in a stereotactic brain tumor biopsy

A fiber optic probe was developed for guidance during stereotactic brain biopsy procedures to target tumor tissue and reduce the risk of hemorrhage. The probe was connected to a setup for the measurement of 5-aminolevulinic acid (5-ALA) induced fluorescence and microvascular blood flow. Along three stereotactic trajectories, fluorescence (n = 109) and laser Doppler flowmetry (LDF) (n = 144) measurements were done in millimeter increments. The recorded signals were compared to histopathology and radiology images. The median ratio of protoporphyrin IX (PpIX) fluorescence and autofluorescence (AF) in the tumor was considerably higher than the marginal zone (17.3 vs 0.9). The blood flow showed two high spots (3%) in total. 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Introduction
In the routine of stereotactic biopsy on suspected tumors located deep in the brain, samples are harvested to determine the type of malignancy.Targets are calculated using the preoperative radiology images with frameless or frame-based techniques.A biopsy needle is then inserted towards the target in the tumor.Biopsies are taken from several precalculated sites and then sent for pathological investigations to obtain a preliminary diagnosis within a waiting time of approximately one hour.The biopsy material is then further examined for a definitive diagnosis within days to weeks before postoperative oncological treatment is decided.The radiology images are not always reliable in terms of defining the right target point.In an analysis of the safety of stereotactic biopsy procedures in 300 patients, the histological diagnosis was reported to have been different from the preoperative clinical diagnosis in 49% of the cases [1].Moreover, during a biopsy procedure the location of the targets may vary relative to the precalculated coordinates due to a brain shift usually caused by the cerebrospinal fluid loss after skull bone and dura opening [2].Brain shift up to 10 mm is reported for stereotactic procedures with the greatest shift observed in the frontal lobe [3].The ambiguity in the images or brain shift makes sampling less accurate and sometimes nondiagnostic.In such cases the biopsy procedure needs to be repeated leading to a longer operation time and potentially increased risk for brain injury and infection.The number of diagnostic samples to the total number of samples (diagnostic yield) is in the range of 74 -100% [4] and is associated with a number of factors including size of the sample [5] and the number of biopsy samples taken.
The main risk with stereotactic biopsy is hemorrhage due to vessel rupture during probe insertion in the brain or at the biopsy site during biopsy sampling (3-8%), and to a lesser extent infection and seizure [4,[6][7][8].Some patients can have a higher risk for hemorrhage due to different diseases, location of the tumor and radiological factors [4,7,9].Observation of persistent blood in the biopsy needle has been suggested as a method for detection of hemorrhage intraoperatively, however, a reliable detection requires postoperative imaging [1].To avoid the complications, it is of benefit to use an intraoperative guidance system to safely reach and identify the target and reduce the risk of hemorrhage due to vessel rupture during the needle insertion and more importantly during the biopsy removal as the tumors have a higher affinity for bleeding.
In order to guide the stereotactic brain biopsy procedures, several techniques including non-invasive imaging or invasive tissue property measurements, have been implemented experimentally or in preclinical settings [10] experimentally or in preclinical settings.Iijima et al. have combined microelectrode recording and image guided stereotactic biopsy of deepseated brain tumors [11].Widhalm et al. have implemented robotic guidance [12] and in a separate work detected the 5-ALA on the stereotactic biopsy samples ex vivo using the fluorescence surgical microscope [13].A similar approach has been reported using fluorescein [14].Several dual-mode optical probes have been described including a probe combining Raman spectroscopy for tumor detection [15] and interstitial white light optical tomography for vessel detection [16,17].A combination of PpIX fluorescence detection by red-excitation and re-emission spectrometry for vessel detection is proposed by Markwardt et al [18,19].Indocyanine green is also a potential method for vessel detection [20].Scolaro et al. have published on a dual-mode needle including optical coherence tomography (OCT) and fluorescence, with the potential to incorporate blood vessel and tumor detection using 5-ALA induced PpIX fluorescence during stereotactic brain biopsy [21].
Tumor and blood vessel detection have earlier been evaluated separately in neurosurgical procedures at Linköping University Hospital.In an earlier study the detection of 5-ALA induced PpIX fluorescence was implemented with double excitation wavelengths (337 and 405 nm) for a more precise quantification in several stereotactic procedures and compared to histology and magnetic resonance (MR) spectroscopy [22,23].A recent fluorescence system adapted to the operating room (OR) environment with single excitation wavelength (405 nm) was later on developed and used in over 50 high grade glioma resections as a stand-alone system [24] or in combination with a surgical fluorescence microscope [25].The tumor tissue could be detected with over 90% sensitivity compared to histopathology [24,25].Moreover, the intensity of PpIX fluorescence could be related to the low and high malignancy grades, infiltrated and non-infiltrated gliosis in the tumor marginal zone.
LDF has been used with a fiber optic probe adapted for deep brain stimulation (DBS) implantations.In this setting the measurement probe was forward-looking and acted as a guide for the DBS lead to record the blood flow and tissue grayness.The LDF technique has a great potential to act as a 'vessel tracking' tool, and has so far been evaluated in over 120 stereotactic DBS lead implantations [26][27][28].
In order to provide optimal guidance for locating the most probable malignant sites, a multi-modal fiber optic probe has been developed.The aim of this study was to introduce and evaluate the concept of tumor detection using a spectroscopy system measuring 5-ALA induced protopophyrin IX (PpIX) fluorescence and microvascular blood flow using an LDF system both connected to one forward-looking probe for guiding brain stereotactic biopsy procedures.

The measurement setup
The measurement setup (Fig. 1(a)) composed an in-house developed fluorescence spectroscopy system and a commercial LDF system, both connected to one probe which was adapted to the stereotactic application.Two pairs of the optical fiber connections from the inhouse fiber-optic probe, were connected to each of the systems.Each system was controlled by a separate computer and LabVIEW (National Instruments Inc., Tex., USA) program which analyzed data in real-time and postoperatively.The measured signals were displayed on a second monitor to the neurosurgeons in the OR.Details of the two systems and the fiber optic probe are described in the following subsections.

Fluorescence spectroscopy
The PpIX was excited with laser light (Oxxius SA, Lannion, France) at 405 nm, 10 mW power (3.5 W/cm 2 ) at the probe distal end.The fluorescence collection was performed using a spectrometer (EPP2000, Stellarnet, Tampa, FL) with 2048-elements charged coupled device camera with a maximum of 8190 photon counts in the wavelength range of 250-850 nm with a resolution of 3 nm, measured dynamic range of 3900 and signal to noise ratio of  620.A longpass filter (Schott CG-GG-475-0.5-3,CVI) eliminates the laser light reflection from the spectrometer.The LabVIEW program controlled the initiation and termination of the laser exposure and measurements.An integration time of 400 ms and three pulses were set in the program.Fluorescence spectra were composed of tissue autofluorescence and the PpIX fluorescence.Further details of the fluorescence spectroscopy system are described in an earlier publication [29].

Laser Doppler flowmetry
The LDF system (PF5000, Perimed AB, Järfälla, Sweden) with a laser light at 780 nm wavelength and 1 mW power at the probe distal end, collected the signals with integration time set to 10 ms, the sampling frequency to 100 Hz and the time constant to 0.03 s.The system measured the microvascular blood flow (also referred to as perfusion) based on the frequency shift in the backscattered light caused by the red blood cells and the total backscattered laser light intensity (TLI) at 780 nm [30].The system measured TLI (0 -10) and perfusion (0 -999) values in arbitrary units (a.u.).The LabVIEW program included measurement and analyzing mode where peak to peak (p-p), mean (m) and standard deviation (S.D.) of the signals selected within a certain time interval could be calculated [31].

The fiber optic probe
The forward-looking fiber optic probe was developed in-house to fit the Leksell Stereotactic System (LSS, Elekta AB, Stockholm, Sweden).The outer circumference of the probe was slightly rounded to avoid injury to the brain tissue during insertion.The probe (lprobe = 190 mm, Ø = 2.2 mm, lcable > 4 m) had one central fiber (Øcore = 600 μm, Øtotal = 640 μm, NA = 0.37) and several surrounding fibers (Fig. 1(b)).Length is denoted with l, diameter with Ø and numerical aperture with NA.The central fiber was connected to the fluorescence laser light and four of the surrounding fibers (Øcore = 200 μm, Øtotal = 240 μm, NA = 0.22) were connected to the fluorescence spectrometer.Two of the adjacent surrounding fibers (Øcore = 125 μm, Øtotal = 250 μm, NA = 0.37) were connected to the LDF system, one to the laser output and one to the input connector.The probe was sterilized prior to surgery using the STERRAD procedure [32].In order to ascertain a comparable signal level with the probes used in the previous studies, the probe was tested in a Motility solution (Perimed AB, Järfälla, Sweden) and on a fluorescence reference.Same measurements were performed before each set of measurements and the systems were calibrated if necessary.

Patients
Three patients (ages 66 -71, two men and one woman) with suspected high grade glioma refereed for stereotactic biopsy procedure were included in the study after written informed consent was received from the patients.The study was approved by the local ethical committee (No.M139-07, 2015/138-32).Approximately 20 mg/kg body weight (20.8 ± 2.4 mg/kg) of Gliolan (Medac GmbH, Hamburg, Germany) powder was dissolved in water and orally given to the patients, 5.5 ± 0.5 hours prior to the measurement start.The neurosurgical procedure and anesthesia were conformant with the routine of stereotactic biopsy at the Department of Neurosurgery, Linköping University Hospital.

Stereotactic imaging and target calculation
The LSS was fixed to the skull of the patients before the imaging according to the clinical routine.The imaging modalities differed as they relied on the appropriate clinical situation.Patient 1 was scanned with CT (Computed tomography) and patients 2 and 3, by 1.5 and 3T MRI, respectively (Table 1).For patients 2 and 3, the target was selected in the Gadolinium contrast enhancing part of the tumor in the T1-weighted sequence images.The corresponding target coordinates (x, y, z, ring and arc angle) together with the entry point defined the trajectories.Surgiplan (Elekta Instrument AB, Sweden) planning software was used for calculating the stereotactic coordinates.Snapshots of the planned trajectories in the three cases are shown in Fig. 2. For the patients included in this study only one trajectory (out of the three planned-see Fig. 3(c) for an example) defined from the cortex to the target in the tumor was used.

Intraoperative optical measurements and tissue sampling
A manually controlled mechanical insertion device developed in-house (Fig. 3(a)) [26] was used for insertion and positioning the probe at precise and controlled sites in steps of 1 mm.Prior to the biopsy needle insertion, the probe was placed into the mechanical insertion device and the LSS, then moved towards the precalculated target with increments of 1 mm.LDF signals were recorded all the way along the entire trajectory but the fluorescence measurements were performed on selected sites on the cortex and then at every mm in the vicinity of and in the tumor (Fig. 3

Biopsy samples and histopathology
The biopsies were taken at the precalculated positions and the target (0 mm) along the trajectories (Table 1).Positions above the target have a minus sign and positions below the target have a plus sign (Fig. 3(b)).For each position, biopsies were taken clockwise from four directions (3, 6, 9, 12 hrs.).A total of 11 biopsy samples approximately 1 -2 mm in size were harvested and analyzed.The biopsies were sent for a smear based section examination during operation and the definite diagnosis according to the WHO grading [33] was placed by an experienced neuropathologist after two weeks.The pathological examination routine is described in an earlier publication [25].The histopathology slides (Fig. 3(e)) were reanalyzed for this study.

Fluorescence
The autofluorescence intensity at wavelength of 510 nm, IAF(510), and the PpIX fluorescence, IPpIX, were calculated on all the captured spectra.IPpIX was calculated by subtracting the interpolated intensity of autofluorescence at 635 nm, IAF(635), from the total fluorescence intensity at 635 nm, I635.IPpIX and IAF(510) values were analyzed separately and as a ratio of IPpIX to IAF(510), as described earlier [29].AF was normalized using its highest value in the same data set (Fig. 5(c)).Signal to noise ratio (SNR) was used as a criterion for defining weak signals.Signals affected by blood were defined by having SNR510  1 with a visible hemoglobin absorption signature.Signals were defined to have been totally blocked by blood when SNR510nm < 1 and SNR635nm < 1.None of the fluorescence spectra measured at the biopsy sites were affected by blood according to this criterion.

Laser Doppler flowmetry
The LDF signals were analyzed by calculating the mean of the perfusion and TLI in a 5 -10 s measurement interval.The movement artifacts caused by the insertion device were used as an identifier for the subsequent measurement site but not included in the analysis [26].The reflection from the fluorescence could be seen in the TLI signals and was excluded from the calculation interval.Figure 4 shows an example of the analysis principle.Since the normal cerebral blood flow is usually less than 50 a.u., values being at least twice as high (> 100 a.u.) were considered as high blood flow spots [26].The TLI signal was normalized by its highest value in the same set of measurements.

Statistics
Pearson regression was used to investigate the goodness of fit (r) and Mann-Whitney U-test to investigate the significant difference between each two data sets using MATLAB (The MathWorks, Inc., Natick, MA, USA).P-values < 0.05 were considered significant.Median values and range were used for statistical representation of the data set.

Results
The dual-mode probe was successfully utilized in the three described stereotactic biopsy procedures.On all occasions clear and strong fluorescence peaks were visible in real-time in the OR. Figure 5 presents the post-processed optical signals for the three patients included in the study.The IPpIX increased in the vicinity and in the tumor, and thereafter decreased gradually (Fig. 5(a),5(b)).Attenuation of AF due to hemoglobin was estimated to less than 5% except for one site explained under case 2 where AF was significantly affected by blood (Fig. 5(c)).The microvascular blood flow was high in 3% of cases in total but did not show any considerable difference in the tumor area relative to the rest of the brain (Fig. 5(a)).

Case 1
The IPpIX was detected from 14 mm and reached its maximum around + 1 to + 7 mm.A slight hemoglobin absorption signature was observed around 26 mm that contributed to about 5% attenuation of the IAF(510).The IPpIX and ratio showed the same trend in the whole trajectory (r = 0.98).The LDF perfusion signal was within the normally perfused interval (0 -100 a.u.) at these points, along the trajectory and in the tumor except for the cortex entry point and the subsequent point (515 and 157 a.u.).Correlation between TLI and IAF(510) was r = 0.98 (n = 42).The tumor margins in the CT images were not sufficiently clear in this case for comparison with the availability of IPpIX.

Case 2
The IPpIX was above zero from 13 mm, increased in the vicinity and through the tumor indicating a diffuse spread of the tumor and continued to be high up to + 15 mm.Hemoglobin absorption signature was observed at 25 mm which attenuated the IAF(510) to 37% of its expected value interpolated between the previous and next point (Fig. 5(c)).The IPpIX and ratio showed the same diffuse trend in the whole trajectory, however, there was some considerable differences (Fig. 5(b)) at some individual measurement points (r = 0.84).The blood flow did not reach any considerably high value except for + 12 to + 14 mm where slightly perfused spots (101 -250 a.u.) were observed.Correlation between TLI and IAF(510) b) a) was r = 0.92 (n = 33).The IPpIX was > 0, from 5 mm before the visible contrast in MRI, i.e., 15 mm.

Case 3
The IPpIX was detected already from 30 mm, it increased around the 1 mm and thereafter reached its maximum around + 10 to + 14 mm.In this case, there was a considerable difference between IPpIX and ratio (Fig. 5 and c) IAF(510) and TLI.Biopsies were taken from the points mentioned in Table 1.

Histopathology
The biopsy targets, their position along the insertion trajectory and the histopathology results are listed in Table 2.The relative portion of gravely atypical tumor cells (TC) in the histopathology slide is denoted with plus and minus.Median of the optical signals for the gliosis and HGT tumor types as confirmed by histopathology at these points are included in Table 3. Comparing the tumor with the gliotic marginal zone, the fluorescence ratio (17.3 [1.7 -23.5] a.u vs. 0.9 [0.1 -1.2]) was higher in the tumor whereas normalized TLI (0.72 vs. 0.21 a.u.) and normalized IAF(510) (0.42 vs. 0.26 a.u.) were lower in the tumor.Only the fluorescence ratio and TLI norm showed a significant statistical difference between the tumor and the gliotic tumor marginal zone (p < 0.05) within this data set.

Discussion
The dual-mode probe connected to fluorescence spectroscopy and LDF system was successfully implemented and evaluated in three patients during stereotactic brain tumor biopsy.Using these systems, the PpIX fluorescence, autofluorescence, microvascular blood flow and TLI were measured along trajectories with 1-mm increments and displayed in real time in the OR.The optical signals were compared to the radiology images and histopathology, postoperatively.Combination of the systems provided a clinically feasible method that is expected to increase the operation safety, efficiency (time gain for the patient and the surgeon) and efficacy (diagnosis accuracy) during brain tumor biopsy procedures.

PpIX fluorescence
The PpIX fluorescence (both IPpIX and fluorescence ratio) clearly showed a considerable difference in the normal brain and the tumor.When compared with histopathology a distinguished level of fluorescence was seen in the tumor and the border zone.Median fluorescence ratio from the same type of histopathology categorization measured during open brain surgery showed a median of 1.1 and 4.8 [25] agreeing with the current results but showing a greater variation in the HGT category due to the less accurate probe positioning and some probable photobleaching effects from the surgical microscope.PpIX fluorescence is reported to be available 0 -10 mm beyond the T1-MR image Gadolinium contrast enhancement by performing volumetric calculations before and after open brain operation [34] or by intra-operative image analysis [35].One suggestion for a future study, is to compare MR images with the optical data using stereotactic procedures which may provide information on the correlation of the PpIX and Gadolinium contrast in MR images with a higher precision compared to the calculations used during open brain surgery.However, such a study requires co-registration of the preoperative and postoperative images to estimate the trajectory which is subject to change in position due to brain shift.In the few studies investigating the effect of ALA dose on the diagnostic performance, no evidence is reported on the improvement or degradation of sensitivity and specificity of fluorescence in the tumor using a lower or higher dose of ALA [24,36].In this study 20 mg/kg ALA was chosen as that is the current conventional dose of Gliolan [37].It should be considered in future to use a lower dose of ALA (5 mg/kg) for stereotactic biopsy as it shows equally reliable diagnostic results as the higher dose but accumulates in the skin in much lower amounts [24].
The described fluorescence spectroscopy system was efficient for performing reliable measurements in the presented data sets.However, in some other data sets measured during stereotactic procedure (not presented in this paper) presence of blood has been disturbing for the fluorescence measurements since blue light is easily absorbed by hemoglobin (total signal blockage at ~50 µm) which will not be possible to rinse away during a stereotactic procedure.Compared to the earlier proposed compensation method for open brain tumor surgery [2] using diffuse reflection spectroscopy (DRS), excitation with a different wavelength that is less absorbed by blood is preferred in the stereotactic biopsy procedures.Using DRS to calculate the light attenuation requires an additional measurement; moreover, the method is applicable only in the presence of a low amount of blood or when the possibility of rinsing exists.Double excitation with 337 and 405 nm has earlier been implemented for this purpose and showed a slight improvement in tissue differentiation compared to single excitation with 405 nm, leading to the conclusion that single excitation with 405 nm was sufficiently efficient [22].However, single excitation with red light (635 nm) has shown promising theoretical and experimental results as it is much less absorbed by hemoglobin than either of 337 and 405 nm wavelengths [18].

Microvascular blood flow and vessel tracking
High blood flow is expected in cortex close to sulci and near ventricles.In the measurements included in the present study, only two highly perfused spots (4 measurement points) were detected along the trajectories, which are likely associated with slightly increased blood vessel size.The blood perfusion did not show any significant difference (p > 0.05) in the tumor that could indicate a considerably higher perfusion in the tumor.It is previously investigated that blood flow measured with LDF is directly related with the vessel size, type and orientation [30,38] and the 0.5 mm insertion step is assumed to suffice for the look-ahead distance of the forward-looking probe [27,39].Other methods of vessel detection are based on re-emission spectroscopy [19] which is an easy to implement module but might not be able to distinguish vessels (blood flow) from minor bleeding during the in vivo measurements.Indocyanine green fluorescence can be used for vessel detection, however, in the actual clinical settings it requires administration of an additional drug and similar to the re-emission spectroscopy, it is not able to detect blood flow [20].OCT is a powerful module which can provide an image of the vessel, its size and position.OCT is already available in a sideviewing configuration suitable for the side-cutting biopsy needles.The disadvantage with OCT is in the complexity of the system fabrication [21].

Autofluorescence and TLI
It is known that brain tumors have different optical properties from the white and gray matter, therefore, according to the published optical properties of the brain [40] the backscattered reflection intensity is expected to decrease in the order of the low grade, white matter, high grade glioma and the gray matter.As illustrated in Fig. 5(c), the TLI (780 nm) and autofluorescence (510 nm) decrease in the tumor with a high correlation even though the analyzed values correspond to different wavelengths.In addition to the lower levels of native fluorophores in the tumors [41], the absorption of the PpIX (for approximately 4%), and the tissue optical properties are expected to influence the intensity of the autofluorescence [42].
In previous measurements during open brain tumor resection it has been shown that the tumor has a lower TLI than the white matter, but higher than or equal to the gray matter [43].The stereotactic measurements, which are more accurate in terms of measurement position and probe distance to the tissue, confirm that the tumor has generally a lower TLI than the white matter.Overall, delineation of the tumor at its borders using TLI or autofluorescence is challenging as the inhomogeneity of the tumors affect their optical properties in a nonuniform pattern all across the tumor.Analysis of the TLI, autofluorescence or any other noncellular specific signal in the tumor is of interest in terms of studying the brain tumor properties or as an additional parameter.These are however not sufficiently accurate for implementation into the clinical routine as the diagnostic reliability does not seem to be as high as the 5-ALA based demarcation in high-grade tumors [44].However, lifetime measurement of autofluorescence has shown a high sensitivity and specificity for identification of low grade (but not high grade) tumors [45].
When analyzing fluorescence in the brain tumor resection the PpIX fluorescence was normalized by the autofluorescence as a reference value since during open brain resection procedures the measurements are more susceptible to be influenced by the probe positioning and interference factors [29,46].During the stereotactic biopsy procedures the circumstances including probe distance and angle to the tissue are identical for the measurements, nevertheless, a ratio of the photosensitizer to the autofluorescence has the advantage of obtaining a dimensionless quantity and increasing the differentiation of the tumor from the surrounding tumor [47].

Intraoperative measurements
The measurements during biopsy procedures were performed with 1-mm increments.Measurement at each position took around 20 s and thus the added time to the routine procedure was around 15 min.The step size of the mechanical device can be decreased to 0.5 mm [27], however, this will also increase the measurement time for a 50 mm long trajectory.In the routine application, a much quicker procedure is desired where the probe can be proceeded continuously.It is also desired to have auditory and visual feedback based on the analyzed parameters embedded in the system control software that displays real-time information in the OR [48].

Conclusion
The forward-looking dual-mode optical guidance probe can make real-time detection of fluorescence possible during the stereotactic biopsy procedures at the same time as the microvascular blood flow is recorded.The setup can help to define the optimal positions for biopsy sampling, give feedback on the malignancy and potentially act as a 'vessel tracker'.This will provide an increase in efficiency, efficacy and safety in stereotactic brain tumor biopsies.

Fig. 1 .
Fig. 1. a) The system design concept and b) configuration of the fibers in the probe.The system was composed of a fluorescence spectroscopy and a laser Doppler flowmetry system.The probe incorporated several optical fibers that were connected to the two systems.Fl: fluorescence, LDF: Laser Doppler flowmetry, em: emission, ex: excitation, AF: autofluorescence, TLI: total backscattered laser light intensity, PpIX: protoporphyrin IX.

Table 1 .Fig. 2 .
Fig. 2. Radiology images from the three patients.The markers show the planned entry and target points where the solid lines are the planned insertion trajectories.The images are oblique and in the same plane as the displayed trajectory.
(d)).Each movement or placement to the next site along the trajectory created an artifact in the LDF signal (Fig. 4(b)).The LDF signals were recorded continuously whereas the fluorescence was measured with three pulses (2.4 s) at each site.Simultaneous measurement of fluorescence and LDF was possible since the LDFs laser light did not interfere with the fluorescence measurements.Thereafter, the optical probe was retracted and the side-cutting biopsy needle (Backlund Catheter Insertion Needle Kit, Elekta Instruments AB, Stockholm, Sweden) with an outer diameter of 2.1 mm was inserted.All together 109 fluorescence and 144 LDF signals were recorded along the three trajectories.

Fig. 3 .
Fig. 3. a) The probe positioned in the mechanical insertion device and the stereotactic frame, b) the concept of measurements along the trajectory towards the tumor, c) MR image with precalculated targets in Surgiplan where the green markers are in displayed plane and the red markers are in a different plane d) example of IPpIX along the insertion trajectory and e) the histopathology slide image from the target point.

Fig. 5 .
Fig. 5. PpIX fluorescence and autofluorescence intensity measured from cortex down to the tumor in the three patient cases.a) Fluorescence ratio and blood perfusion, b) IPpIX, IAF(510) and fluorescence ratio,and c) IAF(510) and TLI.Biopsies were taken from the points mentioned in Table1.

Table 2 .
Summary of the histopathology diagnosis of the biopsy samples taken at the precalculated stereotactic positions.TC: tumor cells, LGT: Low grade tumor, HGT-: High grade tumor with less TCs, HGT+: High grade tumor with more TCs.