The corresponding surgical data has been given with tumor volume and postoperative outcome.

<p>Tumor volume was measured in cm³. Functional tumor localization (abbreviated as fg) was determined by preoperative MRI and classified according to Sawaya <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044885#pone.0044885-Sawaya1" target="_blank">[20]</a>. Surgery was primarily carried out according to the 5-ALA signal, with corresponding iMRI resection controls carried out following disappearances of this signal. The residual tumor volume following each iMRI scan has been indicated in parentheses. The green color code has been used to depict complete resection according to both modalities during the first iMRI scan itself. Yellow has been used to depict complete resection requiring several iMRI scans. Red has been used to depict intentional, incomplete tumor resection.</p

Patient data depicted includes age, sex, presenting symptoms, tumor localization, the maximum dimensions of the tumor, the pre- and postoperative Karnofsky Performance Scale Index (KI) and the Neuropathological diagnosis according to the current WHO Classification System.

<p>All included patients had suspected malignant gliomas (WHO grade III or IV), i.e. evidencing contrast enhancement.</p

Dual intraoperative visualization approach and anatomical view.

<p>On the basis of a typical case of a tumor in the vicinity of an eloquent area, we demonstrate that the risk of missing tumor remnants covered by non-pathological tissue is eliminated through an iMRI control. A, The first iMRI scan carried out following the disappearance of the 5-ALA signal depicted a residual contrast enhancing area (marked by arrows). B, Tumor resection was resumed following re-segmentation and update of the neuronavigation. C, During resection of the intervening layer of non-pathological tissue, the 5-ALA signal reappeared (marked by arrows) and corresponded to the re-segmented contrast enhancing area.</p

Fluorescence guided tumor localization and eloquent area visualization.

<p>On the basis of the same case, we demonstrate the potential dangers, above all of post-operative neurological deterioration, associated with resection carried out according to the 5-ALA signal alone without the safeguard of neuronavigation segmentation and iMRI scans. A, Following resection of the bulk of the tumor, a faint 5-ALA signal was detectable (marked by arrows). B, The corresponding neuronavigation segmentation however, demonstrated that the pyramidal tract was reached (marked by arrows), and that further resection would result in postoperative neurological deterioration. C, The corresponding iMRI control confirmed the close proximity of the resection margin to the pyramidal tract (depicted in pink).</p

Dual intraoperative visualization approach in different functional grade patients.

<p>A, Extent of resection was determined by 5-ALA and the results verified through iMRI. This was defined as one sequence of the procedure. 21 sequences showing complete resection according to 5-ALA were confirmed by iMRI (complete 5-ALA: yes, complete MRI: yes – green bar, first column). 14 sequences showing complete resection according to 5-ALA could not be confirmed by iMRI, which detected residual tumor (complete 5-ALA: yes, complete MRI: no – green bar, second column). 29 sequences showed residual tumor both according to 5-ALA as well as iMRI (complete 5-ALA: no, complete MRI: no – blue bar, second column). The order of these sequences resulted in a p-value = 0.0005 (McNemar). B, Functional tumor localization was categorized according to Sawaya <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044885#pone.0044885-Sawaya1" target="_blank">[20]</a>. Tumors in non-eloquent areas were defined as functional grade I [marked with I]. Functional grade II was defined as tumor localization close to an eloquent brain area [marked with II]. Functional grade III (given as III at the top) was defined as tumor localization in an eloquent brain area. Extent of resection was calculated as a percentage of prior tumor volume. Within the subgroup I the intended 100% resection was achieved by surgery with 5-ALA alone. In the subgroup II 5-ALA alone resulted in a tumor resection of 71.7% (±7.285 sem), whereas additional use of iMRI significantly increased tumor resection to 100% (p-value<0.002; Student's t-test). The subgroup III showed significant difference in extent of tumor resection. The results of tumor resection were 57.6% (±6.01 sem) achieved with 5-ALA alone, whereas a further tumor resection up to 71.2% (±5.257 sem) could be achieved through the additional use of iMRI.</p

Table_1_Tracking cell turnover in human brain using 15N-thymidine imaging mass spectrometry.docx

Microcephaly is often caused by an impairment of the generation of neurons in the brain, a process referred to as neurogenesis. While most neurogenesis in mammals occurs during brain development, it thought to continue to take place through adulthood in selected regions of the mammalian brain, notably the hippocampus. However, the generality of neurogenesis in the adult brain has been controversial. While studies in mice and rats have provided compelling evidence for neurogenesis occurring in the adult rodent hippocampus, the lack of applicability in humans of key methods to demonstrate neurogenesis has led to an intense debate about the existence and, in particular, the magnitude of neurogenesis in the adult human brain. Here, we demonstrate the applicability of a powerful method to address this debate, that is, the in vivo labeling of adult human patients with 15N-thymidine, a non-hazardous form of thymidine, an approach without any clinical harm or ethical concerns. 15N-thymidine incorporation into newly synthesized DNA of specific cells was quantified at the single-cell level with subcellular resolution by Multiple-isotype imaging mass spectrometry (MIMS) of brain tissue resected for medical reasons. Two adult human patients, a glioblastoma patient and a patient with drug-refractory right temporal lobe epilepsy, were infused for 24 h with 15N-thymidine. Detection of 15N-positive leukocyte nuclei in blood samples from these patients confirmed previous findings by others and demonstrated the appropriateness of this approach to search for the generation of new cells in the adult human brain. 15N-positive neural cells were easily identified in the glioblastoma tissue sample, and the range of the 15N signal suggested that cells that underwent S-phase fully or partially during the 24 h in vivo labeling period, as well as cells generated therefrom, were detected. In contrast, within the hippocampus tissue resected from the epilepsy patient, none of the 2,000 dentate gyrus neurons analyzed was positive for 15N-thymidine uptake, consistent with the notion that the rate of neurogenesis in the adult human hippocampus is rather low. Of note, the likelihood of detecting neurogenesis was reduced because of (i) the low number of cells analyzed, (ii) the fact that hippocampal tissue was explored that may have had reduced neurogenesis due to epilepsy, and (iii) the labeling period of 24 h which may have been too short to capture quiescent neural stem cells. Yet, overall, our approach to enrich NeuN-labeled neuronal nuclei by FACS prior to MIMS analysis provides a promising strategy to quantify even low rates of neurogenesis in the adult human hippocampus after in vivo15N-thymidine infusion. From a general point of view and regarding future perspectives, the in vivo labeling of humans with 15N-thymidine followed by MIMS analysis of brain tissue constitutes a novel approach to study mitotically active cells and their progeny in the brain, and thus allows a broad spectrum of studies of brain physiology and pathology, including microcephaly.</p