# Classification of Hyperspectral In Vivo Brain Tissue Based on Linear Unmixing
## 3 Materials and Methods
### In Vivo Human Brain HS Dataset  
The in vivo human brain HS images were captured by using a customized intraoperative HS acquisition system developed in [30], which was part of the European project HELICoID (HypErspectraL Imaging Cancer Detection) (618080) [31]. The system was formed by a push broom HS camera operating in the VNIR (visible and near-infrared) spectral range from 400 to 1000 nm (Hyperspec(r) VNIR A-Series, Headwall Photonics Inc., Fitchburg, MA, USA); an illumination system based on a quartz tungsten halogen (QTH) lamp of 150 W with a broadband emission between 400 and 2200 nm; and a scanning platform to provide the necessary movement for the push broom scanning, capable of covering an effective area of 230 mm. The resulting HS cubes contained 826 spectral bands with a spectral sampling of 0.73 nm, a spectral resolution of 2-3 nm, and a spatial resolution of 128.7 \(\upmu\)m. The HS cube had a maximum size of 1004 \(\times\) 1787 pixels with a maximum image size of 129 \(\times\) 230 mm, where each pixel represents a sample area of 128.7 \(\times\) 128.7 \(\upmu\)m. However, each HS image of the database was manually segmented according to the region of interest, where the exposed brain tissue (parenchymal tissue) was present. The segmented images had a minimum and a maximum of 298 \(\times\) 253 pixels and 611 \(\times\) 527 pixels, respectively. The HS database employed in this study was composed by twenty-six images from sixteen adult patients, and it was described in [32]. Patients underwent craniotomy for resection of intra-axial brain tumor or another type of brain surgery during clinical practice at the University Hospital Doctor Negrin at Las Palmas de Gran Canaria (Spain). Eight different patients were diagnosed with grade IV glioblastoma(GBM) tumor, and eleven HS images of exposed tumor tissue were captured. The remaining patients were affected by other types of tumors or affected by other pathologies that required performing a craniotomy to expose the brain surface. Written informed consent was obtained from all participant subjects, and the study protocol and consent procedures were approved by the Comite Etico de Investigacion Clinica-Comite de Etica en la Investigacion (CEIC/CEI) of the University Hospital Doctor Negrin. To acquire the HS images during the surgical procedures, it was necessary to follow the protocol established in [30]. Craniomy and resection of the dura were performed, and the operating surgeon initially identified the approximate location of the normal brain and tumor (if applicable). The surgeons placed sterilized rubber ring markers on the surface of the brain, where the presence of tumor and normal tissue was identified based on preoperative imaging data. Once the markers were located, the imaging operator captured the HS image, and the clinical expert performed a biopsy of the tissue located within the tumor markers, sending the sample to a pathologist. By this analysis, the physician confirmed the presence or absence of the tumor by histopathological diagnosis and also determined the tumor type and grading. The HS acquisition system was operated by an engineer in charge of positioning the HS camera over the exposed brain surface, controlling the illumination system, and setting the image size to be captured according to the size of the exposed brain surface. The environmental illumination in the operating room did not affect the capture process due to the strong and precise illumination system available in the acquisition system. However, the surgical lighthead had to be turned off during the acquisition procedure, since it could interfere with the acquisition system illumination. The operator must avoid unintentional movements of the acquisition system during the capture. In case of an unexpected movement, the acquisition process was repeated. At the beginning of the surgical procedure, dark and white reference images were captured to perform the HS image calibration as explained in the next section.