## 5 Discussion
The presented fluorescent imaging and fused segmentation system prove extremely capable of discriminating cancerous regions from the remaining tissue and accurately approximating the tumor margins in an almost real-time mode, revealing the potential to be utilized as an efficient, unsupervised, and user-friendly assisting tool for image-guided surgery. The implemented equipment and software enabled the clear identification of the brain tumor margin-penetrating cells and reconstructed the geometrical topology of the tumor extent, offering the capability for numerical measurements regarding the shape, size, area, and depth of cancerous tissue segments and assisting the medical expert in a more detailed diagnosis and targeted treatment. Combining fluorescence imaging with molecular probes targeting tumor-specific markers and advanced image segmentation procedures, the outcome of the presented method could be significantly enhanced and more extensively validated on a larger dataset, further justifying the application of the proposed technique in laboratory assessments. Establishing protocols for safe administration of fluorescent molecules, these would be introduced into glioma tissues or cells either before surgery or applied to the tumor intraoperatively; typical applications encompass the areas of fluorescence-guided surgery (FGS) and confocal laser endomicroscopy (CLE). As a result, the specific imaging modality could remarkably improve decision-making protocols in the course of operations for brain tumor resection and significantly facilitate brain surgery neuropathology during an operation. Today, surgical operations are still primarily conducted under white light endoscopy; therefore, in addition to white-light imaging, fluorescence cameras or surgical microscopes can be used including both wavelength-specific excitation sources, such as lasers, LEDs, or filtering of a polychromatic light source or combinations of these approaches together with wavelength-specific imaging detectors [61]. Several challenges remain that can be addressed holistically in terms of the imaging hardware, photophysics of dyes, and tissue composition. Any digital imaging system (optical, X-ray, gamma-ray) must be quantum noise limited (Poisson limited); equivalently, quantum noise must prevail over the electronic noise of the system to avoid non-linearities. In the case of molecular imaging, the intensity and quality of the acquired signals are critical factors for the efficient utilization of fluorescence throughout image-guided surgery procedures. It is imperative that the fluorescence intensities in surgically challenging tissue structures are increased enough so that they can be systematically separated from neighboring tissue components and large signal-to-background ratio levels are achieved. In addition, light absorption and scattering from endogenous molecules are key characteristics that prevent fluorescent emissions from penetrating through tissue. Therefore, dye excitation at the maximum level and increased emission detection performance are of extreme significance. The extent of fluorescent emissions is also significantly affected by photophysical properties, namely, the absorption coefficient, i.e., the measure of the distributed light absorption in a dye, and the quantum yield, i.e., an index that represents the capability of a dye to transform absorbed light into a fluorescent emission. As a result, different tissues exhibit different background signals, due to the presence of different endogenous dyes, such as flavins, flavoproteins, collagen, and elastin, nicotinamide adenine dinucleotide (NAD)+hydrogen (H) (NADH), and other redox controllers. Again, high fluorescence intensities in surgically challenging anatomies would minimize the detrimental background signals, by maintaining high-signal-to-background ratios.