gms | German Medical Science

Objective: In recent years we could show that high resolution multiphoton (MF) microscopy can differentiate glioma from adjacent brain in native tissue samples. Although MF is in principle harmless to the living tissue and is clinically used for ophthalmic and dermatologic imaging, it has not yet been applied for real-time in vivo imaging during neurosurgical procedures.

Method: We applied a new generation of MF microscopes, the MPTflex™ Multiphoton Laser Tomograph (Jenlab, Germany). Excitation of native samples was performed using a 40fs pulsed laser with a tuneable wavelength of 700–850 nm. The MF microscope allowed fluoroscence intensity- and fluorescence lifetime imaging (FLIM) in sample volumes of 800x800x300 µm. Image acquisition took about 20s per microscopic field. The instrument could be fitted with a small resterilizable endoscopic tip or a larger resterilizable coverglass. The microscopes head is mounted on a flexible arm and fitted with a handle and breaks which resemble those of a conventional neurosurgical operating microscopes. We first examined cultured cells glioma cells (U87) and stem cell like subpopulations (10,1080 and 1095) as monolayer culture and in a mouse-tumor model. Besides native glioma tissue and adjacent brain tissue were examined. All samples were than processed for conventional histopathological examination.

Results: Morphologic fluorescence intensity imaging as well as FLIM of cultured tumor cells, harvested tumor-bearing mouse brains and native tissue samples using the MPTflex™ Multiphoton Laser Tomograph could clearly differentiate between glioma and adjacent brain tissue. Finally we applied the instrument in vivo during resection of a glioblastoma multiforme, which constituted the first intraoperative application of a MF microscope worldwide. Intraoperative image quality was comparable to ex vivo examinations of native tissue samples and in principle allowed for real-time identification of brain tumor tissue on a cellular scale.

Conclusions: We here present the first intraoperativ application of a MF microscope. It allowed in vivo identification of tumor tissue on a cellular level in real-time. However the instrument is not yet optimized for the neurosurgical setting and the microscopic fields are small. Therefore the technology could be combined with technologies for intraoperative imaging with a lower resolution like OCT or 5-ALA guided surgery.