gms | German Medical Science

Objective: The possibility of in situ tissue diagnostics for tumor operations in neurosurgery would be great benefit to the patient's outcome. Multiphoton microscopy is a method that generates histological images from the molecular composition of the tissue without any staining. The description of qualitative and quantitative histological features in the multi-photon microscopic image of various tumor entities was the subject of this study and aimed to establish the basis for a rapid intraoperative diagnosis.

Methods: 419 human tissue samples of normal and tumorous brain tissue from routine operations were used as cryosections. Of each sample, at least 100 multimodal multiphoton images (100 μm x 200 μm) were taken. The intensities for CARS (coherent anti-Stokes Raman scattering) TPEF (endogenous two photons excited fluorescence) and SHG (second harmonic generation) were simultaneously recorded. Gray and white matter, glioblastoma multiforme, glioma (WHO III) and five different metastatic entities were analyzed and evaluated for their respective characteristics in multiphoton microscopy. HE, immunohistochemical staining and histopathological diagnosis served as references.

Results: Even with a pixel size of 1 μm and an acquisition time of 700 ms, the known features of the tumor tissue types could be displayed in the multiphoton microscopic. A pixel size of 0.2 microns also allowed the identification of subcellular features. Normal tissue was characterized by an ordered and regular tissue. Gray matter was represented by large cells with polar inclusions (TPEF) and white matter was essentially displayed by many axons (CARS). The brain tumors and metastases showed increased cell density and particularly large cells with perinuclear inclusions (CARS TPEF). Single axons could be found in 20% of the samples of brain tumors, these were solitary and significantly thinner. In all cases, metastases showed marked network structures in the SHG channel, which were absent in the other tissues.

Conclusion: Multimodal multiphoton microscopy enables high-resolution histological images in a fraction of the time of staining. The high information rate of the images allows the differentiation of normal and different pathological tissue on the basis of specific characteristics. The results suggest that multiphoton microscopy may in the future enable intraoperative histopathological diagnosis and has the potential for in situ discrimination of normal and tumorous tissue.