Pseudo-HE images derived from CARS/TPEF/SHG multimodal imaging in combination with Raman-spectroscopy as a pathological screening tool

Background: Due to the steadily increasing number of cancer patients worldwide the early diagnosis and treatment of cancer is a major field of research. The diagnosis of cancer is mostly performed by an experienced pathologist via the visual inspection of histo-pathological stained tissue sections. To save valuable time, low quality cryosections are frequently analyzed with diagnostic accuracies that are below those of high quality embedded tissue sections. Thus, alternative means have to be found that enable for fast and accurate diagnosis as the basis of following clinical decision making. Methods: In this contribution we will show that the combination of the three label-free non-linear imaging modalities CARS (coherent anti-Stokes Raman-scattering), TPEF (two-photon excited autofluorescence) and SHG (second harmonic generation) yields information that can be translated into computational hematoxylin and eosin (HE) images by multivariate statistics. Thereby, a computational HE stain is generated resulting in pseudo-HE overview images that allow for identification of suspicious regions. The latter are analyzed further by Raman-spectroscopy retrieving the tissue's molecular fingerprint. Results: The results suggest that the combination of non-linear multimodal imaging and Raman-spectroscopy possesses the potential as a precise and fast tool in routine histopathology. Conclusions: As the key advantage, both optical methods are non-invasive enabling for further pathological investigations of the same tissue section, e.g. a direct comparison with the current pathological gold-standard.