Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.

Coherent anti-Stokes Raman scattering (CARS) microscopy provides fine resolution imaging and displays morphochemical properties of unstained tissue. Here, we evaluated this technique to delineate and identify brain tumors. Different human tumors (glioblastoma, brain metastases of melanoma and breast cancer) were induced in an orthotopic mouse model. Cryosections were investigated by CARS imaging tuned to probe C-H molecular vibrations, thereby addressing the lipid content of the sample. Raman microspectroscopy was used as reference. Histopathology provided information about the tumor's localization, cell proliferation and vascularization. The morphochemical contrast of CARS images enabled identifying brain tumors irrespective of the tumor type and properties: All tumors were characterized by a lower CARS signal intensity than the normal parenchyma. On this basis, tumor borders and infiltrations could be identified with cellular resolution. Quantitative analysis revealed that the tumor-related reduction of CARS signal intensity was more pronounced in glioblastoma than in metastases. Raman spectroscopy enabled relating the CARS intensity variation to the decline of total lipid content in the tumors. The analysis of the immunohistochemical stainings revealed no correlation between tumor-induced cytological changes and the extent of CARS signal intensity reductions. The results were confirmed on samples of human glioblastoma. CARS imaging enables label-free, rapid and objective identification of primary and secondary brain tumors. Therefore, it is a potential tool for diagnostic neuropathology as well as for intraoperative tumor delineation.

In order to provide an alternative to day-old chick culling in the layer hatcheries, a noninvasive method for egg sexing is required at an early stage of incubation before onset of embryo sensitivity. Fluorescence and Raman spectroscopy of blood offers the potential for precise and contactless in ovo sex determination of the domestic chicken (Gallus gallus f. dom.) eggs already during the fourth incubation day. However, such kind of optical spectroscopy requires a window in the egg shell, is thus invasive to the embryo and leads to decreased hatching rates. Here, we show that near infrared Raman and fluorescence spectroscopy can be performed on perfused extraembryonic vessels while leaving the inner egg shell membrane intact. Sparing the shell membrane makes the measurement minimally invasive, so that the sexing procedure does not affect hatching rates. We analyze the effect of the membrane above the vessels on fluorescence signal intensity and on Raman spectrum of blood, and propose a correction method to compensate for it. After compensation, we attain a correct sexing rate above 90% by applying supervised classification of spectra. Therefore, this approach offers the best premises towards practical deployment in the hatcheries.