gms | German Medical Science

Objective: Non-linear optical microscopy (NLOM) is a powerful technique that enables confocal imaging of brain tissue using two-photon fluorescence (TPF), second harmonic generation and coherent anti-Stokes Raman scattering (CARS). Several optical processes can be excited to build a multimodal image of the nervous tissue avoiding the use of staining. This technique is therefore especially interesting for in vivo application in humans. Nevertheless, it requires irradiation of the tissue with high optical peak power, which could lead to photodamage of the tissue. For safe in-vivo use, the potential of this novel technique to induce damage on brain tissue was evaluated.

Method: The NLOM system was composed of a laser scanning microscope and two ps NIR fiber lasers. The characterization of photodamage was performed on rehydrated mouse brain cryosection to mimic the fresh tissue, on ex-vivo fresh mouse brain and in a cranial window on living mice by serial irradiations (up to 4000) of an area 80x120 µm². Vibrational spectroscopy and staining of the sections after irradiation enabled biochemical and histological characterization of damage.

Results: Irradiation was found to produce a strong increase of green TPF intensity (up to 4 times the background) and a decrease in the CARS signal. The intensity of the induced TPF was used as marker to calculate the damage threshold and revealed a non-linear relationship of damage vs. laser power. The fluorescence could be detected also after fixation, suggesting the formation of stable fluorescent compounds inside the tissue. Analysis of the tissue by means of Raman and FT-IR spectroscopy underlined alterations in the lipid and protein content, as well as formation of new carbon compounds. Histological and IHC stainings showed that the damage was not correlated with a certain cellular compartment or cell type. The experiments on fresh ex-vivo mouse brain tissue and in-vivo displayed the same qualitative behavior. Photodamage thresholds were found to range from less than 100 consecutive irradiations for a laser power of 50 mW, to more than 2000 for 25 mW.

Conclusions: The strong increase of green TPF induced by photodamage of brain tissue, can be detected during the imaging itself, providing an internal reference for the level of damage and enabling damage detection in its earliest stage. The dose to produce damage was found at least 10 fold higher than the required dose for imaging, suggesting that the use of NLOM for diagnostic purposes is safe in our setup.