Objective: Two Photon Microscopy (TPM) is an imaging method producing high definition images out of the volume of samples, e.g. of human tissue. Up to now the technology and application parameters limit the method to research application. A new approach will enable the TPM to make the step into a method for clinical application.
Material and methods: To realize the imaging by TPM a near infrared femtosecond-laser was used to excite endogenous fluorophores in the skin. The advantage of the two-photon-excitation is that the fluorescence is located only in the laser focus. By moving the laser beam through the tissue, the fluorescence signal can be detected with a resolution of 0.5µm. The combination of the position of the laser beam and the signal strength of the two-photon-excited fluorescence (TPEF) leads to a digital image.
The new feature of the new technology presented here is the lateral scanning regime that increases the up to now very limited field of view of the TPM to 10mm. Optical distortion, vignetting and other disadvantages are eliminated. In order to realize this method a special scan lens has been developed and strictly controlled high definition linear lateral scanning stages are used.
Additionally to the TPEF the second harmonic generation (SHG) in the tissue, especially in the collagen fibers, can be detected by a second channel in the TPM.
Results: The fusion image of both signals leads to a false colored images which allow for a clear separation between the cellular parts of the skin (e.g. epidermis) and the fibrous dermis, leading to a clear demarcation of the basal membrane and resemble a histological like image. The results of the measures will be presented and discussed with different tissue images comparing TPM images and H&E stained histological specimen.
Conclusion: With this new lateral scanning technology it is possible to analyze tissue samples of human skin by Two Photon Microscopy with an image size that is appropriate for the diagnosis. The lateral scanning is an excellent method to survey specimens with an improved image quality without distortion. The prospects of the new method and the upcoming applications to be realized in the near future will be presented.
Acknowledgement: The work was carried out with the support of Bundesministerium für Forschung und Technologie (FKZ 13N8786).
