Multiphoton CARS imaging tool promising for skin biopsies

Enabling label-free, 3-D optical biopsies from the skin that provide information on morphology at a subcellular level as well as lipid and water content, JenLab (Jena, Germany), together with the Charite Hospital and APE GmbH (both in Berlin, Germany), accomplished clinical coherent anti-Stokes Raman scattering (CARS) imaging in patients with dermatological disorders.

Previously, the only available tools for CARS imaging were microscopes for the investigation of cells and ex-vivo tissue. CARS tomography that didn't provide information on the distribution of autofluorescent molecules and collagen would not be sufficient for pathologists to decide whether or not tissue is abnormal. But combining multiphoton fluorescence/SHG tomography (MPT) and CARS imaging would provide the necessary information and subcellular resolution.

Dubbed MPT-CARS, the certified clinical hybrid tomograph combines JenLab's DermaInspect clinical multiphoton tomograph and the add-on CARS module based on APE’s optical parametric oscillator (OPO). The OPO provides the required second laser beam for CARS imaging. The CARS signal relies on the wavelength difference between DermaInspect’s tunable femtosecond pump laser beam (710–920 nm) and the OPO’s Stokes laser beam (1000–1300 nm) that has to be tuned to match vibrational modes of the molecule of interest.

In order to image the intratissue lipid distribution in patients suffering from psoriasis, cancer, and other skin diseases, the MPT-CARS wavelengths were tuned to 811 and 1053 nm, respectively. The CARS signal from lipids arises in the red spectral range (660 nm) from the symmetric C-H₂ stretch vibration band near 2845 cm^-1 and could be detected with a red-sensitive photomultiplier. Collagen was detected in the blue spectral region due to SHG and the biomolecules NAD(P)H, flavins, keratin, elastin and melanin in the green/yellow. The combined mean power of both beams did not exceed 50 mW, as required by the European Notified Body for the approval of medical products. The standard acquisition time for an optical section (0.4 × 04 mm²) was 7 seconds. Up to 20 sections at different tissue depths were obtained per region of interest with a piezo-driven high NA objective.

The distribution of topically applied lipid-water-emulsions was investigated in time-lapse studies that are of high interest for cosmetics and pharmaceutical companies. Further clinical studies are currently being performed within the German BMBF project ChemoPraevent to further evaluate the potential of MPT-CARS for early diagnosis of skin diseases, optimization of treatment procedures, the evaluation of the efficacy of cosmetics, and the investigation of side effects of pharmaceutical drugs such as chemotherapy agents.