The ultimate challenge for early diagnostics is labelfree high-resolution intratissue imaging without taking physical biopsies. A novel hybrid femtosecond laser tomograph provides in vivo optical biopsies of human skin based on non-linear excitation of autofluorescence and the detection of lipids and water by CARS. Applications include skin cancer detection, biosafety tests of intradermal nanoparticles, and the testing of anti-aging products. μmOverlay (CARS/autofluorescence) of the outermost skin layer (stratum corneum) of a healthy male volunteer

We present combined epi-coherent anti-Stokes Raman scattering (CARS) and multiphoton imaging with both chemical discrimination and subcellular resolution on human skin in vivo. The combination of both image modalities enables label-free imaging of the autofluorescence of endogenous fluorophores by two-photon excited fluorescence, as well as imaging of the distribution of intercellular lipids, topically applied substances and water by CARS. As an example for medical imaging, we investigated healthy and psoriasis-affected human skin with both image modalities in vivo and found indications for different lipid distributions on the cellular level.

We report on in vivo coherent anti-Stokes Raman scattering spectroscopy (CARS), two-photon fluorescence and second-harmonic-generation imaging on human skin with a novel multimodal clinical CARS/multiphoton tomograph. CARS imaging is realized by a combination of femtosecond pulses with broadband continuum pulses generated by a photonic crystal fiber. The images reveal the microscopic distribution of (i) non-fluorescent lipids, (ii) endogenous fluorophores and (iii) the collagen network inside the human skin in vivo with subcellular resolution. Examples of healthy as well as cancer-affected skin are presented.

The majority of existing coherent anti-Stokes Raman scattering (CARS) imaging systems are still huge and complicated laboratory systems and neither compact nor user-friendly nor mobile medically certified CARS systems. We have developed a new flexible multiphoton/CARS tomograph for imaging in a clinical environment. The system offers exceptional 360° flexibility with a very stable setup and enables label free ‘in vivo histology’ with chemical contrast within seconds. It can be completely operated by briefly trained non-laser experts. The imaging capability and flexibility of the novel in vivo tomograph are shown on optical biopsies with subcellular resolution and chemical contrast of patients suffering from psoriasis and squamous cell carcinoma.

A preliminary clinical trial using state‐of‐the‐art multiphoton tomography (MPT) and optical coherence tomography (OCT) for three‐dimensional (3D) multimodal in vivo imaging of normal skin, nevi, scars and pathologic skin lesions has been conducted. MPT enabled visualization of sub‐cellular details with axial and transverse resolutions of <2 μm and <0.5 μm, respectively, from a volume of 0.35 × 0.35 × 0.2 mm3 at a frame rate of 0.14 Hz (512 × 512 pixels). State‐of‐the‐art OCT, operating at a center wavelength of 1300 nm, was capable of acquiring 3D images depicting the layered architecture of skin with axial and transverse resolutions ∼8 μm and ∼20 μm, respectively, from a volume of 7 × 3.5 × 1.5 mm3 at a frame rate of 46 Hz (1024 × 1024 pixels). This study demonstrates the clinical diagnostic potential of MPT/OCT for pre‐screening relatively large areas of skin using 3D OCT to identify suspicious regions at microscopic level and subsequently using high resolution MPT to obtain zoomed in, sub‐cellular level information of the respective regions (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)