Bernhard Messerschmidt

This manuscript reports on the first two-photon, label-free, metabolic imaging of biological tissues in vivo at histological resolution on an extremely compact, fiber-optic endomicroscopy platform. This system provides new opportunities for performing non-invasive and functional histological imaging of internal organs in vivo, in situ and in real time. As a routine clinical procedure, traditional histology has made significant impacts on medicine. However, the procedure is invasive and time consuming, suffers random sampling errors, and cannot provide in vivo functional information. The technology reported here features an extremely compact and flexible fiber-optic probe ~2 mm in diameter, enabling direct access to internal organs. Unprecedented two-photon imaging quality comparable to a large bench-top laser scanning microscope was achieved through technological innovations in double-clad fiber optics and miniature objective lenses (among many others). In addition to real-time label-free visualization of biological tissues in situ with subcellular histological detail, we demonstrated for the first time in vivo two-photon endomicroscopic metabolic imaging on a functioning mouse kidney model. Such breakthroughs in nonlinear endoscopic imaging capability present numerous promising opportunities for paradigm-shifting applications in both clinical diagnosis and basic research. A compact, flexible probe based on nonlinear optics offers doctors and researchers a noninvasive way to directly image internal organs. Histology using optical microscopy has been widely employed for diagnosing disease, but it typically takes a few days to obtain results and it does not provide functional information. Now, Xingde Li at Johns Hopkins University and coworkers have developed a 2-millimeter-diameter probe that uses two-photon imaging to obtain images of organs inside the body that are comparable in quality to those obtained by a laser scanning microscope. They realized this through innovations in double-clad fiber optics, a miniature objective lens and short pulse management. The team demonstrated the potential of their probe by using it to obtain metabolic imaging of a functioning mouse kidney model. The probe is promising for both diagnosing disease and basic research.

We present a compact multimodal fiber probe that enables the simultaneous recording of nonlinear imaging modalities like coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excited auto-fluorescence (TPEF) for biomedical applications. The probe is based on a gradient index lens design and a multi-core fiber supplying the excitation laser light. The multi-core fiber preserves the spatial relationship between the entrance and output; therefore, the laser scanning procedure can be shifted from the distal to the proximal end of the probe. No moving parts or electric power are required in situ. The generated sample signals can be collected in the backward (epi) direction and transferred to a detection setup with a multimode fiber integrated in the probe head. The first CARS/SHG/TPEF multimodal tissue images recorded with the introduced fiber probe will be presented.

Multimodal non-linear microscopy combining coherent anti-Stokes Raman scattering, second harmonic generation, and two-photon excited fluorescence has proved to be a versatile and powerful tool enabling the label-free investigation of tissue structure, molecular composition, and correlation with function and disease status. For a routine medical application, the implementation of this approach into an in vivo imaging endoscope is required. However, this is a difficult task due to the requirements of a multicolour ultrashort laser delivery from a compact and robust laser source through a fiber with low losses and temporal synchronization, the efficient signal collection in epi-direction, the need for small-diameter but highly corrected endomicroobjectives of high numerical aperture and compact scanners. Here, we introduce an ultra-compact fiber-scanning endoscope platform for multimodal non-linear endomicroscopy in combination with a compact four-wave mixing based fiber laser. The heart of this fiber-scanning endoscope is an in-house custom-designed, single mode, double clad, double core pure silica fiber in combination with a 2.4 mm diameter NIR-dual-waveband corrected endomicroscopic objective of 0.55 numerical aperture and 180 µm field of view for non-linear imaging, allowing a background free, low-loss, high peak power laser delivery, and an efficient signal collection in backward direction. A linear diffractive optical grating overlays pump and Stokes laser foci across the full field of view, such that diffraction-limited performance is demonstrated for tissue imaging at one frame per second with sub-micron spatial resolution and at a high transmission of 65% from the laser to the specimen using a distal resonant fiber scanner.