Grintech (Germany)

Intravital imaging of bone marrow provides a unique opportunity to study cellular dynamics and their interaction with the tissue microenvironment, which governs cell functions and metabolic profiles. To optically access the deep marrow of long bones, we previously developed a microendoscopy system for longitudinal two-photon fluorescence imaging of the murine femur. However, this does not provide information on cell functions or metabolism, for which quantification fluorescence lifetime imaging (FLIM) has proven to be a versatile tool. We present and characterize FLIMB, an adapted GRIN-based microendoscopic system capable of performing reliable, co-registered TCSPC-based two-photon excited FLIM and fluorescence imaging in the femur of fluorescent reporter mice, at sub-cellular resolution. Using FLIMB, we demonstrate metabolic imaging via NAD(P)H-FLIM and intracellular Ca2+ signaling via FRET-FLIM in immune cell subsets, in the femoral marrow. This method retains the power to study molecular mechanisms underlying various cell functions in tissue context thus providing new insights into bone biology.

We present here the concept of a MEMS-mirror based nonlinear endomicroscopic probe for coherent anti-Stokes Raman scattering (CARS), two-photon excitation fluorescence (TPEF), and second harmonic generation (SHG). The rigid probe head is 5 mm in diameter and 4 cm in length, offering a large field of view (FOV) with a high numerical aperture (NA). It incorporates a double-core fiber delivering two excitation wavelengths of CARS in isolated cores, with a large cladding area to increase the collection efficiency of the nonlinear signal from the tissue. A diffractive grating element is included in the probe to compensate for the spatial offset of two emission cores.

Nonlinear optical endoscopy is an attractive technique for biomedical imaging since it promises to give access to high resolution imaging in vivo. Among the various techniques used for endoscopic contrast generation, coherent anti-Stokes Raman scattering (CARS) is especially interesting. CARS endoscopy allows molecule specific imaging of unlabeled samples. In this contribution, we describe the design, implementation, and experimental characterization of a rigid, compact CARS endoscope with a spatial resolution of 750 nm over a field of view of roughly 250 μm. Omission of the relay optics and use of a gradient index lens specifically designed for this application allow one to realize these specifications in an endoscopic unit which is 2.2 mm wide over a length of 187 mm, making clinical applications during surgical interventions possible. Multimodal use of the endoscope is demonstrated with images of samples with neurosurgical relevance.

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.

High power diode lasers have become very attractive as an intelligent tool for direct material processing as well as pump sources for miniaturized solid state lasers and fiber lasers since optical beam transformation systems generate the required beam circularization from the originally highly asymmetric brightness of the output beam. This presentation focuses on reliable microassembling processes to realize micro-optical beam transformation systems for high power diode laser bars and stacks for the efficient coupling of the beam into optical fibers at reasonable costs. The modular semi automatic micro assembly set up realized consists of a six axes alignment system with an accuracy in the submicron range for the three spatial areas as well as microrad for the three rotational axes, a semi-automatic glue dispenser as well as respective grippers and magazines which are necessary for the handling of the micro optical components. The established automatic processes for the mounting of the fast axis collimation lenses are of particular interest for the assembly of e.g. fiber coupled modules of diode laser stacks whereas more than 20 fast-axis collimations are necessary.