Micro-optics are increasingly used for minimally invasive in vivo imaging, in miniaturized microscopes and in lab-on-a-chip devices. Owing to optical aberrations and lower numerical apertures, a main class of microlens, gradient refractive index lenses, has not achieved resolution comparable to conventional microscopy. Here we describe high-resolution microlenses, and illustrate two-photon imaging of dendritic spines on hippocampal neurons and dual-color nonlinear optical imaging of neuromuscular junctions in live mice.

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.

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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.