Feasibility studies of multimodal nonlinear endoscopy using multicore fiber bundles for remote scanning from tissue sections to bulk organs.

Here, we report on the development and application of a compact multi-core fiber optical probe for multimodal non-linear imaging, combining the label-free modalities of Coherent Anti-Stokes Raman Scattering, Second Harmonic Generation, and Two-Photon Excited Fluorescence. Probes of this multi-core fiber design avoid moving and voltage-carrying parts at the distal end, thus providing promising improved compatibility with clinical requirements over competing implementations. The performance characteristics of the probe are established using thin cryo-sections and artificial targets before the applicability to clinically relevant samples is evaluated using ex vivo bulk human and porcine intestine tissues. After image reconstruction to counteract the data's inherently pixelated nature, the recorded images show high image quality and morpho-chemical conformity on the tissue level compared to multimodal non-linear images obtained with a laser-scanning microscope using a standard microscope objective. Furthermore, a simple yet effective reconstruction procedure is presented and demonstrated to yield satisfactory results. Finally, a clear pathway for further developments to facilitate a translation of the multimodal fiber probe into real-world clinical evaluation and application is outlined.

Multimodal nonlinear endomicroscopic imaging probe using a double-core double-clad fiber and focus-combining micro-optical concept.

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.

Reliable profile reconstruction of GRIN lenses produced by ion-exchange processes.

We propose a method that allows for a fast and accurate reconstruction of the refractive index profile of radially symmetric gradient index lenses fabricated by ion-exchange processes. The presented method enables the reconstruction of the profile up to the 10th polynomial order without direct spatially resolved refractive index measurements. It requires as input a working distance measurement at the paraxial limit and an accurate wavefront aberration measurement at full aperture. In addition, the approach combines the information about the optical behavior with the knowledge about the overall mass density changes of the glass rods during the ion exchange production processes to refine the reconstruction. Finally, the reconstruction of multiple profiles produced with different boundary conditions is demonstrated and confirms the functionality of the method.

Chip-on-the-tip compact flexible endoscopic epifluorescence video-microscope for in-vivo imaging in medicine and biomedical research.

We demonstrate a 60 mg light video-endomicroscope with a cylindrical shape of the rigid tip of only 1.6 mm diameter and 6.7 mm length. A novel implementation method of the illumination unit in the endomicroscope is presented. It allows for the illumination of the biological sample with fiber-coupled LED light at 455 nm and the imaging of the red-shifted fluorescence light above 500 nm in epi-direction. A large numerical aperture of 0.7 leads to a sub-cellular resolution and yields to high-contrast images within a field of view of 160 µm. A miniaturized chip-on-the-tip CMOS image sensor with more than 150,000 pixels captures the multicolor images at 30 fps. Considering size, plug-and-play capability, optical performance, flexibility and weight, we hence present a probe which sets a new benchmark in the field of epifluorescence endomicroscopes. Several ex-vivo and in-vivo experiments in rodents and humans suggest future application in biomedical fields, especially in the neuroscience community, as well as in medical applications targeting optical biopsies or the detection of cellular anomalies.

Design and evaluation of new color-corrected rigid endomicroscopic high NA GRIN-objectives with a sub-micron resolution and large field of view.

We demonstrate new GRIN-based endomicroscopic objectives for high resolution single photon fluorescence imaging modalities. Two endoscopic optical design approaches are presented in detail utilizing firstly diffractive and secondly refractive optical elements for the color correction in a spectral range from 488 nm to 550 nm. They are compared with their precursor device experimentally and by simulation. Inherent aberrations for off-axis field points could be lowered remarkably compared with the values of the state-of-the-art system by increasing the intrinsic optical complexity but maintaining the outer spatial dimensions. As a result, those presented objectives predict a diffraction-limited imaging of objects up to 300 µm in diameter with a numerical aperture of 0.8 while keeping an overall outer diameter of the assembly at 1.4 mm. Lastly, confocal fluorescence imaging experiments focus on the comparison between the numerical predicted and the practically achieved quality parameters.