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.

Design and test of a rigid endomicroscopic system for multimodal imaging and femtosecond laser ablation.

Significance: Conventional diagnosis of laryngeal cancer is normally made by a combination of endoscopic examination, a subsequent biopsy, and histopathology, but this requires several days and unnecessary biopsies can increase pathologist workload. Nonlinear imaging implemented through endoscopy can shorten this diagnosis time, and localize the margin of the cancerous area with high resolution. Aim: Develop a rigid endomicroscope for the head and neck region, aiming for in-vivo multimodal imaging with a large field of view (FOV) and tissue ablation. Approach: Three nonlinear imaging modalities, which are coherent anti-Stokes Raman scattering, two-photon excitation fluorescence, and second harmonic generation, as well as the single photon fluorescence of indocyanine green, are applied for multimodal endomicroscopic imaging. High-energy femtosecond laser pulses are transmitted for tissue ablation. Results: This endomicroscopic system consists of two major parts, one is the rigid endomicroscopic tube 250 mm in length and 6 mm in diameter, and the other is the scan-head (10×12×6 cm3 in size) for quasi-static scanning imaging. The final multimodal image accomplishes a maximum FOV up to 650 µm, and a resolution of 1 µm is achieved over 560 µm FOV. The optics can easily guide sub-picosecond pulses for ablation. Conclusions: The system exhibits large potential for helping real-time tissue diagnosis in surgery, by providing histological tissue information with a large FOV and high resolution, label-free. By guiding high-energy fs laser pulses, the system is even able to remove suspicious tissue areas, as has been shown for thin tissue sections in this study.

Field curvature reduction in miniaturized high numerical aperture and large field-of-view objective lenses with sub 1 µm lateral resolution.

In this paper the development of a miniaturized endoscopic objective lens for various biophotonics applications is presented. While limiting the mechanical dimensions to 2.2 mm diameter and 13 mm total length, a numerical aperture of 0.7 in water and a field-of-view (FOV) diameter of 282 µm are achieved. To enable multimodal usage a wavelength range of 488 nm to 632 nm was considered. The performed broad design study aimed for field curvature reduction when maintaining the sub 1 µm resolution over a large FOV. Moreover, the usage of GRadient-INdex (GRIN) lenses was investigated. The resolution, field curvature improvement and chromatic performance of the novel device were validated by means of a confocal laser-scanning-microscope.

Design parameters for Airy beams in light-sheet microscopy.

We derive analytical expressions for the length, thickness, and curvature of an Airy light sheet in terms of basic parameters of the cubic phase and the paraxially defined focusing optics that form the beam. The length and thickness are defined analogously to the Rayleigh range and beam waist of a Gaussian beam, hence providing a direct and quantitative comparison between the two beam types. The analytical results are confirmed via numerical Fresnel propagation simulations and discussed within the context of light-sheet microscopy, providing a comprehensive guide for the design of the illumination unit.

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.

Nonlinear optical endomicroscopy for label-free functional histology in vivo.

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.

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.

In vivo fluorescence imaging with high-resolution microlenses.

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.