This PDF file contains the front matter associated with SPIE Proceedings Volume 10854, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

Conference Presentation for "Training the next generation to interpret IVM images"

Conference Presentation for "Collaborative opportunities between engineers and pathologists: defining standards and requirements for implementation"

Environmental Enteric Dysfunction (EED) is a poorly understood condition of the small intestine that is prevalent in regions of the world with inadequate sanitation and hygiene. EED affects 25% of all children globally and causes over a million deaths each year. The condition is associated with increased intestinal permeability, bacterial translocation, inflammation and villous blunting. The loss of absorptive area and intestinal function leads to nutrient malabsorption, with long term outcomes characterized by stunted growth and neurocognitive development. Currently, the only way to directly evaluate the morphology of the intestine is endoscopy with mucosal biopsy. Yet because EED is endemic in low and middle-income countries, endoscopy is untenable for studying EED. As a result, the diagnosis of EED and the assessment of the efficacy of EED interventions is hampered by an inability to evaluate the intestinal mucosa. Our lab has previously developed a technology termed tethered capsule OCT endomicroscopy (TCE). The method involves swallowing an optomechanically-engineered pill that generates 3D images of the GI tract as it traverses the lumen of the organ via peristalsis, assisted by gravity. In order to study the potential of using TCE to investigate EED, we initiated and conducted a TCE study in adolescents at Aga Khan Medical Center in Pakistan. To make swallowing easier, the tethered capsule’s size was reduced from 11x25 mm to 8x22 mm. Villous morphologic visualization was enhanced by building a notch (x mm deep, y mm wide) in the capsule’s imaging window. To date, 26 Pakistani subjects with ages ranging from 14 to 18 y/o (16.4 +/- 1.0) have been enrolled and imaged. A total of 19 subjects were able to swallow the capsule. Of those, 9 successfully passed through the pylorus, allowing successful microscopic imaging of the entire duodenum. There were no adverse events in any of the cases. Maximum villous height and width were measured from 3 randomly chosen, representative frames from each Pakistan subject as well as a matching number from US controls. Preliminary results, comparing Pakistani vs US villous morphology, indicated that subjects from Pakistan have shorter (628.6 +/- 22.0 um and 492.3 +/- 13.2 um, respectively, p< 0.0001) and wider duodenal villi (244.9 +/- 8.8 um and 293.4 +/- 13.2 um, respectively, p< 0.0001). These findings suggest that OCT TCE of the duodenum may be a useful tool for evaluating villous morphology in EED.

Colorectal cancer, the third most common type of cancer globally, has ~1.4 million new cases and 694,000 deaths annually. Endoscopic photoacoustic (PA) imaging is a non-invasive imaging modality that provides structural, functional and molecular information while maintaining a deep depth. Several groups have reported different designs of endoscopic PA imaging systems that represent a significant step forward for the characterization of gastrointestinal (GI) cancer. However, these imaging systems are still not adequate for in-vivo clinical translation due to insufficient field-of-view, large probe diameters, and slow imaging speed. Endoscopic dual-modality photoacoustic and ultrasound imaging has the potential for early detection of cancer in the gastrointestinal tract. Currently, slow imaging speed is one of the limitations for clinical translation. In this study, we demonstrate an integrated endoscopic PA and US imaging system. Utilizing a high repetition rate pulsed laser, optimized rotary joint as well as proximal scanning method, this integrated imaging system is able to obtain morphological tissue information and vasculature of the GI tract simultaneously at a high imaging speed up to 50 frames/s (the fastest speed reported to date). We conducted in-vivo animal studies to demonstrate the performance of our imaging system for evaluating the GI tract. The results obtained from the in-vivo rat experiment showed that the typical layered architecture and vasculature can be identified by this integrated system with a high imaging speed.

Early detection of cancer lesions is a critical factor for improving disease prognosis. Detection rates might be improved by catheter based optical coherence tomography (OCT), which enables three dimensional high-resolution images acquisition of organ surfaces and luminal walls. However, OCT lacks in specificity; this limitation can be overcome combining it with targeted fluorescence. To this end, we developed a miniature motorized endoscopic probe with an outer diameter of 1.35mm and a rotation speed of 3,200rpm. Combining near-infrared fluorescence (NIRF) imaging with our OCT system we were able to specifically locate a cell-type by targeting its membrane receptors with fluorescently labelled monoclonal antibodies (mAbs). Exploiting double clad fiber (DCF) coupler technology the OCT and NIRF excitation signals were delivered to the endoscope tip through its single mode core, while the emitted fluorescence signal was efficiently collected by the DCF inner cladding. The catheter was demonstrated to acquire in situ images in a xenograft mouse model of human colorectal cancer, by metabolized mAb labelled with a near-infrared fluorophore (IRDye800CW). While circumferentially scanning the sample with the endoscope, the NIRF signal served as navigation tool to identify the tumour location; once a suspicious region was identified, B-scans and NIRF were acquired. NIRF and OCT images proved to complement each other by revealing molecular contrast within the surrounding tissue architectural context. Moreover, high degree of heterogeneity in the malignant tissue was revealed by the NIRF images. The excellent contrast provided by endoscopic immuno-NIRF-OCT demonstrated its potential to reduce erroneous sampling of tissue.

Multimodal endoscopes are highly complex and difficult to miniaturize, thus packing multiple optical systems into them requires ingenuity, creativity and a special set of optical design and optomechanical skills. While multimodal and miniaturized endoscope systems are becoming a hot topic nowadays, few designs have been explained in enough detail for the scientific community to utilize this knowledge. We present our approach to the design and manufacturing of an endoscopic prototype that encapsulates Optical Coherence Microscopy (OCM), Multi Photon Microscopy (MPM), and White Light navigation. In order to successfully encapsulate all three optical modalities in the endoscope, two optical paths were enabled in the same optical elements through the use of dichroic surfaces. One of the optical paths had to be bent to allow high resolution microscopy. The optics within the endoscope are aligned by the custom distal ferrule and a lens stacking technique. Additionally, a compact piezo actuator tube is used to scan the field of view for all imaging modalities, while 3D printed parts have been designed to accommodate the endoscope’s specific needs. To manufacture the prototype, custom 3D printed holders are used to facilitate the positioning, handling and cleaning of such small lenses and system parts. These holders were proven to be very beneficial and useful for the integration of the endoscope. While it is challenging to assemble such small endoscopes by hand, there are some techniques that can be used to facilitate the process, allowing encapsulation of multiple functionalities in the same optical system.

Non-linear optical microscopy proves to be an indispensable tool in natural sciences and becomes more and more attractive for clinical applications. Coherent Raman scattering, for instance, has the potential to become an in-vivo fast label-free histology tool as its chemical selectivity provides quantitative information on lipids and proteins locations and concentrations in tissues. Along with these techniques, second-harmonic generation of collagen and 2-photon excitation fluorescence broaden even more the non-linear imaging ability as collagen fibers represent an important role in human body construction. Whilst 2-photon excitation fluorescence allows to study auto-fluorescence (ex. NADH and NADHP molecules), and to excite a vast range of chromophores. However, absorption and scattering limit significantly the nonlinear imaging depth into tissues. As a solution, we offer a flexible, compact, and multimodal nonlinear endoscope (2.2 mm outer diameter, 35 mm rigid length) based on a resonantly piezo scanned hollow-core negative curvature double-clad fiber. The fiber design allows distortion-less, background-free delivery of femtosecond excitation pulses and the back-collection of nonlinear signals through the same fiber. The double-cladding of this fiber attends 10^5μm of silica collection surface which allows for a 4-fold collection improvement compared to previously used Kagomé hollow core fibers. Having a good control on the resonantly scanning fiber the endoscope can perform nonlinear imaging up to 8 frames per second over a field of view of 400μm. We demonstrate 2photon, SHG and CARS imaging in ex vivo gastric human tissue samples and in-vivo 2-photon fluorescence imaging of GFP-labeled neurons in mouse brain.

Video endoscopy remains the most common means of examining the upper gastrointestinal tract. However, white-light widefield imagery is limited in diagnostic sensitivity and specificity for conditions such as Barrett’s esophagus. To provide cross-sectional imaging that yields more accurate diagnosis, optical coherence tomography (OCT) has been implemented for upper GI screening and diagnosis in several form factors, such as inflatable balloons and tethered capsules. We now present an alternative configuration for esophageal OCT. Our OCT probe is contained within an articulating paddle that attaches to the end of an upper GI endoscope via flexible cuff. This arrangement allows regions of interest to be visually identified by the physician operating the endoscope and targeted for OCT imaging by application of the paddle. The probe housing was 3D printed using biocompatible dental resin. The flexible cuff was also 3D printed using a silicone-based resin to allow a tight fit to the endoscope. The optical probe was a rotating fiber-optic design, consisting of a gradient-index lens and prism on the distal end, wound steel torque coil, a polymer sheath, and a fiber-optic rotary junction. The OCT system was a spectral domain configuration with custom spectrometer operating at 20,000 A-lines per second. The illumination source was a superluminescent diode centered at 1310 nm. A clinical pilot study at the University of North Carolina Endoscopy Center is scheduled to begin imminently.

Upper endoscopy is a standard technique for imaging, sampling, and treating gastrointestinal tissue. Endoscopy is frequently requiring the subjects who undergo the procedure be consciously sedated. Sedation necessitates that the endoscopy procedure be conducted in a specialized setting to mitigate complications should they arise. Endoscopy is further problematic for infants and young children (aged 0-24 months) who sometimes need to be anesthetized. These issues motivate alternative methods for upper gastrointestinal tract visualization and biopsy that do not require conscious sedation/anesthesia. To address this need, we have developed a double lumen 6.5 Fr transnasal introduction catheter (TNIC). During transnasal insertion, real-time OCT imaging provides confirmation of the anatomical location of the device. Once in the stomach, a safe and high-density liquid metal fills a balloon at the distal tip of the TNIC, allowing it to passively transit through stomach into the small intestine. Once properly positioned, OCT-guided instruments for imaging and biopsy can be inserted through main lumen of the TNIC, performing many of the functions of conventional endoscopy and advanced endomicroscopy. To test the feasibility of the TNIC, we conducted a clinical study using the first version of the device in 4 unsedated normal volunteers. Results showed detailed OCT endomicroscopy images of the esophagi and duodena. These results suggest that TNIC may be an effective, less invasive method for the diagnosis of upper GI tract conditions.

Environmental enteric dysfunction (EED) is a pathological condition of the small intestine that is endemic to low- and middle-income countries (LMICs). EED is thought to interfere with nutrient absorption and enteropathogen exclusion, resulting in altered immune response, increased infection, and limited neurological and physical development. Biopsy of the small intestine is the current diagnostic gold standard for diagnosis yet is untenable due to lack of availability in these countries. Endoscopic biopsy is further problematic since EED-related stunting can only be reversed if diagnosed in the first two years of life when endoscopy must be conducted under anesthesia in advanced medical care settings. Thus, there is an unmet need for a minimally invasive technology for obtaining small intestinal biopsies in unsedated infants in LMICs. To address this need, we have developed an OCT image-guided trans-nasal cryobiopsy device. The device comprises a dual-lumen 1.2 mm outer diameter (OD) probe, terminated by a metal tip, through which Freon is injected. The device is introduced through the lumen of a novel liquid-metal transnasal imaging tube that passively transits to the small intestine. M-mode OCT image guidance is used to determine when the metal tip is in contact with the mucosa so that cryobiopsies may be efficiently acquired. We have conducted feasibility experiments using this device in 10 swine in vivo, demonstrating residual bleeding that is comparable to conventional excisional biopsy, tissue sampling volumes that are greater than or equal to those of conventional biopsy, and high-quality histopathology. These results suggest that this transnasal cryobiopsy technique may be suitable for infants in low-resource settings where EED is prevalent, due to its simplicity and its ability to be used in unsedated subjects.

The early detection of cancer brings increased success in the treatment of cancer patients. A prototype sub-millimeter diameter high resolution fibre endoscope for the in-vivo imaging in oral, lung, cervix, ovarian and pancreas sites for the early detection and delineation of cancers is currently in its early stages of development. The endoscope is to utilize a combination of rotary and pullback motion to allow a wide field-of-view while capturing high-resolution (10 to 20 um) RGB images. In this system an RGB laser module uses the core of a dual-clad fibre for illumination and the inner cladding for detection to achieve real time in-vivo reflectance imaging . Signal detection for each laser (RGB) has been tested using a white card printed with black lines of varying widths. The contrast between the white and black portions of the card and the Signal to Noise Ratio (SNR) for the pullback mechanism of the system were determined. The card contrast values for red, green and blue light were calculated to be 25.0, 15.6 and 8.3 respectively, while the SNR values were 180, 155, and 154 respectively. These values suggest that the performance of the system is wavelength dependent. The imaging performance characteristics of the endoscope with rotary and pullback motion combined will be further quantified, and results and images will be presented.

Diagnosis of peripheral lung nodules through transbronchial biopsy is highly prone to sampling errors due to the inability of current techniques to accurately locate and/or sample lesions. Volumetric optical imaging techniques such as optical coherence tomography (OCT) have the potential to address this issue, however, current imaging catheter designs cannot achieve sufficiently high-resolution, or diffraction-limited imaging; focusing elements bear spherical aberrations and multilayered structures with asymmetric curvatures in the optical path cause astigmatism. In this work, we propose a new class of optical imaging catheters – termed nano-optic endoscope – that use metalenses to achieve diffraction-limited endoscopic imaging at greatly extended depth-of-focus through negating non-chromatic aberrations and chromatic dispersion engineering. A metalens consists of a 2-dimentional array of subwavelength-spaced scatterers with specific geometric parameters and distribution that locally shift the phase of the incident light and modify its wavefront. The metalens ability to arbitrarily and accurately modify the phase allows the nano-optic endoscope to overcome spherical aberrations and astigmatism. Remarkably, the tailored chromatic dispersion of the metalens in the context of spectral interferometry is utilized to maintain high-resolution imaging beyond the input field Rayleigh range, overcoming the compromise between transverse resolution and depth-of-focus. Endoscopic imaging is demonstrated ex vivo in resected human airway specimens and in vivo in sheep airways. Fine pathology such as irregular glandular pattern, the hallmark of adenocarcinoma, is readily visualized in high-resolution images captured by the nano-optic endoscope. The versatility and design flexibility of the nano-optic endoscope significantly elevate endoscopic imaging capabilities that will likely impact clinical applications.

Tissue with fibrillar architecture, such as collagen or muscle fiber, exhibits birefringence. In addition to the scalar amount of birefringence, the orientation of the birefringence axis, i.e. the fast optic axis, provides important information on the physical orientation of the fibrillar tissue components. We have previously demonstrated local optic axis mapping using bench-top fiber-based polarization-sensitive optical coherence tomography (PS-OCT), by compensating for the transmission through fiber and system elements, imperfect system alignment, and preceding tissue layers. Using depth-multiplexed PS-OCT, the compensation considers both retardation and diattenuation and is applied in the wavenumber domain, preserving the full axial resolution of the system. Here, we extend our approach to catheter-based imaging. Analyzing a reflection signal from the distal tip of the optical probe, we decompose the recovered system transmission into a static component and a varying catheter transmission to accurately correct for the rotation-dependent transmission through the catheter. Catheter-based local optic axis mapping is validated with a custom-made birefringence phantom. Imaging ex-vivo human bronchus demonstrates the utility of reconstructing the local optic axis orientation to assess airway smooth muscle (ASM), which is oriented approximately orthogonal from the surrounding tissue, offering strong optic axis orientation contrast. Thickening and contraction of the ASM is considered a primary cause of breathing difficulties, and the capacity to clearly image the ASM could lead to an improved understanding of diseases such as asthma.

Utilizing spatial wavelength encoding, spectrally encoded endoscopy (SEE) makes it possible to create miniature, small diameter endoscopic probes that can allow easy access to hard-to-reach locations within the body. Previously described SEE probes have been side-viewing, which limits their use for guiding the navigation of narrow passages. Forward-viewing SEE (FVSEE) probes are advantageous as they provide a look ahead that facilitates navigation and surveillance of a wider field of view (FOV). In this work, we present a novel FVSEE probe. The 500-µm illumination optics are designed in such a way that the shortest wavelength (460 nm) propagates along the optical axis, while an angle of approximately 56° is formed between the longest wavelength (720 nm) and the optical axis. Two-dimensional illumination was accomplished by rotating the illumination optics at a speed of 15 rps using a miniature torque coil. Reflected light from the sample was collected by 8 multimode detection fibers that were arranged into a circular array around the illumination optics. The proximal ends of the detection fibers were polished at a 17° angle, resulting in a total angle of detection of approximately 100°. Light coming out from the distal end of the detection fibers, which were rearranged into a linear array, was detected using a custom spectrometer with a tall-pixel linear CCD camera. The FVSEE probe was used to conduct a preclinical imaging of a swine joint. The results were compared to a commercial chip-on-the-tip mini-endoscope and showed a better spatial resolution and a wider FOV using the FVSEE probe.

The tympanic membrane plays a key role in the human hearing by translating air pressure waves into bone vibrations, and its function and dynamics are directly linked to various pathologies and hearing disorders. Current methods for imaging tympanic membrane dynamics, including stroboscopic holography and Doppler OCT would be challenging for in-vivo applications due to high system complexity or the need for point-by-point scanning. Here, we demonstrate in-vivo imaging of the tympanic membrane dynamics of a human volunteer using interferometric spectrally encoded endoscopy (iSEE). Briefly, in iSEE, spectral interference between a reference signal and the reflectance along a spectrally encoded transverse line is captured by a high speed (20 kHz) spectrometer. Using single-axis scanning across the membrane provides a two-dimensional interferometric data that is later analyzed using specialized software. The imaging probe includes a single optical fiber, optics for light delivery and scanning of the tympanic membrane, and a dedicated port for transmitting the excitation acoustic signals comprised of multiple single-frequency stimuli. Measuring the full vibration patterns of the tympanic membrane would help scientists to study signal transduction into the middle ear and observe the three-dimensional acoustic motion of the membrane. From a clinical perspective, the study could be used for developing a compact system that could be incorporated into conventional clinical otoscopes for providing functional information noninvasively with unprecedented resolution and sensitivity.

Fluorescence endoscopy is emerging for guiding biopsy and early cancer detection in the gastrointestinal tract. A multimodal scanning fiber endoscope (mmSFE) with 2 fluorescence labeled peptides is designed to image overexpressed biomarkers associated with esophageal adenocarcinoma (EAC) and high-grade dysplasia (HGD), thus detecting early neoplasia. Quantification of multiplexed fluorescence images is critical, which ‘red-flags’ suspicious regions and supports diagnosis. The Target/Background (T/B) ratio is calculated to quantitatively evaluate fluorescence images. However T/B ratios are based on fluorescence intensities alone, so adding morphological features can be critical to providing evidence for diagnosis. Moreover, currently the T/B ratio is calculated for a single fluorescence channel. A protocol for multiplexed fluorescence quantification is needed. Materials and Methods: Peptides targeting EGFR and ErbB2 are labeled with NIR fluorescence Cy5 and IRdye800, respectively. A mmSFE with 2.4mm flexible shaft and wide-field forward-view imaging is designed to image these two near-infrared fluorophores with an additional reflectance channel for anatomical identification. In first-in-human clinical trials the two peptides are topically sprayed, briefly incubated, and then rinsed in subjects at high risk of EAC. Both fluorescence channels were captured simultaneously with the co-registered reflectance channel at 30Hz. After removing artifacts, frames were manually selected using morphological features. T/B ratios were then calculated for all fluorescence channels in selected frames. Results and Conclusions: T/B ratios for HGD subjects are greater than for healthy subjects in at least one of the fluorescence channels. Future work will design algorithms to automatically select suspicious regions based on morphological features. More analysis will be performed based on co-registered fluorescence channels.

The development of high resolution, 3D imaging endomicroscopy has recently been gaining interest due to needs in in vivo studies of neuronal dynamics and interactions in deep brain tissue. However, most types of endomicroscopy techniques adopting micro-objectives can hardly perform 3D imaging due to the lack of minimally invasive scanning mechanism. Here, we present a GRIN-lens based fluorescence endomicroscopy capable of obtaining pseudo 3D image of a fluorescent sample without scanning the probe. We use a confocal laser scanning microscope (CLSM) to scan the proximal end of a coherent fiber bundle and obtain an extended depth of focus at the distal end. Preliminary results using fluorescent beads showed that a single bead can be detected throughout hundreds of microns depth. In the detection path, we use two reflecting pinholes of different size to form two confocal and one non-confocal images of the proximal end of the fiber bundle - one pinhole conjugates to the single core being illuminated and the other pinhole conjugates to the surrounding cores. The intensity ratio of an object in these three images almost explicitly depends on its distance from the distal end of the fiber bundle. Thus, we can determine the relative depths of objects in a volume sample in a single acquisition. Future works will include calibrating the setup to obtain the true depth instead of relative depth and applying this technique to brain samples.

HiLo technique provides an effective means of eliminating signals from out-of-focus in widefield fluorescence microscopy by synthesizing two images sequentially acquired with uniform and structured illumination. However, light scattering within the sample often deteriorates the optical sectioning effect. Here, we demonstrate that optical sectioning can be improved by combining HiLo technique and confocal slit detection. Light scattering is reduced by using the rolling shutter of a CMOS scanner as a virtual detector slit. Synchronizing the camera rolling shutter with a scanning hybrid-illumination laser line results in a HiLo endomicroscopy with confocal line detection at a high frame rate of 60 fps. In endomicroscopy, an expanded laser beam passes through a beam splitter and is reflected by a spatial light modulator which toggles between two illumination patterns, grid and uniform. The illumination patterns are focused by a cylindrical lens and then delivered through a fiber bundle probe that transfers the laser line to the tissue and collects emitted fluorescence. To check the axial depth sectioning strength, a thin, uniform fluorescent plane is illuminated. The fullwidth half maximum of the axial distance scanning is 19 μm for the proposed HiLo confocal as opposed to 28.5 μm for the line scanning confocal and 51 um for the widefield HiLo. Experiments on imaging phantoms and ex vivo tissues demonstrate that the optical sectioning ability of the HiLo confocal endomicroscopy is improved when compared to its counterparts.

Molecular imaging using fluorescence microendoscopy offers promise for the optical biopsy of cancer to inform precision medicine. However, microscopic resolution generally comes with the trade-off of a tiny field of view and tunnel vision. Micro-image mosaicking offers the capability of stitching together larger scenes of the tissue to aid visualization and interpretation. The development of hyperspectral microendoscopes provides motivation for adapting mosaicking algorithms to process a plurality of simultaneous channels. We present an algorithm that mosaics hyperspectral microendoscopic video by correlating channels of consecutive frames as a basis for calculating image alignments. A typical raster path to produce suitable data for mosaicking images the same location several times redundantly in different frames, making this algorithm well-suited for analyzing video-rate data. To complement this data rate, we employ parallel processing via GPUs to alleviate computational bottlenecks and approach video-rate mosaicking speeds. This implementation lays the foundation for real-time multi-channel mosaicking to accompany video-rate hyperspectral microendoscopic probes.

Optical fiber bundles are the backbone of modern, ultra-thin, clinical fluorescence microendoscopes. Each core in the bundle acts as a pixel, allowing image transmission from inside hard to reach spaces in the body. Each core relays light from a given location in 2D space and therefore the bundle is thought to yield only a 2D image. However, we show that these fiber bundles do in fact transmit 3D information about the scene, by way of intensity distribution within the cores. Our key observation is that the intra-core intensity distribution depends on the angular coupling efficiency of incoming light. Normally incident light tends to couple into the fundamental mode of the core, whereas oblique light couples to higher order modes that carry most of their energy near the core/cladding interface. By leveraging this phenomenon, we are able to extract 3D information from fiber bundle-based microendoscopes in the form of a light field. We show that this light field 3D information can be visualized in many ways, from stereo images to full-parallax animations, refocusing and depth mapping. Our light field fiber bundle imaging technique is single-shot, resistant to fiber bending and does not require any physical modification to stock optical fiber bundles. In this talk we will outline the mathematical model of our technique and present several examples of light field fluorescence imaging through bare optical fiber bundles.

Confocal, multi-photon, and wide-field endomicroscopy often use coherent fiber-optic bundles to facilitate in vivo imaging. The narrow diameter and flexibility of these bundles allow excellent tissue access, but fabrication processes place a practical limit on fiber packing density, restricting the number of resolvable points in an image. Furthermore, the hexagonal packing of discrete fibers creates inter-fiber gaps that prevent some regions of the object from being imaged. We have combined compressed sensing (CS) principles with dispersive optics to simultaneously address these two fundamental limitations of the fiber bundle architecture. We previously reported a CS approach to improve the spatial resolution of bundle based imaging systems by recovering multiple resolvable points within each fiber (Dumas et al., Proc. SPIE 2018). This manuscript will discuss and integrate approaches for recovering object details that lie behind inter-fiber gaps with our CS-based method for resolving intra-fiber detail. First, we show that modifying our CS model to consider the whole field of view rather than a discrete point for each fiber can partially recover inter-fiber detail. Next, we outline how a dispersive component at the distal end of the bundle can be used to spectrally shift object detail such that information from all locations on the sample are transmitted through the bundle. We then implement image compounding techniques with our CS approach to produce a more continuous image. We demonstrate that our platform can produce images of biological samples with 65,536 resolved pixels using a fiber bundle with only 3,700 fiber cores.

We present a proof of concept for microscopy through a multimode fiber using single pixel imaging. We present two implementations, one using galvo scanners and a diffuser plate and one using a digital micromirror device (DMD). Using these setups we can generate thousands of distinct speckle patterns at the distal end of a 50 micron core fiber as illumination patterns for single pixel imaging. We show that the correlation between speckle patterns can be made as low as 30% and the repeatability as high as 98% for a sample of 3200 patterns, and show example single pixel images using a distal detector.

Non-Gaussian beams can provide extended depth of focus (DOF) at constant and potentially uncompromised transverse resolution, as well as a degree of self-reconstruction after beam shadowing, which may be present in tissue imaging. Hence such beams are being developed for imaging systems throughout many disciplines, including endoscopic imaging, where they hold great potential. General possibilities include up to more than 20-fold extension of DOF, tunable working distance, imaging around obstacles and integrated all-fiber designs. In all-fiber based optical imaging systems; however, these advantages are limited by system design considerations. Trade-offs between miniaturization, extended DOF, SNR, and fiber availability arise, and estimating the effects of design modifications can be difficult and time consuming. We model zero-order quasi-Bessel illumination and detection for a range of common probe and sample materials based on an analytic solution of the Fresnel diffraction integral and compare the results to Gaussian beams. We show that these beams, on scales that match optical fiber dimensions, generally have an upper limit for the spot size above which their distinct advantages over Gaussian beams fade. Similarly, we show the existence of a lower limit of practical performance of quasi-Bessel beams, where the imaging SNR penalty compared to a Gaussian beam becomes significant. Additionally to general theoretic considerations we discuss designs, modeling and characterization of all-fiber imaging probes. This work provides an accessible overview for researchers to estimate what potential benefit non-Gaussian beams can introduce into their optical imaging system.

Lensless endoscopes have generated a great deal of interest in the development of minimally invasive probes for imaging in sensitive and hitherto inaccessible regions as found in deep brain imaging. Lifting the requirement for the opto-mechanical elements at the distal end of the fiber reduces the footprint of the endoscope down to the fundamental limit, the fiber itself. Our approach, using specially designed multicore fibers allows us to i) generate two-photon fluorescence contrast with femtosecond pulses, ii) image at high speeds with resonant scanners, iii) simple and non-interferometric calibration schemes and iii) exhibits a high resilience to spatio-temporal distortion of the focus due to fiber bending. In this contribution, we will discuss how novel designs of the MCFs can lift several of the instrumental complexity typically associated wavefront shaping and high speed imaging. We show that the use of sparse arrays of fiber cores can provide pixelation-free imaging with no artifacts when employed in the wavefront domain as opposed to conventional fiber bundles. Furthermore, we examine the unique properties of the MCFs which allow for fast and non-interferometric calibration schemes and can tolerate severe bending with an intact focus. The inclusion of a secondary cladding on the MCF allows us high sensitivity detection though the fiber (NA = 0.6) whilst preserving the advantages of a sparse MCF. The combination of these new developments brings us towards the application of these ultrathin probes in realistic imaging conditions.

Endomicroscopy is a technique for obtaining real-time images in vivo, eliminating the need to biopsy a tissue sample. A simple fluorescence endomicroscope can be constructed using a fiber bundle, camera, LED and filters, and individual images can be mosaicked as the probe is moved across the tissue to increase the image size. However, to improve image contrast optical sectioning is required for the removal of returning out-of-focus light. Commonly, this is done using the confocal technique, requiring more expensive laser sources and mechanical scanning mirrors which limits the frame rate. Structured illumination microscopy (SIM) instead uses line patterns projected onto the sample to allow for computational optical sectioning. This eliminates the need for point scanning and allows an incoherent light source, such as an LED, to be used, at the cost of some loss of signal-to-noise ratio. However, as SIM requires multiple images to be combined, motion of the probe results in severe image artefacts, preventing the use of mosaicking techniques. We report a SIM endomicroscope using a digital micro-mirror device (DMD) to generate line patterns at high speed, and with the ability to change the patterns on the fly. Combined with a high-speed camera, this reduces motion artefacts significantly, but not sufficiently to allow for video mosaicking techniques. We therefore demonstrate further reduction of artefacts by orienting the illumination patterns parallel to the direction of motion and performing inter-frame registration and correction. This offers potential for low cost, versatile, optically-sectioned endomicroscopy.

Bacteria-mediated cancer imaging and immunotherapy is emerging fields. However, most of the study related to targeting and monitoring of therapeutic response after bacterial therapy was done with tissue analysis after sacrificed the animal. To evaluate the theranostic efficacy of fluorescence encoded bacteria in vivo, the tumor should be made on the subcutaneous area due to the limitation of light penetration of the tissue material. However, the subcutaneous xenograft model doesn’t reflect the actual tumor microenvironment. In this study, we monitored theranostic bacteria in orthotopic tumor model with lab-built wide-field fluorescence endoscopy (WFE) and commercialized confocal endomicroscopy (CFEM) in vivo. First, orthotopic mouse colon cancer models were made by using endoscopic cancer cell implantation methods. Seconds, fluorescence emitting theranostic bacteria was constructed by using Salmonella strain. The bacteria were injected into the orthotopic colon tumor-bearing mice via tail vein injection. Finally, we serially monitored fluorescence signal emitted from theranostic bacteria with WFE and CFEM. Multiscale fluorescence image showed the accumulation of bacteria within the tumor area compared to normal tissue contrast. We expected that this endoscopic fluorescence imaging approaches can be used in the direct monitoring of bacterial immunotherapy in vivo.