En-face optical coherence tomography/fluorescence endomicroscopy for minimally invasive imaging using a robotic scanner," J.Abstract. We report a compact rigid instrument capable of delivering en-face optical coherence tomography (OCT) images alongside (epi)-fluorescence endomicroscopy (FEM) images by means of a robotic scanning device. Two working imaging channels are included: one for a one-dimensional scanning, forward-viewing OCT probe and another for a fiber bundle used for the FEM system. The robotic scanning system provides the second axis of scanning for the OCT channel while allowing the field of view (FoV) of the FEM channel to be increased by mosaicking. The OCT channel has resolutions of 25∕60 μm (axial/lateral) and can provide en-face images with an FoV of 1.6 × 2.7 mm 2 . The FEM channel has a lateral resolution of better than 8 μm and can generate an FoV of 0.53 × 3.25 mm 2 through mosaicking. The reproducibility of the scanning was determined using phantoms to be better than the lateral resolution of the OCT channel. Combined OCT and FEM imaging were validated with ex-vivo ovine and porcine tissues, with the instrument mounted on an arm to ensure constant contact of the probe with the tissue. The OCT imaging system alone was validated for in-vivo human dermal imaging with the handheld instrument. In both cases, the instrument was capable of resolving fine features such as the sweat glands in human dermal tissue and the alveoli in porcine lung tissue. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Significance: Confocal laser scanning enables optical sectioning in fiber bundle endomicroscopy but limits the frame rate. To be able to better explore tissue morphology, it is useful to stitch sequentially acquired frames into a mosaic. However, low frame rates limit the maximum probe translation speed. Line-scanning (LS) confocal endomicroscopy provides higher frame rates, but residual out-of-focus light degrades images. Subtraction-based approaches can suppress this residue at the expense of introducing motion artifacts.Aim: To generate high-frame-rate endomicroscopy images with improved optical sectioning, we develop a high-speed subtraction method that only requires the acquisition of a single camera frame.Approach: The rolling shutter of a CMOS camera acts as both the aligned and offset detector slits required for subtraction-based sectioning enhancement. Two images of the bundle are formed on different regions of the camera, allowing both images to be acquired simultaneously.Results: We confirm improved optical sectioning compared to conventional LS, particularly far from focus, and show that motion artifacts are not introduced. We demonstrate high-speed mosaicing at frame rates of up to 240 Hz. Conclusion:High-speed acquisition of optically sectioned images using the new subtraction based-approach leads to improved mosaicing at high frame rates.

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.

Significance: Confocal laser scanning enables optical sectioning in clinical fiber bundle endomicroscopes, but lower-cost, simplified endomicroscopes use widefield incoherent illumination instead. Optical sectioning can be introduced in these simple systems using structured illumination microscopy (SIM), a multiframe digital subtraction process. However, SIM results in artifacts when the probe is in motion, making the technique difficult to use in vivo and preventing the use of mosaicking to synthesize a larger effective field of view (FOV). Aim: We report and validate an automatic motion compensation technique to overcome motion artifacts and allow generation of mosaics in SIM endomicroscopy. Approach: Motion compensation is achieved using image registration and real-time pattern orientation correction via a digital micromirror device. We quantify the similarity of moving probe reconstructions to those acquired with a stationary probe using the relative mean of the absolute differences (MAD). We further demonstrate mosaicking with a moving probe in mechanical and freehand operation. Results: Reconstructed SIM images show an improvement in the MAD from 0.85 to 0.13 for lens paper and from 0.27 to 0.12 for bovine tissue. Mosaics also show vastly reduced artifacts. Conclusion: The reduction in motion artifacts in individual SIM reconstructions leads to mosaics that more faithfully represent the morphology of tissue, giving clinicians a larger effective FOV than the probe itself can provide.