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Significance: The current gold standard for monitoring small intestinal transplant (IT) rejection is endoscopic visual assessment and biopsy of suspicious lesions; however, these lesions are only superficially visualized by endoscopy. Invasive biopsies provide a coarse sampling of tissue health without depicting the true presence and extent of any pathology. Optical coherence tomography (OCT) presents a potential alternative approach with significant advantages over traditional white-light endoscopy. Aim: The aim of our investigation was to evaluate OCT performance in distinguishing clinically relevant morphological features associated with IT graft failure. Approach: OCT was applied to evaluate the small bowel tissues of two rhesus macaques that had undergone IT of the ileum. The traditional assessment from routine histological observation was compared with OCT captured using a handheld surgical probe during the days post-transplant and subsequently was compared with histophaology. Results: The reported OCT system was capable of identifying major biological landmarks in healthy intestinal tissue. Following IT, one nonhuman primate (NHP) model suffered a severe graft ischemia, and the second NHP graft failed due to acute cellular rejection. OCT images show visual evidence of correspondence with histological signs of IT rejection. Conclusions: Results suggest that OCT imaging has significant potential to reveal morphological changes associated with IT rejection and to improve patient outcomes overall.

Significance: Raman spectroscopy has been developed for surgical guidance applications interrogating live tissue during tumor resection procedures to detect molecular contrast consistent with cancer pathophysiological changes. To date, the vibrational spectroscopy systems developed for medical applications include single-point measurement probes and intraoperative microscopes. There is a need to develop systems with larger fields of view (FOVs) for rapid intraoperative cancer margin detection during surgery. Aim: We design a handheld macroscopic Raman imaging system for in vivo tissue margin characterization and test its performance in a model system. Approach: The system is made of a sterilizable line scanner employing a coherent fiber bundle for relaying excitation light from a 785-nm laser to the tissue. A second coherent fiber bundle is used for hyperspectral detection of the fingerprint Raman signal over an area of 1 cm2. Machine learning classifiers were trained and validated on porcine adipose and muscle tissue. Results: Porcine adipose versus muscle margin detection was validated ex vivo with an accuracy of 99% over the FOV of 95 mm2 in ∼3 min using a support vector machine. Conclusions: This system is the first large FOV Raman imaging system designed to be integrated in the workflow of surgical cancer resection. It will be further improved with the aim of discriminating brain cancer in a clinically acceptable timeframe during glioma surgery.

Significance: Differential phase contrast (DPC) is a well-known imaging technique for phase imaging. However, simultaneously acquiring multidepth DPC images is a non-trivial task. We propose simultaneous multiplane DPC imaging using volume holographic microscopy (VHM). Aim: To design and implement a new configuration of DPC-VHM for multiplane imaging. Approach: The angularly multiplexed volume holographic gratings (AMVHGs) and the wavelength-coded volume holographic gratings (WC-VHGs) are used for this purpose. To obtain asymmetric illumination for DPC images, a dynamic illumination system is designed by modifying the regular Köhler illumination using a thin film transistor panel (TFT-panel). Results: Multidepth DPC images of standard resolution chart and biosamples were used to compare imaging performance with the corresponding bright-field images. An average contrast enhancement of around three times is observed for target resolution chart by DPC-VHM. Imaging performance of our system is studied by modulation transfer function analysis, which suggests that DPC-VHM not only suppresses the DC component but also enhances high-frequency information. Conclusions: Proposed DPC-VHM can acquire multidepth-resolved DPC images without axial scanning. The illumination part of the system is adjustable so that the system can be adapted to bright-field mode, phase contrast mode, and DPC mode by controlling the pattern on the TFT-panel.

We demonstrate a propagating-path uniformly scanned light sheet excitation (PULSE) microscopy based on the oscillation of voice coil motor that can rapidly drive a thin light sheet along its propagation direction. By synchronizing the rolling shutter of a camera with the motion of laser sheet, we can obtain a uniform plane-illuminated image far beyond the confocal range of Gaussian beam. A stable 1.7-μm optical sectioning under a 3.3 mm×3.3 mm wide field of view (FOV) has been achieved for up to 20 Hz volumetric imaging of large biological specimens. PULSE method transforms the extent of plane illumination from one intrinsically limited by the short confocal range (μm scale) to one defined by the motor oscillation range (mm scale). Compared to the conventional Gaussian light sheet imaging, our method greatly mitigates the compromise of axial resolution and successfully extends the FOV over 100 times. We demonstrate the applications of PULSE method by rapidly imaging cleared mouse spinal cord and live zebrafish larva at isotropic subcellular resolution.

Blockage of healthy blood vessels by blood clots can lead to serious or even life-threatening complications. The use of a combined ultrasound (US) and photoacoustic (PA) imaging was explored for blood clot monitoring during microbubble-assisted sonothrombolysis. PA imaging is an emerging hybrid imaging modality that has garnered the attention of the biomedical imaging community in recent years. It enables the study of the composition of a blood clot due to its sensitivity toward optical absorption. Here, in vitro imaging of the side of a blood clot facing the microbubbles was done over time. The US and PA signal-to-noise (SNR) ratio value changes during microbubble-assisted sonothrombolysis were studied for two different local environments: blood clot in deionized water and blood clot in blood. In the first case, US and PA SNR values increased by 4.6% and reduced by 20.8%, respectively after 30 min of sonothrombolysis treatment. After 10 min of sonothrombolysis treatment of the blood clot in blood, the US and PA SNR values increased by 7.7% and 38.3%, respectively. The US and PA SNR value changes were recorded in response to its local environment. This technique can be used to determine the final composition of the blood clot which may, in turn, help in the administration of clot-dissolving drugs. MOE (Min. of Education, S’pore) Published version

pA stable multimodal system is developed by combining two common-path digital holographic microscopes (DHMs): coherent and incoherent, for simultaneous recording and retrieval of three-dimensional (3-D) phase and 3-D fluorescence imaging (FI), respectively, of a biological specimen. The 3-D FI is realized by a single-shot common-path off-axis fluorescent DHM developed recently by our group. In addition, we accomplish, the phase imaging by another single-shot, highly stable common-path off-axis DHM based on a beam splitter. In this DHM configuration, a beam splitter is used to divide the incoming object beam into two beams. One beam serves as the object beam carrying the useful information of the object under study, whereas another beam is spatially filtered at its Fourier plane by using a pinhole and it serves as a reference beam. This DHM setup, owing to a common-path geometry, is less vibration-sensitive and compact, having a similar field of view but with high temporal phase stability in comparison to a two-beam Mach-Zehnder-type DHM. The performance of the proposed common-path DHM and the multimodal system is verified by conducting various experiments on fluorescent microspheres and fluorescent protein-labeled living cells of the mossitalicPhyscomitrella patens/italic. Moreover, the potential capability of the proposed multimodal system for 3-D live fluorescence and phase imaging of the fluorescent beads is also demonstrated. The obtained experimental results corroborate the feasibility of the proposed multimodal system and indicate its potential applications for the analysis of functional and structural behaviors of a biological specimen and enhancement of the understanding of physiological mechanisms and various biological diseases./p.

Previously introduced bundles of hundreds or thousands of microfibers have the potential to extend optical access to deep brain regions, sampling fluorescence activity throughout a three-dimensional volume. Each fiber has a small diameter (8 μm) and follows a path of least resistance, splaying during insertion. By superimposing the fiber sensitivity profile for each fiber, we model the interface properties for a simulated neural population. Our modeling results suggest that for small (