Optical Coherence Tomography (OCT) has the ability to acquire cross-sectional images under tissue surfaces in real-time, which can provide real-time tissue characterization. One of the possible applications is improving the accuracy of white-light based diagnosis in the digestive system. In this work, we integrate OCT with a robotic surgical endoscope, using OCT local scanning to form complementary information of the white light endoscopic camera. Meanwhile, the white light camera can help to roughly guide the OCT probe to the potential pathological area. To accelerate the local scanning, we explore different volumetric scanning strategies to find a good trade-off between efficiency and 3D recovering quality. Based on stabilization and segmentation of the OCT images using deep learning techniques navigation information is extracted for autonomous control, as well as tactile information. The proposed method allows an increase of field of view (FoV) for OCT imaging in large lumen under dynamic displacement caused by tissue motion.

This Conference Presentation, “Optical guidance for radiation therapy,” was recorded at SPIE Photonics Europe 2022 held in Strasbourg, France.

Patients with pancreatic adenocarcinoma have a short survival time and many are not eligible for surgery due to vascular proximity or involvement. Hence, downstaging therapies that can effectively clear the tumor from the vessel, are desirable. We hypothesize photodynamic ablation via the endovascular route can effectively clear vessels from such tumor involvement. To evaluate endovascular light delivery for Photodynamic therapy (PDT) as a potential approach, proof-of-concept studies were performed, including in silico Monte Carlo light simulations, and in vivo, testing in a porcine model. Monte Carlo simulations were carried out in an anatomic realistic model based on segmentation of a porcine pancreas and its blood vessels, where light sources were placed into the superior mesenteric and splenic arteries. Tissue response was evaluated based on the photodynamic threshold model taking local fluence, photosensitizer and tissue sensitivity into consideration. The simulations showed that while limiting the irradiance on the intima to non-thermal levels, pancreatic tissue destruction up to several mm is feasible within clinically acceptable irradiance times for BPD-MA mediated PDT. In vivo studies used normal pigs where pre-and-post PDT contrast-enhanced CT scans (24-48h) were performed to obtain anatomical details and to evaluate gross tissue response. The splenic, hepatic and superior mesenteric arteries were reached via the femoral access route. A guidewire was inserted under fluoroscopy and exchanged with a prototype endovascular catheter for intravascular irradiation. We found it feasible to reach the target areas, however optimal positioning of the irradiators within the prototype catheter was somewhat challenging. Light delivery of several hundreds of J.cm-2 is feasible albeit requiring long irradiation and vessel occlusion times. No vessel perforations were noted on histopathology, however some expected necrosis in smooth muscle cells. The maximum radius of necrosis beyond the vessel's outer diameter reached several mm. This is potentially acceptable for downstaging patients and performing surgical tumor resection.

Liposomes have revolutionized the field of photomedicine. Photodynamic therapy (PDT) using Visudyne®, a liposomal photosensitizer formulation, has helped many patients globally. Since the FDA approved Visudyne® in 2002, countless studies have examined strategies to further improve the therapeutic index of lipid-based photosensitizing nanoconstructs. While liposomes can improve the pharmacokinetics of hydrophobic photosensitizers, they could also modulate cellular uptake and singlet oxygen production. Furthermore, it is evident that there are other immunological and toxicological considerations for the design of liposomal drugs. Accordingly, there is now an emerging trend to engineer carrier-free nanodrugs. Here, we developed a pure-drug nanoparticle using the clinically used verteporfin photosensitizer (termed nanoVP) for photodynamic applications. We validated the effects of nanoVP in three contexts: 1) cytotoxic PDT, 2) subtherapeutic PDT, and 3) dark toxicity. Using a brain cancer murine model, we showed that light activation of nanoVP reduced tumor volume by up to 54% compared to liposomal VP. Fluorescence imaging revealed that nanoVP had a superior tumor-to-liver tissue ratio (~0.92) compared to liposomal VP (~0.4). We further studied nanoVP-mediated PDT at subtherapeutic doses to achieve photodynamic priming (PDP). PDP has been shown to enhance drug delivery, activate antitumor immunity, and sensitize tumors to chemotherapy. This approach is particularly relevant in the brain, where high doses of PDT can result in edema, neurotoxicity, and even animal death. Using a rat model, we demonstrated that nanoVP-assisted PDP improved blood-brain barrier permeability and accumulation of a model drug (Evans Blue dye) in rat brains by >5 fold. Minimal to no brain damage was observed. Lastly, under dark conditions, we validated that nanoVP significantly reduced viability while liposomal VP stimulated cancer cell growth. Results from this work demonstrate the utility of nanoVP for cancer treatment. The development of pure-drug photosensitizing nanoparticles for photodynamic applications could further revolutionize the field of photomedicine.

Early in their development, tumours stimulate new blood vessel growth through a range of mechanisms to meet their metabolic needs, leading to marked differences in the absorption spectra of normal and tumour tissue that could be exploited for early cancer detection. To extract quantitative biomarkers such as haemoglobin concentration and oxygenation from optical images, we combine novel spectral imaging methods with advanced computational analysis and biophysical modelling, applied in both preclinical cancer models and early phase clinical trials in patients. In this talk, I will focus on one aspect of these studies, applying multi- and hyper-spectral imaging during endoscopic surveillance of the gastrointestinal tract for detection of early oesophageal cancer.

This Conference Presentation, “Surgical spectral sensing and imaging,” was recorded at SPIE Photonics Europe 2022 held in Strasbourg, France.

A major goal for optical imaging techniques is to provide quantitative information in real-time during minimally invasive treatments, where the experience and expertise of the practitioner still play a central role for the successful outcome of the procedure. In this context, Single Snapshot imaging of Optical Properties (SSOP) is an imaging technology based on sinusoidal structured light that has already been demonstrated to have real-time capabilities for wide-field imaging of biological tissues. In this work, we present an endoscopic implementation of SSOP that provides high quality imaging capabilities over a large field of view (70 mm x 70 mm). The instrument is based on a rigid two channels endoscope that can be further adapted for robotized manipulation with systems such as “Da Vinci”. From the optical design point of view, the structured illumination through the first channel of the endoscope is achieved with a laser source coupled to a custom optical path where high definition 2D sinusoidal patterns printed on a glass substrate are used for the generation of high spatial frequency images. The acquisition of the SSOP frames is performed through the second endoscope channel with a tri-sensor CMOS camera covering an RGB channel (for the anatomical view of the surgical field), and two NIR channels selected for optimal oxygenation wavelength coverage (i.e. 665 nm and 860 nm). Real-time imaging is still achievable despite the presence of a deep-learning-based processing architecture and the adoption of a 3D profile correction algorithm, thanks to a custom low-level GPGPU implementation for the visualization and processing which allows us to optimize the total computational time to enable high frame rate acquisitions (>10 fps). The imaging performances of a handheld version of the system will soon be assessed through pre-clinical trials on swine models before moving to the surgical robot version.

Multiple exposure speckle imaging (MESI) allows to map relative blood flows at the surface of biological tissues. MESI is an extension of laser speckle contrast imaging (LSCI). It relies on the computation of speckle contrast K for several exposure times T, allowing to discriminate the contribution of static scatters (bulk tissues) and moving scatterers (red blood cells). The MESI model describes K(T) as a function of tc, rho, beta, and v. These variables are respectively the decorrelation time of the moving scatterers, the relative contribution of static scatterers to the speckle pattern, a normalization factor for the imaging parameters and the contribution of noises to the speckle contrast. In LSCI theory, tc is commonly assumed to be inversely proportional to the flow. The acquisition of the speckle data at multiple exposures and the subsequent non-linear fit on a pixel-wise basis are instrumentally complex and time-intensive tasks that prevent real-time computation of the flow maps. In the study, we evaluated the feasibility of machine learning analysis of MESI data to bypass the non-linear fitting procedure based on the synthetic exposure acquisition. Synthetic exposures limit acquisition bias due to imperfect illumination normalization and are less sensitive to camera noises except for low illumination conditions or imaging of fast flows. A residual convolutional neural network was adopted to predict the blood flow map based on a database of representative speckle images of channels in a microfluidic chip with calibrated flows. The MESI database contains images with different exposure times for different flow and different channel diameters. The database was spitted into a training and testing data set with a 50:50 ratio. Preliminary results showed that blood flow mapping using deep learning can achieve moderate accuracy and yield a more stable prediction with high noise-resistant ability, compared to pixel-wise non-linear fit.

This Conference Presentation, “Choosing a reflectance spectroscopy technique for a clinical application: Instrumentation and data analysis considerations for fiber-optic and wide-field techniques,” was recorded at SPIE Photonics Europe 2022 held in Strasbourg, France.

Cancers of the upper gastrointestinal (GI) tract remain a major contributor to the global cancer risk. This study uses a diffuse reflectance spectroscopic probe for tissue characterisation. An approach to reconstruct a dense 3D model of tissue surface from stereo optical videos is proposed. The reconstructed tissue model is used to aid histology correlation, and thus tissue classification. The extracted tissue depth information is combined with the 3D DRS probe position offering an enhanced augmented visualisation of the specimen. The recovery of 3D tissue structure and probe location will enable the accurate deployment of surgical guidance in minimal invasive surgery.

Lymphedema is characterized by the accumulation of protein-rich fluid in the intersitium (i.e., dermal backflow (DBF)), causing swelling. It is commonly seen due to iatrogenic damage to the lymphatics after surgical and radiotherapeutic treatment of cancer. An important microsurgical treatment is lymphovenous bypass (LVB) surgery, during which a lymphatic vessel is anastomosed to a vein to bypass the site of lymphatic flow obstruction. Pre-operative imaging of the lymphatic vessels is a prerequisite for surgical planning. Photoacoustic imaging (PAI) can potentially visualize both lymphatic vessels (with the aid of indocyanine green; ICG) and the receiving veins, with high resolution, in depth, in the presence of superficial DBF. We investigated lymphatic vessel imaging using light emitting diode (LED) based PAI (Acoustic X, Cyberdyne Inc., Tsukuba, Japan) in patients with secondary limb lymphedema. Ten patients were enrolled in the study. ICG-mediated near-infrared fluorescence lymphography (NIRF-L) revealed multiple locations of lymphatic vessels on the healthy and affected limb, with and without the presence DBF in various stages. Subsequently, interleaved PAI (820 and 940 nm) and ultrasound images were acquired in these locations and successful vessel depiction was determined. The ratio between the PA signal at 940 and 820 nm was used to differentiate between ICG-labelled lymphatics and blood vessels. We demonstrated that dual-wavelength, LED-based PAI can visualize lymphatic and blood vessels even in the presence of DBF. These findings suggest that LED-based PAI has potential for pre-operative lymphedema assessment, especially in cases with extensive DBF pattern hindering adequate assessment with NIRF-L.

This Conference Presentation, “Translation of higher harmonic generation microscopy for intra-operative pathology in the clinic,” was recorded at SPIE Photonics Europe 2022 held in Strasbourg, France.

Multiphoton microscopy (MPM) is a technology that can generate real-time, depth-resolved subsurface images of skin with histologic resolution and sensitivity based on endogenous molecular and chemical contrast. In skin, MPM contrast is derived from second harmonic generation (SHG) of collagen and two-photon excited fluorescence (TPEF) of autofluorescent co-factors NAD(P)H and FAD, elastin, keratin, and melanin. In addition to the specificity provided by the detection of the SHG signal from collagen, MPM can visualize specific skin fluorophores based on their fluorescence lifetime detection. Over the past several years, our group and others have demonstrated the MPM strong potential for a broad range of applications from advancing the understanding of skin biology to non-invasive diagnosis of skin diseases and monitoring therapy effects. However, a routine implementation of this technology in clinical research and practice requires advancing the instrumentation to allow for easier access by clinicians and for more efficient imaging in terms of speed and scanning area. Our group recently introduced benchtop innovations and developed a fast large area multiphoton exoscope (FLAME) that can provide rapid, real-time, depth-resolved images of skin, over macroscopic areas (cm-scale) with microscopic resolution (0.5-1m) and chemical contrast (selective detection of melanin). This presentation will highlight the latest advances in the FLAME development, including its conversion into a compact, portable device, highly optimized for clinical skin imaging with enhanced sensitivity and specificity. The technical abilities of this imaging platform will be demonstrated along with the results of a study that establishes clinical safety and imaging performance in 20 volunteers with normal skin. The results emphasize the significance of macroscopic imaging in the context of skin heterogeneity in terms of pigment distribution and dermal photodamage.

The retina might be a promising target to identify early alterations associated with Alzheimer’s Disease (AD). Recent publications show promising results in the detection of retinal amyloid in AD patients in-vivo as well in post-mortem retinal tissue. The aim of this study is to confirm previously published findings using fluorescent retinal imaging and curcumin as labelling fluorophore. In total, 40 patients were enrolled (26 AD, 14 controls) and the subjects’ amyloid assessment was based on CSF analysis and/or amyloid PET. We administered three different curcumin formulations: Longvida, Theracurmin and Novasol. Blue fluorescence (λ = 486 nm) retinal baseline and follow-up images of 2 to 6 retinal regions were performed. The resulting images were visually assessed in a multidisciplinary setting and a selection of images were quantitatively analyzed (only from participants receiving Longvida and Novasol). The visual analysis of baseline images showed no increased fluorescence in AD patients compared to controls. Furthermore, no difference was found comparing pre- and post-curcumin images within AD and control patients. The quantitatively analysis confirmed the visual analysis, identifying similar amount of fluorescence spots in AD and control patients, even after curcumin intake. Despite previous studies assessing retinal amyloid in AD patients with fluorescent retinal imaging using curcumin, we could not confirm the retinal changes described in previous studies. We were unable to reproduce the discrimination of AD patients from controls based on fluorescent retinal amyloid visualization.

For patients with suspected lung cancer, fast and accurate tissue diagnosis is important for optimal treatment allocation. Currently, multiple biopsies are taken, without any feedback on the biopsy quality. Immediate histopathological feedback has the potential to improve the biopsy quality and to reduce the total number of biopsies, thereby potentially reducing adverse events and repeated diagnostic procedures, and prevent delay in start oncological treatment. A promising imaging technique for rapid histopathological feedback on lung biopsies is third and second harmonic generation (THG/SHG) and two-photon excited autofluorescence (2PEF) microscopy, which is non-invasive, label-free and provides 3D images with a high, sub-cellular resolution, within seconds. In a previous study, we showed that using THG/SHG/2PEF microscopy, we could successfully reveal alveolar structures and histopathology hallmarks of unprocessed lung tissue, including cell morphology and general tissue architecture (collagen and elastin organization) [1]. Here, we used for the first time, to the best of our knowledge, a compact, mobile THG/SHG/2PEF microscope (Flash Pathology B.V.) in the clinic to image fresh bronchoscopic lung biopsies. So far, we imaged 75 biopsies of 34 patients, each within a few minutes. Independent lung pathologists assessed the lung biopsies based on the THG/SHG/2PEF images, determining the biopsy representativity and the histopathological diagnosis, which were compared with the standard histopathological assessment of these biopsies. In this way, we tested the ability of the microscope to provide fast feedback to the endoscopist. In addition, we show the added value of using deep learning algorithms for rapid assessment of bronchoscopic lung biopsies. [1] L.M.G. van Huizen, et al. Translational Biophotonics (2020)