Optical and optoacoustic imaging have now progressed beyond critical boundaries in contrast and penetration depth of optical methods, enabling the use of light for a variety of research and clinical applications. The talk discusses advances in multi-spectral optoacoustic tomography (MSOT) and optoacoustic mesoscopy and microscopy that impart unprecedented optical imaging performance in visualizing anatomical, physiological, and molecular biomarkers invivo. In particular, we illuminate how MSOT visualizes biomarkers that relate to inflammatory and metabolic processes in a safe and portable manner, and the role of these biomarkers in addressing unmet clinical needs. Applications including imaging of breast cancer, brown fat activation, or inflammatory conditions of the skin and bowels are showcased as examples of the clinical potential. We discuss how MSOT is appropriate for disseminated, frequent and longitudinal imaging, and how applications in hybrid mode, together with ultrasonography, offer novel potential for advancing diagnostics and theranostics. We further highlight recent progress in the label-free imaging of molecules using Mid-IR Optoacoustic Microscopy (MiROM), a novel modality that pushes the limits of visualization in optical microscopy. Finally, we discuss progress in fluorescence molecular imaging (FMI), which not only has the potential to improve surgical procedures, but now also contributes to early diagnostics, such as the detection of cancerous lesions in patients with Barrett’s esophagus. Please verify that (1) all pages are present, (2) all figures are correct, (3) all fonts and special characters are correct, and (4) all text and figures fit within the red margin lines shown on this review document. Complete formatting information is available at http://SPIE.org/manuscripts Return to the Manage Active Submissions page at http://spie.org/submissions/tasks.aspx and approve or disapprove this submission. Your manuscript will not be published without this approval. Please contact authorhelp@spie.org with any questions or concerns. 12146

Imaging modalities capable of visualizing the human brain have led to major advances in neuroscience. Functional magnetic resonance imaging has enabled a better understanding of brain function and connectivity abnormalities in brain diseases and has become a workhorse in neuroimaging. Nuclear imaging technologies such as positron emission tomography and single-photon emission computed tomography assisted with radiolabelled tracers have further enabled molecular diagnosis and monitoring of brain diseases. Limitations of these established technologies, such as the high cost or the use of ionizing radiation as well as accessibility, have fostered the development of new imaging approaches that can complement or enhance their performance. Optoacoustic tomography (OAT) provides unique capabilities to study cerebral function by mapping changes in several hemodynamic parameters within the brain. It further provides molecular imaging capabilities that can facilitate disease diagnosis and treatment monitoring. However, OAT imaging of the human brain is severely hampered by acoustic attenuation and aberrations of ultrasound waves propagating through the skull. Herein, we performed transcranial OAT imaging through the temporal bone of an adult healthy volunteer based on a previously reported spherical ultrasound array. We validated the OAT results by using head-to-head time-of-flight (TOF) magnetic resonance angiography (MRA) and T1-weighted structural MRI. The superior middle cerebral vein in the temporal cortex was identified in the OAT images matching the observed location in TOF-MRA. This basic demonstration anticipates the development of new hardware and reconstruction algorithms, ultimately enabling accurate OAT imaging of the human brain cortex.

Background: Spinal cord injury (SCI) compromises muscle function; when the pelvic floor muscles (PFM) are involved continence is affected. Women with partial injury rely on PFM rehabilitation therapy (PFMT) to aid continence, but the current lack of absolute measures to quantify training effects by monitoring changes in muscle oxygenation and perfusion hampers rehabilitation. We report clinical translation of a near infrared spectroscopic (NIRS) system to enable women with partial SCI to apply transvaginal optical detection of physiologic changes during PMFT as a point of care tool to quantify the effects of their PFM rehabilitation. Methods: The NIRS interface incorporates a circumferential grid of 6 LED emitters and 4 photodiode receivers with interoptode distances of 20 and 35 mm. The system was developed iteratively by engineers working with clinicians and patient care advocates to be clinically applicable for women following spinal cord injury. The continuous wave system uses 2-wavelengths (nominal 760nm, 850nm) and a sampling rate of 50Hz. Placed in the vagina during PMFT, the system monitors changes in oxy and deoxy-hemoglobin concentration (O₂Hb/HHb) in real time at multiple points in the PFM. The slope of reoxygenation recovery post contraction is a measure of muscle oxidative capacity. Results: A point of care system was successfully developed that provides a means of detecting chromophore change occurring in the PFM during muscular contraction. The chromophore changes monitored also allow an absolute measure, HbDiff half-recovery time (½RT) to be derived. Comparison of these data over time provides a means of evaluating PMFT regimens for a training effect. In preparation for application in subjects with SCI, the reproducibility of SMVCs was monitored successfully in a pilot study where a volunteer conducted a series of 4 SMVCs on 15 occasions. Discussion: Skeletal muscle recovery from exercise-induced oxygen deficit indicates oxidative capacity; this equates with muscular fitness. SMVC is a robust measure of muscle strength and endurance, and HbDiff in occlusion free ½RT analysis reflects metabolic changes within muscle better than O2Hb. This clinical translation of NIRS provides a hand held system for women to use to quantify physiologic changes in their PFM; this will aid women with partial spinal cord injury in whom PFM function may either be too weak to be detected by physical exam or manometry, and may only be present unilaterally. Hence the merit of an optical system able to provide quantifiable measures of reoxygenation recovery as a measure of PFM fitness. A quantitative physiologic measure for evaluating for PMFT training effect is currently lacking Conclusions: The collaborative development of this self-contained transvaginal NIRS system, and prior proof that optical detection of a validated quantifiable oxygenation parameter is feasible in the pelvic floor, now allows clinical evaluation using this point of care system to aid women with partial spinal cord injury. The aim is to enable them to use this system to optimize their PFM rehabilitation therapy as means of enhancing their continence and quality of life.

RGB optical imaging is a marker-free, contactless, and non-invasive technique that is able to monitor hemodynamic brain response following neuronal activation using task-based and resting-state procedures. As opposed to functional task-based analyses, resting-state functional connectivity aims to identify the low frequency cortical hemodynamic fluctuations during patient rest that are linked to resting-state networks. Using intraoperative optical imaging, the main issues of using resting-state procedures come from the partial access to the brain cortex, whereas fMRI or fNIRS resting-state models used whole brain imaging. Task-based fMRI brain maps were compared to intraoperative optical functional brain maps by registering these maps to a preoperative anatomical MRI volume. The objective is to improve the patient care process before, during and after neurosurgery. With the task-based procedure, the RGB brain map showed a good correspondence with task-based fMRI (DICE = 0:75). With the resting-state procedure, the RGB brain map showed a good correspondence with task-based fMRI (seed correlation method: DICE = 0:58 and ICA method: DICE = 0:75).

Prostate cancer is a significant healthcare problem in many western countries. In response to the current need for more accurate and less invasive real-time diagnosis systems, we are developing a thin optical probe that uses Raman spectroscopy to detect prostate cancer in vivo and in real-time. We present results from an ex vivo study on fresh biopsy cores seconds after collection. Findings show that our system can identify prostate cancer from benign tissue, as well as differentiate tissue with different Gleason patterns. We aim to apply this technique, in the near future, for real-time PCa identification during routine biopsy appointments.

Protoporphyrin IX (PpIX) is a fluorophore now used to identify tumoral tissues. The tissue is usually excited at one wavelength, e.g., 405 nm, and the fluorescence signal generated by this molecule and other fluorophores (the baseline) is used to estimate the amount of PpIX. However, fluorophores too close to PpIX impair the estimation and resulting classifications. Thus, we handle this issue by suggesting an efficient multi-excitation wavelengths method, free from any a priori on the baseline. Our method aims to distinguish healthy tissues from tumor margins, while being more robust to baseline variability. It keeps an ability to distinguish healthy from tumor tissues up to 87% in cases where existing methods’ ability drops near 0%.

Near-Infrared (NIR) imaging of the fluorophore Indocyanine Green (ICG) is only provided clinically via rigid surgical scopes. NIR-ICG using Artificial Intelligence Methods (AIM) has been demonstrated for the real-time classification of rectal cancer [1], but more proximal intestinal applications need a capable flexible NIR endoscope, which is challenging due to the weak quantum yield of ICG and bidirectional energy attenuation over distance. We developed a 2m long, 6.4mm diameter flexible Y-bundled fiberoptic prototype that transmits excitation light (785nm) via a single fiber and collects emitted ICG fluorescence (800-850nm) via multimode fibers. Emission signals from an ICG with Dimethyl sulfoxide (DMSO) solution (7.5μM) excited by an 60mW source and isolated using an 840nm bandpass filter were detectable, with significant signal noise from both sample and bundle tip lens reflections needing further refinement. ICG with Bovine Serum Albumin (BSA) depots in bovine-colon mimicking colorectal polyps exhibited reduced reflection while optimizing ICG concentration (determined empirically by comparing fluorescence intensities of incremental concentrations versus surgical use recommendation (4.6μM)) augmented emission signal strength. Additionally, a long-pass filter (800nm) at the collection tip further cleaned reflections, although the distal excitation lens performance needs further design consideration. Source power intensity, NIR-exposure time and tip-target distance were also proven significant parameters with likely considerable clinical relevance. Initial development of a novel flexible NIR imaging device capable of carrying raw NIR energy for AIM algorithmic calculations has proven feasible although challenges remain regarding signal cleaning/augmentation as well as computerized image construction.

Timely assessment of bone perfusion in orthopaedic trauma surgery plays an important role in successful treatment outcome. For guiding accurate debridement of bones with impaired blood supply, fluorescence-guided surgery (FGS) technique have gained increasingly popularity. Compared to other imaging modalities like computed tomography and nuclear magnetic resonance imaging that are time consuming and less practical during surgery, fluorescence imaging can be performed intraoperatively and is able to visualize the bone blood flow in real time. In order to link the blood flow fluorescence imaging to quantitative bone perfusion numbers, in this study we are using a modified fluorescent microsphere (FM) approach called microsphere quantification using imaging cryomacrotome (mQUIC). Bone perfusion is assessed by identifying the density of deposited microspheres in reconstructed imaging volumes, which are proportional to the regional blood flow. In the rabbit model presented here, cryoimaging was used to scan femurs injected with three colors of microspheres corresponding to three conditions: baseline, post-osteotomy and post-periosteal stripping. Image processing, such as top-hat transform and object-based colocalization, was used to enable accurate counting of FMs to produce their 3D-localization within the bones. FM density volumes were converted to bone perfusion units (mL/min/100g) using the reference organ technique. This study provides a groundwork for direct comparison with our DCE-FI technique for measuring bone perfusion in orthopaedic trauma surgery models.

Near Infrared (NIR) camera systems in conjunction with Indocyanine Green (ICG) have been clinically applied for intraoperative tissue perfusion assessment. However signal interpretation remains human operator dependent, prone to inter-user variability and to distance-related signal attenuation. In this work, fluorescence signal emission from ICG-human albumin solutions were quantified at a variety of cameratarget distances and speeds of camera movements. Utilising an electromagnetic field generator and sensors, these characteristics were spatially resolved and logged in real time. Such spatial mapping capability was also investigated in a clinical operation to confirm clinical applicability. Four clinical (two open, Elevision Medtronic, EMO, Ireland and Spy-Phi, Stryker, SSO, US and two laparoscopic, Elevision, Medtronic, EML, Ireland and Pinpoint, Stryker, PNL, USA, with both zero and 30 degree lenses) NIR systems were evaluated. Fluorescence imagery from each system regarding wells filled with ICG solutions were compared using bespoke region-of-interest (ROI) annotation, tracking and quantificative software with the cameras of each both still and moving at different distances from these targets. Chronologically-synchronised logged data featuring spatial coordinates and fluorescence intensities were plotted. Subsequently distance-intensity scatters were synthesised on which appropriate curves were fitted. Peak intensity, ideal optical (field of view) distance (IOD), and peripheral image fluorescence signal loss were defined. In vivo, tumour-to-scope distance spatial mapping capability was demonstrated with a single sensor setup during transanal fluorescent endoscopic examination of a rectal tumour. Three systems (SSO, PN, EML) displayed inverse distance intensity curves which were fitted to inverse square functions while EMO demonstrated static intensity at all distances. EML displayed directionality with a sigmoid curve when moving towards the wells and an inverse square function moving away. The IOD for the thirty-degree scope was further away (PNL 24.7cm vs 14cm) than the 0 degree despite having similar distances where their intensity maxed out (10cm and 8.27cm for the 30 and 0 degree respectively). While all laparoscopic systems displayed peripheral vs central well fluorescence signal hypoattenuation (p<0.001), open systems (EMO and SSO) were shown to hypoattenuate intensity centrally versus peripherally. In vivo assessment successfully logged a tumour to camera distance of 5.3cm during realworld clinical use. Fluorescence detection performance differs between systems in situations corresponding to real-world use. Such behaviours can be mathematically described and modelled enabling system-specific best use guidance and also should inform algorithms developed for the purpose of automated quantitative/artificially intelligent ICG perfusion angiogram classification.

Purpose: Keratoconus is an eye disorder that results in progressive thinning of the cornea. Corneal biomechanical data can be used for early diagnosis of keratoconus disease. Measurements show the first signs of corneal pathological changes while anterior surface topographic maps don’t. In cases of severe keratoconus, the measurements of corneal biomechanical parameters can be modified due to eye lid tension that can influence the evaluation of the keratoconus progression. Aim of this study was to determine differences in corneal biomechanical parameters in keratoconus patients depending on the stage of keratoconus. Method: In our research participated 144 keratoconus patients (256 eyes) and 61 control group patients (122 eyes). We evaluated the corneal thickness (Oculus Pentacam) and corneal biomechanical response with Oculus Corvis. We analyzed the Corvis biomechanical index (CBI), tomographic biomechanical index (TBI), and corneal elevation deviation index (BAD D). The results were compared between the control group and the different stages of the keratoconus patients and were analyzed with ANOVA (two factor) and linear regression. Results: As the keratoconus progresses, corneal thickness decreases (r=-0,98), corneal elasticity decreases (r=0,94), elevation height increases (r=0,77) and the deviation of the corneal elevation from the normative values increases (r=0,98). Conclusion: The results show a strong negative correlation between the stage of keratoconus and the decrease in corneal thickness. Corneal thickness, compared with the control group, decreases statistically significantly (p <0,05), starting with keratoconus stage 2, but CBI, TBI and BAD D corneal biomechanical index values - starting with keratoconus 0-1 stage.

Pseudoxanthoma elasticum and Fabry disease are rare multi-systemic diseases with characteristic skin manifestations. Their rarity and complicated diagnostic process causes disability, decreased quality of life, and increased medical costs for the patients before the diagnosis. Therefore, a new non-invasive diagnostic approach will be developed using multispectral imaging to analyze images of Pseudoxanthoma elasticum skin manifestations and angiokeratomas which are characteristic of Fabry disease. For image acquisition an imaging prototype will be used utilizing 526 nm, 663 nm and 964 nm multispectral LEDs for diffuse reflectance imaging and 405 nm LEDs for autofluorescence excitation, as well as Nuance camera with spectral range 450 – 950 nm. Spectral reflectance and autofluorescence images will be analyzed to determine informative/efficient parameters suitable for identification and diagnostics of rare diseases.

The optical imaging described here is a marker-free, contactless, and non-invasive technique that is able to monitor hemodynamic brain response following neuronal activation during neurosurgery. However, a robust quantification is complicated to perform during neurosurgery due the critical context of the operating room, which makes the calibration and adjustment of optical devices more complex. To overcome this issue, tissue-simulating objects that mimic the properties of biological tissues are required for the development of detection or diagnostic imaging systems. In this study, we evaluated the performance of quantification of chromophore concentration changes measured by experimental setups using two phantoms: a liquid and a numeric brain-simulating phantom. These phantoms mimicked an exposed cerebral cortex as well as the slow concentration changes that occur after neuronal stimulation and the periodic changes due to heartbeat.

The modern medical diagnostical system often use the method of multispectral processing of images based on the initial polychromic image transformation into the consequence of monochrome sub-images. Each of them is formed only by light of the narrow selected spectral sub-range. The best results of multispectral processing can be obtained if an acoustooptic tunable filter (AOTF) is used as a selective element. However, the quantitative estimation of AOTF applicability in such medical diagnostic systems is necessary. This estimation can be realized only using spectral and spatial resolving powers. In order to define them, the corresponding criteria must be formulated. The expressions for determination of the spectral resolving power are also discussed. The image digitization principles which are necessary to determine the amount of information contained in the image, are considered. It has been shown that the diffraction principle of digitization is corresponded to Raileigh criterion which can be applied in the limited cases. It is shown that the optimal digitization principle is statistical one, on the basis of which the information criteria for both kinds of resolving power are formulated. The formulations of these criteria proposed by the authors, are listed. The experimental device for spectral resolving power measurements according to the proposed criterion, is also described. The functional circuit and operation of the device are discussed. The proposed criteria take into consideration not only number of gray scale levels which must be recognized in each resolved element (spatial or spectral) but also the noise accompanying the transmitted signal.