Label-free intravital imaging and imaging of fresh, unstained, resected tissue specimens during surgery offers a wealth of new biomarkers for assessing the tumor microenvironment and diagnosing disease. By generating new excitation wavelengths and manipulating the light stimulus in new ways, Simultaneous Label-free Auto-fluorescence Multi-harmonic (SLAM) microscopy can achieve fast and simultaneous visualization of the rich intrinsic molecular, metabolic, and structural information. These results suggest the broad potential of this stain-free, slide-free, technology and methodology for real-time intraoperative molecular-guided surgical applications.

We developed a new dual-channel optical instrument to detect and enumerate rare fluorescently labeled circulating cells and clusters in vivo. The instrument (termed ‘diffuse in vivo flow cytometry’; DiFC) allows sampling of large circulating blood volumes with diffuse light, while maintaining a negligible false alarm rate. DiFC allows accurate enumeration of circulating cells at concentrations as low as 1 cell per mL. We demonstrate the use of DiFC in monitoring of early dissemination of circulating tumor cells and clusters in a multiple myeloma xenograft model in mice. We anticipate that DiFC will have many other applications in the study of hematogenic metastasis in mice.

Full-Field Optical Coherence Tomography (FFOCT) is an effective technique for cancer detection through tissue anatomy imaging. Dynamic Cell Imaging (DCI) complementarily records the temporal variations of the FFOCT signal induced by the intracellular activity, thus allowing identifying normal, cancer and immune cells in e.g. breast, liver or lung. Cell cultures and resections are studied over time to characterize the involved endogenous biomarker at the subcellular scale. Bimodal FFOCT is also evaluated on breast cancer detection at Peking University People’s Hospital (Beijing) on 200 samples. To further speed-up cancer assessment during surgeries, a computer-aided diagnostic system based on Deep Learning is investigated.

Early characterization of microscopic tumor could play a large role in the treatment of ovarian cancer. The best indicator of cancer free survival is complete tumor resection i.e., no visible disease. Even in this case, some patients fare poorly due to unseen residual disease. Intraoperative visualization of this disease could enable improved tumor removal and improved outcomes. Here we describe a microendoscope able to detect several molecular markers of ovarian cancer in real time with cellular resolution and demonstrate its utility on a mouse model of cancer with peritoneal dissemination.

To exploit fully the clinical potential of molecular fluorescence contrast agents, there is growing interest in the development of quantitative fluorescence imaging systems for surgical use. Here, we leverage cone-beam CT (CBCT) imaging and surgical navigation technology to generate spatial priors for diffuse optical fluorescence tomography (DOFT). A software pipeline is described to incorporate imaging and localization data for tetrahedral mesh generation, non-contact surface flux mapping, and sub-surface region segmentation. Liquid phantom experiments quantified the benefits of spatial priors over an unguided approach. An in vivo animal model demonstrated system performance for tumour delineation using a multi-modal liposomal agent.

Near Infrared Spectroscopy (NIRS) is noted as a noninvasive, cheap, continuous monitoring tool that provides excellent positive and negative predictive values, leading to an improvement of flap salvage. Nevertheless, it is unable to monitor deep layers in the tissue without very large distance between source and detector which is impossible in some configurations. Time-Resolved approach allows us to address such buried flaps. To do so, we developed a Time-Resolved (TR) system and a specific optical stethoscope probe that works in the 750-800 and 850nm wavelengths. This system was tested on 32 pigs bearing abdominal flaps buried on the left lateral abdominal muscle (~1cm deep of skin, fat and highly absorbing muscle). Arterial or venous occlusions were performed and we monitored the blood oxygenation in the flaps using our TR system and compared it with NIRS or LICOX measurements (that measure the tissue pressure in Oxygen, Integra laboratory). Our results show that while the detection of occlusion depends on its depth in NIRS, we were able to detect occlusions whatever the sample (from the surface down to 1.23 mm) using the Resolved Time signals. In addition, the Oxy and Deoxy concentrations information provided by TR made it possible to identify the type of occlusion, which LICOX does not always allow. Acknowledgement: This study is made possible by the support of the ANR FLAPS Monitoring ANR-15-CE19_0010 (Agence Nationale de Recherche, France).

Fluorescence guided surgery, especially aminolevulinic acid induced protoporphyrin IX, has had significant impact for improving the gross total resection of gliomas. The fluorescence is mainly observed visually by the surgeon, and tissue properties like specular reflections or scattering may hide malign tissue which is then left untreated. To overcome these limitations, we propose using fluorescence lifetime imaging of protoporphyrin IX detected via a modulated time-of-flight camera. Here the fluorophore acts as a molecular sensor and can be used to investigate its chemical microenvironment. Tissue phantoms and ex-vivo human brain cancer biopsies were imaged providing additional information compared to intensity-based imaging.

Experimental setup geometry in Monte Carlo (MC) simulations is often simplified to shorten computation times. We investigate the effect of these simplifications on the accuracy of the spatial frequency domain (SFD) reflectance and propose a framework that allows fast and accurate simulations of realistic experimental geometries. We also introduce a new detection scheme in the MC method that eliminates the often overlooked errors arising from the Hankel transform of the spatially discretized reflectance profiles to SFD reflectance. Finally, we propose and evaluate an artificial neural network-based framework capable of estimating high-definition maps of optical properties in real-time.

This presentation overviews clinically-compatible multispectral fluorescence lifetime imaging (FLIM) techniques developed in our laboratory and their ability to operate as stand-alone tools, integrated in a biopsy needle and in conjunction with the da Vinci surgical robot. We present clinical studies in patients undergoing surgery that demonstrate the potential of these techniques for intraoperative delineation of brain tumors and brain radiation necrosis as well as head and neck cancer including image-guided augmented reality during trans-oral robotic surgery (TORS). Challenges and solutions in the clinical implementation of these techniques are discussed.

A proof-of-concept camera for fluorescence lifetime imaging is presented. The camera is based on a novel 32x32-pixel time-gated image sensor and is designed specifically for sub-nanosecond near-infrared fluorescence lifetime imaging. Imaging lifetimes can bring some important benefits to fluorescence imaging and the properties of this camera render it a good candidate for use in fluorescence-guided surgery or pre-clinical imaging. We demonstrate its capabilities by imaging the lifetime of several fluorescent dies in cuvette and also show its in vivo capabilities by performing imaging of NIR fluorescent phantoms in a mouse.

Multispectral imaging (MSI) collects morphological (spatial) and biochemical (spectral) information from tissue, potentially allowing us to more effectively delineate disease. Here, a multispectral endoscope was developed that is capable of capturing such morphological and biochemical information during transsphenoidal surgery. Subjects due to undergo transsphenoidal surgery to resect pituitary adenoma were enrolled for experimental imaging in a pilot clinical study (MAPS). Images were captured before, during and after resection of the adenoma. Here, we present the results from these first-in-human tests, including evaluation of the image quality and classification potential of the multispectral image cubes.

There is growing interest in fluorescence imaging in the short wave infrared (SWIR or NIR-II), range for biomedical applications. Although potential benefits of imaging in this regime have yet to be established, early reports suggest these characteristics provide significant advantages for fluorescence guided surgical applications such as vascular imaging or identification of sub-surface tumors. In this report, we evaluate the performance of SWIR fluorescence imaging in phantoms, human, and a large animal model, and compare it to NIR-I imaging of the same surgical field. To our knowledge, these are the first reports of SWIR fluorescence imaging in humans and large animals.

Fluorescence guidance systems are used for different indications, and the relevant range of concentrations and the relevant dye used varies considerably for human clinical trials. Most systems are designed for high concentration ICG imaging, where 0.1 mg/kg is injected IV and perfusion is imaged, yet other applications such as delayed uptake (or second temporal window) ICG imaging can have tissue concentrations an order of magnitude or more below this. Additionally, IRDye800 is used in both antibody imaging and small protein imaging in clinical trials, but injected doses vary from therapeutic to microdose levels.

Accidental nerve injury is a major morbidity associated with many surgical interventions. Fluorescence image-guided surgery to aid in the precise visualization of vital nerve structures could greatly improve patient outcomes. Herein we report our efforts to synthesized a focused library of 66 far red to NIR fluorophores. Nerve specificity screening using murine models resulted in greater than half of the library with the nerve to background ratio >2. Nerve specificity and scalability of several lead compounds have also been confirmed in swine studies. Further modification of the lead compounds enabled red-shifted emission to 800 nm while maintaining nerve specificity.

Numerous strategies to enhance the specificity of image guided surgery are ongoing, including tumor activatable antibody probes that de-quench upon targeted endocytosis. We have engineered cetuximab (Cet; anti-EGFR Mab) with an optimal fluorophore:quencher ratio yielding the highest degree of activation. A non-specific sham IgG probe was used to account for non-specific activation. We show that specificity of pancreatic cancer imaging is highest with the co-administered probe pair. Using IRDye coupled nanoliposomes functionalized with Cet or sham IgG, non-invasive in vivo quantitation of EGFR expression in glioblastoma, which enabled the generation of high-contrast tumor images for applications in image guided surgery.

We reported the uptake value of a near-infrared imaging agent in an orthotopic glioma mouse models. The imaging agent was IRDy680®. The mice were injected with a trace amount of this imaging agent and imaged on a magnetic resonance imaging device that is coupled with a fluorescence imaging tomography system. By applying a reconstruction method, at each time point, we got a single value for the uptake of this imaging agent in the tumor region. The mean of the uptake values in the mice is reported here.

Complete removal of malignant tissue during primary breast cancer resection is a critical prognostic indicator of local recurrence and overall patient survival, making intraoperative tumor margin assessment essential. Resected specimen staining is a conceptually simple approach; however, fluorescence imaging is dominated by non-specific fluorescent probe uptake in normal tissues requiring a novel approach. Herein, we utilize topical application of dual probe staining strategy termed dual probe difference specimen imaging (DDSI) where a biomarker-targeted fluorescent probe and an untargeted, spectrally-distinct fluorescent probe are topically applied to the fresh resected specimen facilitating calculation of a difference image.

Our group has developed a topical tissue staining technique referred to as dual difference specimen imaging (DDSI) in order to identify targeted receptors in tissue margins of ex vivo breast tissue. We describe the development, characterization and utility of quantum dot antibody complexes (QDACs) administered topically to perform DDSI on breast xenografts. Several staining variables (temperature during staining and QDAC concentration) were tested in order to optimize current tissue staining protocols. Increasing the temperature during tissue staining improved the tumor-to-muscle contrast and measure of diagnostic potential compared to tissues stained at room temperature. Future investigation will be focused on detecting several targeted receptors in breast tissue with DDSI.

Developing novel imaging devices to improve fluorescence-guided surgery require tissue-mimicking optical phantoms with adjustable and well-characterised optical properties for validation. In this study, we introduce soft tissue-mimicking fluorescence phantoms (TMFP) based on gel wax and quantum dots. We investigated and demonstrated: 1) the ability to independently tune the scattering, absorption and fluorescence of the phantoms; 2) the stability of the TMFP fluorescence signal over time; and 3) the potential of the proposed phantoms for the validation of imaging systems.

Breast cancer patients that experience complete removal of the primary tumor, or negative surgical margins (NSMs), benefit from decreased rates of local recurrence and increased survival. However, intraoperative margin detection is limited to visualization, palpation, and experience to identify malignant vs. healthy tissue. As a result, roughly 1/3 of patients treated with breast conserving surgery (BCS) have residual cancer cells left at the resection border, or positive surgical margins (PSMs). Fluorescence image-guided surgery (FIGS) is a promising alternative for intraoperative margin detection, providing surgeons with real-time feedback on tumor location, increasing the likelihood of achieving NSMs. Our past work has demonstrated that the use of self-assembled hyaluronic acid (HA) nanoparticles improves the delivery of indocyanine green (ICG) to breast tumors, enhancing intraoperative tumor signal and contrast. This study built upon these findings by assessing the surgical efficacy of ICG-loaded HA nanoparticles (NanoICG) for the image-guided resection of orthotopic iRFP+/luciferase+ 4T1 breast tumors in BALB/c mice. Tumors were resected with FIGS in mice treated with ICG or NanoICG and compared to bright light surgery (BLS) or sham controls. Tumor growth and recurrence were monitored with bioluminescence imaging. NanoICG improved complete resection and prolonged tumor-free survival. Additionally, NanoICG provided greater intraoperative contrast in malignant tissue than ICG or BLS. Furthermore, NanoICG demonstrated a greater ability to identify small, occult lesions than ICG. Overall, the use of NanoICG for the fluorescence image-guided resection of breast tumors could potentially decrease PSM rates and improve complete tumor removal.

Improved methods to determine tumor localization and disease extension are critical factors in the management of pancreatic ductal adenocarcinoma (PDAC). Thus, contrast-enhanced fluorescence-guided detection of these lesions could improve the treatment of PDAC. A current limitation to fluorescence enhancement of PDAC is non-specific signal due to the clearance of the imaging dye, e.g. indocyanine green, like the liver, spleen or other local, intraperitoneal organs where metastatic spread may be present. We report surgical imaging agents that provide strong enhancement of PDAC compared to healthy tissue, but also have significantly reduced nonspecific background signal in clearance organs of the peritoneal cavity.

Accurate, real-time visualization is critical for efficient, effective and safe surgery. Although optical imaging using near-infrared (NIR) fluorescence has been used for visualization of anatomic structures and physiologic functions in open and minimally invasive surgeries, its efficacy and adoption remain suboptimal due to the lack of specificity and sensitivity. Herein, we report a novel class of compounds, which are exclusively metabolized in liver or kidney, rapidly excreted into to biliary or urinary systems, and emitted two different NIR fluorescence spectrums.

Although reflectance confocal microscopy has increased sensitivity of BCC detection, specificity remains moderate (60-70%), due to poor endogenous differential contrast between tumors and normal structures. Fluorescence confocal microscopy, using PARPi-FL, a small fluorescent molecule that targets PARP1 in nuclei, may improve specificity. We demonstrated more intense PARPi-FL staining in 25/30 (83.33%) BCC compared to normal in tissue sections and its permeability through intact ex vivo human skin. PARPi-FL has acquired an IND status for oral cancer detection in vivo and may be eventually translated as a topical contrast agent on human skin in vivo for improving specificity for detecting BCCs.

The emerging clinical use of targeted fluorescent agents heralds a shift in intraoperative imaging practices that overcome the limitations of human vision. However, in contrast to established radiological methods, no appropriate performance specifications and standards have been established in fluorescence molecular imaging. Moreover, the dependence of fluorescence signals on many experimental parameters and the use of wavelengths ranging from the visible to short-wave infrared (400–1,700 nm) complicate quality control in fluorescence molecular imaging. In this talk, the experimental parameters that critically affect fluorescence molecular imaging accuracy will be discussed. Furthermore, the concept of high-fidelity fluorescence imaging as a means for ensuring reliable reproduction of fluorescence biodistribution in tissue will be introduced.

Molecular image-guided surgery has the potential for translating the tools of molecular pathology to real-time guidance in surgery. The strengths and opportunities must be continued but are hampered by important weaknesses and threats within the field. Key issues to solve are related to the inability of macroscopic imaging tools to resolve microscopic biological disease heterogeneity and the limitations in microscopic systems matching surgery workflow, and also parsing out true molecular specific uptake from simple enhanced permeability and retention. These are very technical concerns which can likely be solved with added technical focus. Some solid steps have been demonstrated in the use of fluorescent labeling commercial antibodies and separately in microdosing studies with small molecules.

Sentinel lymph node status is a critical prognostic factor in breast cancer treatment and is essential to guide future adjuvant treatment. The possibility of missed micrometastases by using conventional methods created a demand for the development of more accurate approach to guide pathology. This study demonstrates the promise of paired-agent approach to significantly improve the sensitivity of cancer detection for breast cancer sentinel lymph node biopsy, and suggests that fewer than 1000 cells may be potentially observable in a whole human lymph node.

Although sodium fluorescein (NaFl) has been under investigation for use as a fluorescent tumor marker during brain surgery for decades, recent clinical observations have shown significant accumulation of the dye in normal brain tissue. To explain these observations, we fully characterized the behavior of NaFl in blood, brain, and tumor tissue. Upon administration, only a portion of NaFl binds to blood proteins and the remaining unbound dye crosses into normal brain tissue readily, impairing tumor-to-normal contrast. A larger, pegylated form of fluorescein was also studied and showed no penetration of the barrier in normal brain and improved tumor discrimination.

Near-infrared fluorescence imaging is a promising intraoperative technique for real-time visualization of vital structures and tumor tissue during surgery. This manuscript describes the applications and limitations of NIR fluorescence imaging, and provides a general overview of two novel fluorescent agents and the process of clinical translation. The process of clinical translation of novel fluorescent agents is an essential part in the evolution of NIR fluorescence guided surgery. Treatments and surgeries are constantly advancing, which can occasionally cause challenges or difficulties for the surgeons. Poor visualization of tumors during surgery is one of the major challenges surgeons often face in oncologic patients, mainly due to the improved neo-adjuvant treatment patients receive. In these cases, NIR fluorescence imaging with the use of tumor-targeted fluorescent agents can play an essential role and help provide better results or outcomes. However, before this technique can be implemented in standard of care, optimal tumor-targeted fluorescent agents need to be developed and novel fluorescent agents need to undergo a successful process of clinical translation.

Since 2011, several clinical trials in intraoperative molecular imaging of lung cancer have been performed. Most recently, a Phase II clinical trial has been initiated to test the efficacy of folate receptor targeted imaging of pulmonary adenocarcinomas. This is the largest trial to-date in lung cancer. As of August 2018, more than 65 patients have been enrolled in the study evaluating a novel near-infrared (NIR) contrast agent that targets folate receptor alpha on pulmonary tumors. This receptor is present in virtually all lung adenocarcinomas. Several of the key findings include the dye can detect tumors at 2 cm depth of penetration, it can identify synchronous lesions and it can identify cancer cells at the margins.

Introduction: Glioblastoma (GBM) is the most common and devastating primary brain tumor, with ill-defined margins that makes maximal tumor resection with minimal morbidity a challenge. Epidermal growth factor receptor (EGFR) is the most frequently amplified gene in GBM and is associated with overexpression. We hypothesize that fluorescence labeled anti-EGFR monoclonal antibodies (mAb), panitumumab-IRDye800 (pan800) and cetuximab-IRDye800 (cet800), could be leveraged to enhance tumor contrast during surgical resection and improve patient outcome. Methods: 50mg fluorescently labeled corresponding study drugs, pan800 and cet800 respectively, were administered 1-2 days in glioblastoma patients with contrast enhancing (CE) tumors prior to surgery following 100 mg loading dose of unlabeled cetuximab or panitumumab. Near-infrared fluorescence imaging of tumor and histologically negative peri-tumoral tissue was performed intraoperatively and ex vivo. Fluorescence was measured as mean fluorescence intensity (MFI), and tumor-to-background ratios (TBRs) were calculated by comparing MFIs of tumor and histologically uninvolved tissue. Results: Despite heterogeneous drug uptake across all resected brain tissues, mean fluorescence intensity (MFI) correlated strongly (R^2=0.97) with tumor volume among histologically confirmed tumor tissues. The smallest detectable tumor size in a closed-field setting was 4.2 x 2.7 mm^2 (8.2 mg) for pan800 and 8.5 x 6.6 mm^2 (70mg) for cet800. Tumor tissues from pan800 infusion had significantly higher mean TBR (8.1 ± 4.6) than cet800 infused ones in intraoperative imaging (3.3 ± 2.7; P = 0.004). NIR fluorescence from both test drugs provided high contrast to identify as few as a cluster of (5 ± 1) tumor cells in macroscopic imaging of whole sections of paraffin embedded tissues. Sensitivity and specificity of MFI for viable tumor detection was calculated and fluorescence was found to be highly sensitive (64.4% for pan800, 73.0% for cet800) and specific (98.0% for pan800, 66.3% for cet800) for viable tumor tissue while normal peri-tumoral tissue showed minimal fluorescence. No related grade-2 adverse events were observed 30 days beyond the infusion of either study drugs. Conclusion: EGFR antibody based imaging for contrast-enhanced glioblastomas proved safe in human patients and specific intratumoral delivery of NIR fluorescence provided high optical contrast and resolution for intraoperative image-guided resection.

In fluorescence guided surgery, visualization of tumour present at a subsurface level, such as soft tissue sarcomas, is not straightforward. Resection of these tumours is performed using ‘wide local excision’ where a single, complete mass is removed with an intact zone of normal tissue. We aim to determine best practice methodology for NIR-guided surgical dissection for human trials with multiple wavelength imaging combined with traditional white light imaging. By measuring the fluorescence of both ABY-029 and ICG, we compare the performance of two commercially available systems, Solaris and SPY-Elite, in gelatine phantom models of sarcoma and sarcoma xenograft murine model.

The use of fluorescence guided surgery has increased greatly since the introduction of Novadaq’s SPY system for use in cardiac bypass surgery in 2005. The subsequent release of Firefly, the fluorescence imaging technology on the da Vinci Surgical System was obtained in 2011 and has helped improve access of fluorescence imaging to surgeons around the world. Fluorescence imaging is now an option provided by many medical device manufacturers. An overview of successes and challenges in the field will be presented.

Near-infrared (NIR) fluorescence combined with targeting epidermal growth factor receptor (EGFR) overexpression for surgical guidance in many cancers is gaining momentum. ABY-029 is an anti-EGFR Affibody molecule conjugated to IRDye 800CW that is FDA approved as an exploratory Investigational New Drug (eIND 122681) and is being investigated in a number of promising first-in-human clinical Phase 0 trials for recurrent glioma, soft-tissue sarcoma, and head and neck cancers. Here, we summarize our experience using ABY-029 for surgical resection by describing tissue contrast and correlation of ABY-029 fluorescence to EGFR tissue expression.

The most common complication of autologous breast reconstructions using a deep inferior epigastric artery (DIEP) flap after mastectomy is fat necrosis due to ischemia. DIEP flap perfusion assessment can be optimized by using near-infrared fluorescence (NIRF) imaging with indocyanine green (ICG). Several studies demonstrated diminished incidence of fat necrosis, partial flap loss and other complications. Studies with large patient numbers and standardized outcomes are lacking. To determine the correlation of incidence of fat necrosis and NIRF imaging with ICG we performed: 1) a review to evaluate relevant literature, and 2) a pilot study of NIRF imaging. With the aim to decide whether a randomized controlled trial (RCT) should be started. Seven studies were included in the review, showing NIRF imaging was feasible and incidence of fat necrosis was diminished. However, most studies were retrospective and with small patient numbers. In the pilot study, 18 flaps, assessed with NIRF imaging were prospectively included and compared to 18 retrospectively included flaps solely assessed based on clinical findings. There were no significant differences in patient and surgery characteristics. Incidence of complications and fat necrosis decreased from 30% to 6% (p-value: 0.07) in the NIRF imaging group. No strong conclusions can be drawn from the pilot study given the low patient number, neither from the reviewed studies. But based on the results a multicenter RCT would be recommended to determine the actual value of NIRF imaging for perfusion assessment of DIEP flaps. An RCT could also aid in wider implementation of this accessible technique in a standardized matter.

Radiation medicine (radiation therapy, diagnostic radiology) and photomedicine (therapeutic, diagnostic) have evolved independently. However, there are multiple uses of optical technologies to enable radiation medicine and, conversely, technologies employing ionizing radiation play important roles in photomedicine. In some cases, the respective technologies simply provide complementary information (e.g. MRI plus fluorescence imaging to guide surgery). However, there are also deeper synergies, both technological (e.g. Xray image guidance of light-based treatments) and mechanistic (e.g. Xray activated phototherapy, optical readout of Xray-tissue interactions). Light- and radiation-sensitive nanoparticles are also playing increasing roles in these emerging synergistic approaches, with potential high clinical impact.

Chemo- and/or radiotherapy remain treatment standards for cancer. Clinical challenges including poor tumour specificity and cause dose-dependent side effects. However, the radiotherapy dose can be reduced by using a nanoconstruct approach. To achieve this, we combined X-ray radiation and other treatment modalities by using a range of specially designed nanocarriers. These include scintillating inorganic nanoparticles, gold nanoparticles, Poly Lactic-co-Glycolic Acid (PLGA) and liposomes. Photosensitive verteporfin molecules were either conjugated or loaded inside the nanoconstructs. These unique nanoconstructs enables the effective ablation of cancer cells in vitro and control of tumour growth in vivo at a low X-ray dose.

Photodynamic therapy (PDT) has emerged as one of the important therapeutic options in management of cancer and other diseases. Most photosensitizers are highly hydrophobic and require delivery systems, through which the therapy efficiency could be improved. Here different nanoparticles, including gold nano-sphere, gold nanorods and titanium dioxide (TiO2) nanopaticles, were introduced as passive carriers to delivery photosensitizer for photodynamic therapy.

X-ray luminescence computed tomography (XLCT) and X-ray fluorescence computed tomography (XFCT) are two emerging technologies in X-ray imaging. In these modalities, images are formed through detection of secondary emissions (light in XLCT, or secondary X-rays in XFCT) following X-ray excitations. XLCT and XFCT enable us to leverage the widely used X-ray imaging for simultaneous in vivo molecular and functional imaging. Depending on the geometry of the excitation X-ray beam (pencil-, fan-, and cone-beam or coded apertures), optimal tradeoff between imaging efficiency and spatial resolution can be achieved. The novel imaging principles of XLCT/XFCT make it possible to achieve a spatial resolution comparable to that of anatomical X-ray imaging. Here, we summarize our studies in this area in the past decade and discuss their prospects.

Our research group discovered that the luminescence of water during irradiations of radiations at lower energy than the Cerenkov-light threshold. The phenomenon can be used for range estimations of particle therapies. The luminescence increased linearly with the radiation energies thus the phenomenon can be used for dose estimation of radiations. In addition, we found the source of the luminescence of water recently from the light spectrum measurement. Because luminescence of water lower energy than the Cerenkov-light threshold is not included in any simulation software, the process needs to be added to avoid the misunderstanding of the physical phenomenon related to optical imaging.

During radiotherapy, X-ray beams induce Cherenkov light emission in tissue as part of the dose delivery. This light can be used for dosimetry. The Cherenkov light levels are challenging to detect in clinical environments. Thus, imaging sensors for this application must be highly sensitive and must be able to time gate faster than a microsecond. In this study, the use of a SPAD camera was examined. The SPAD camera sensors could be a viable alternative for Cherenkov imaging, as compared to current imaging methods that are mostly focused around image intensifier-based cameras.

The linearity between TSET radiation dose and Cherenkov signal can be affected by factors including perspective direction, tissue curvature and tissue optical properties. In this study, we investigate the effect of tissue optical properties on detected Cherenkov emission from tissue surface using Monte Carlo modelling. We simulate the propagation of Cherenkov photons generated in the tissue of optical properties over the wavelength range of 400-1000nm. Fractional Cherenkov photons escaping the air-tissue interface are scored as a function of lateral radius and escaping angle.

Solid tumors often exhibit abnormal morphology which can result in tumor hypoxia and has been correlated with poor prognosis. External beam radiation therapy (EBRT) delivers daily targeted radiation doses over the course of weeks to kill cancerous cells. A non-contact optical method for measuring in vivo oxygen levels during EBRT treatments has been developed to provide early indications of hypoxic tumor environments. A pixel-map of the pO2 can be generated using a time-gated intensified camera to measure both Cherenkov emissions, generated by interactions between tissue and high-energy electrons, and Cherenkov-excited luminescence of oxygen-sensitive phosphorescent compounds.