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Oxygen sensing with light has been developing for many decades using injectable molecules called Oxyphors, which are pegylated, dendrimer-encapsulated metalloporphyrins that have a phosphorescence emission lifetime that is a direct reporter of the local oxygen partial pressure (pO2). In recent years, the ability to image this emission from tissue with Cherenkov light excitation during high-energy X-ray-based radiation therapy has been shown and developed for research studies. The main value of this type of lifetime-based pO2 sensing, termed Cherenkov-Excited Luminescence Imaging (CELI) is in its ability to image values of pO2 from within the tissue during radiation therapy using tracers that are systemic and biologically compatible. Spatial mapping of pO2 can realized either as surface imaging or deep tissue tomography through a few centimeters. The spatial resolution is radiation dose-dependent but can be near 0.1 mm, based upon radiation doses expected in a fractionated treatment plan. When imaging tumors with a broad beam irradiation, histograms of pO2 values across the surface have been demonstrated illustrating microscopic sensitivity to the ranges of oxygen levels present, and the ability to track these microscopic histograms during daily fractionated radiation therapy is possible. The pO2 distributions provide for sensitivity to the hypoxic fraction of the tumor—a unique capability of oxygen imaging that has microscopic spatial sampling. Comparisons of the CELI pO2 method to other oxygen-sensing methods, as well as the ability to use the CELI technique as a tool to examine the optimization of radiation therapy treatment technique is ongoing.

X-ray induced molecular luminescence (XML) is a phenomenon that can be utilized for clinical, deep-tissue functional imaging of tailored molecular probes. In this study, a survey of common or clinically approved fluorophores was carried out for their megavoltage X-ray induced excitation and emission characteristics. We find that direct scintillation effects and Cherenkov generation are two possible ways to cause these molecules’ excitation. To distinguish the contributions of each excitation mechanism, we exploited the dependency of Cherenkov radiation yield on X-ray energy. The probes were irradiated by constant dose of 6 MV and 18 MV X-ray radiation, and their relative emission intensities and spectra were quantified for each X-ray energy pair. From the ratios of XML, yield for 6 MV and 18 MV irradiation we found that the Cherenkov radiation dominated as an excitation mechanism, except for aluminum phthalocyanine, which exhibited substantial scintillation. The highest emission yields were detected from fluorescein, proflavin and aluminum phthalocyanine, in that order. XML yield was found to be affected by the emission quantum yield, overlap of the fluorescence excitation and Cherenkov emission spectra, scintillation yield. Considering all these factors and XML emission spectrum respective to tissue optical window, aluminum phthalocyanine offers the best XML yield for deep tissue use, while fluorescein and proflavine are most useful for subcutaneous or superficial use.

Purpose High dose-rate conditions, coupled with problems related to small field dosimetry, make dose characterization for FLASH-RT challenging. Most conventional dosimeters show significant dependence on dose rate at ultra-high dose rate conditions or fail to provide sufficiently fast temporal data for pulse to pulse dosimetry. Here fast 2D imaging of radioluminescence from a water and quinine phantom was tested for dosimetry of individual 4 μs linac pulses. Methods A modified clinical linac delivered an electron FLASH beam of >50 Gy/s to clinical isocenter. This modification removed the X-ray target and flattening filter, leading to a beam that was symmetric and gaussian, as verified with GafChromic EBT-XD film. Lateral projected 2D dose distributions for each linac pulse were imaged in a quinine-doped water tank using a gated intensified camera, and an inverse Abel transform reconstruction provided 3D images for on-axis depth dose values. A total of 20 pulses were delivered with a 10 MeV, 1.5 cm circular beam and beam with jaws wide open (40 × 40 cm2 ), and a 3D dose distribution was recovered for each pulse. Beam output was analyzed on a pulse by pulse basis. Results The Rp , Dmax and the R50 measured with film and optical methods agreed to within 1 mm for the 1.5 cm circular beam and the beam with jaws wide open. Cross beam profiles for both beams agreed with film data with > 95 % passing rate (2%/2mm gamma criteria). The optical central axis depth dose agreed with film data, except for near the surface. A temporal pulse analysis revealed a ramp-up period where the dose per pulse increased for the first few pulses and then stabilized. Conclusions Optical imaging of radioluminescence was presented as a valuable tool for establishing a baseline for the recently initiated electron FLASH beam at our institution.

Purpose The novel scintillator‐based system described in this study is capable of accurately and remotely measuring surface dose during Total Skin Electron Therapy (TSET); this dosimeter does not require post‐exposure processing or annealing and has been shown to be re‐usable, resistant to radiation damage, have minimal impact on surface dose, and reduce chances of operator error compared to existing technologies e.g. optically stimulated luminescence detector (OSLD). The purpose of this study was to quantitatively analyze the workflow required to measure surface dose using this new scintillator dosimeter and compare it to that of standard OSLDs. Methods Disc‐shaped scintillators were attached to a flat‐faced phantom and a patient undergoing TSET. Light emission from these plastic discs was captured using a time‐gated, intensified, camera during irradiation and converted to dose using an external calibration factor. Time required to complete each step (daily QA, dosimeter preparation, attachment, removal, registration, and readout) of the scintillator and OSLD surface dosimetry workflows was tracked. Results In phantoms, scintillators and OSLDs surface doses agreed within 3% for all data points. During patient imaging it was found that surface dose measured by OSLD and scintillator agreed within 5% and 3% for 35/35 and 32/35 dosimetry sites, respectively. The end‐to‐end time required to measure surface dose during phantom experiments for a single dosimeter was 78 and 202 sec for scintillator and OSL dosimeters, respectively. During patient treatment, surface dose was assessed at 7 different body locations by scintillator and OSL dosimeters in 386 and 754 sec, respectively. Conclusion Scintillators have been shown to report dose nearly twice as fast as OSLDs with substantially less manual work and reduced chances of human error. Scintillator dose measurements are automatically saved to an electronic patient file and images contain a permanent record of the dose delivered during treatment.

The 3D printing of fluorescent materials could help develop, validate, and translate imaging technologies, including systems for fluorescence-guided surgery. Despite advances in 3D printing techniques for optical targets, no comprehensive method has been demonstrated for the simultaneous incorporation of fluorophores and fine-tuning of absorption and scattering properties. Here, we introduce a photopolymer-based 3D printing method for manufacturing fluorescent material with tunable optical properties. The results demonstrate the ability to 3D print various individual fluorophores at reasonably high fluorescence yields, including IR-125, quantum dots, methylene blue, and rhodamine 590. Furthermore, tuning of the absorption and reduced scattering coefficients is demonstrated within the relevant mamalian soft tissue coefficient ranges of 0.005–0.05 mm−1 and 0.2–1.5 mm−1, respectively. Fabrication of fluorophore-doped biomimicking and complex geometric structures validated the ability to print feature sizes less than 200 μm. The presented methods and optical characterization techniques provide the foundation for the manufacturing of solid 3D printed fluorescent structures, with direct relevance to biomedical optics and the broad adoption of fast manufacturing methods in fluorescence imaging.

Luminescent probes based on silicon nanocrystals (SiNCs) have many advantages for bioimaging compared to more conventional quantum dots: abundancy of silicon combined with its biocompatibility; tunability of the emission color of SiNCs in the red and NIR spectral region to gain deeper tissue penetration; long emission lifetimes of SiNCs (hundreds of μs) enabling time-gated acquisitions to avoid background noise caused by tissue autofluorescence and scattered excitation light. Here we report a new three-step synthesis, based on a low temperature thiol-ene click reaction that can afford SiNCs, colloidally stable in water, with preserved bright red and NIR photoluminescence (band maxima at 735 and 945 nm for nanocrystals with diameters of 4 and 5 nm, respectively) and long emission lifetimes. Their luminescence is insensitive to dioxygen and sensitive to pH changes in the physiological range, enabling pH sensing. In vivo studies demonstrated tumor accumulation, 48 hours clearance and a 3-fold improvement of the signal-to-noise ratio compared to steady-state imaging.

Background Total skin electron therapy (TSET) utilizes high-energy electrons to treat malignancies on the entire body surface. The otherwise invisible radiation beam can be observed via the optical Cherenkov photons emitted from interactions between the high-energy electron beam and tissue. Methods and materials With a time-gated intensified camera system, the Cherenkov emission can be used to evaluate the dose uniformity on the surface of the patient in real time. Fifteen patients undergoing TSET in various conditions (whole body and half body) were imaged and analyzed. Each patient was monitored during TSET via in vivo detectors (IVD) in nine locations. For accurate Cherenkov imaging, a comparison between IVD and Cherenkov profiles was conducted using a polyvinyl chloride board to establish the perspective corrections. Results and discussion With proper corrections developed in this study including the perspective and inverse square corrections, the Cherenkov imaging provided two-dimensional maps proportional to dose and projected on patient skin. The results of ratio between chest and umbilicus points were in good agreement with in vivo point dose measurements, with a standard deviation of 2.4% compared to OSLD measurements. Conclusions Cherenkov imaging is a viable tool for validating patient-specific dose distributions during TSET.

PURPOSE The aim of this study was to create an optical imaging-based system for quality assurance (QA) testing of a dedicated Co-60 total body irradiation (TBI) machine. Our goal is to streamline the QA process by minimizing the amount time necessary for tests such as verification of dose rate and field homogeneity. METHODS Plastic scintillating rods were placed directly on the patient treatment couch of a dedicated TBI 60 Co irradiator. A tripod-mounted intensified camera was placed directly adjacent to the couch. Images were acquired over a 30-s period once the cobalt source was fully exposed. Real-time image filtering was used; cumulative images were flatfield corrected as well as background and darkfield subtracted. Scintillators were used to measure light-radiation field correspondence, dose rate, field homogeneity, and symmetry. Dose rate effects were measured by modifying the height of the treatment couch and scintillator response was compared to ionization chamber (IC) measurements. Optically stimulated luminesce detector (OSLD) used as reference dosimeters during field symmetry and homogeneity testing. RESULTS The scintillator-based system accurately reported changes in dose rate. When comparing normalized output values for IC vs scintillators over a range of source-to-surface distances, a linear relationship (R2 = 0.99) was observed. Normalized scintillator signal matched OSLD measurements with

Background Daylight-activated PDT has seen increased support in recent years as a treatment method for actinic keratosis and other non-melanoma skin cancers. The inherent variability observed in broad-spectrum light used in this methodology makes it difficult to plan and monitor light dose, or compare to lamp light doses. Methods The present study expands on the commonly used PpIX-weighted effective surface irradiance metric by introducing a Monte Carlo method for estimating effective fluence rates into depths of the skin. The fluence rates are compared between multiple broadband and narrowband sources that have been reported in previous studies, and an effective total fluence for various treatment times is reported. A dynamic estimate of PpIX concentration produced during pro-drug incubation and treatment is used with the fluence estimates to calculate a photodynamic dose. Results Even when there is up to a 5x reduction between the effective surface irradiance of the broadband light sources, the effective fluence below 250 μm depth is predicted to be relatively equivalent. An effective threshold fluence value (0. 70 J e f f / c m 2 ) is introduced based on a meta-analysis of previously published ALA-PpIX induced cell death. This was combined with a threshold PpIX concentration (50 nM) to define a threshold photodynamic dose of 0.035 u M J e f f / c m 2 . Conclusions The threshold was used to generate lookup tables to prescribe minimal treatment times to achieve depth-dependent cytotoxic effect based on incubation times and irradiance values for each light source.

We report a high light-throughput spectroscopic dosimeter system that is able to non-invasively measure luminescence signals singlet oxygen (1 O2 ) produced during photodynamic therapy (PDT) using a CW (continuous wave) light source. The system is based on a compact, fiber-coupled, high collection efficiency spectrometer (>50% transmittance) designed to maximize optical throughput but with sufficient spectral resolution (~7 nm). This is adequate to detect 1 O2 phosphorenscence in the presence of strong luminescence background in-vivo. This system provides simultaneous acquisition of multiple spectral data points, allowing for more accurate determination of luminescence baseline via spectral fitting and thus the extraction of 1 O2 phosphorescence signal based solely on spectroscopic decomposition, without the need for time-gating. Simultaneous collection of photons at different wavelengths improves the quantum efficiency of the system when compared to sequential spectral measurements such as filter-wheel or tunable-filter based systems. A prototype system was tested during in-vivo PDT tumor regression experiments using benzoporphyrin derivative (BPD) photosensitizer. It was found that the treatment efficacy (tumor growth inhibition rate) correlated more strongly with 1 O2 phosphorescence than with PS fluorescence. These results indicate that this high photon-collection efficiency spectrometer instrument may offer a viable option for real-time 1 O2 dosimetry during PDT treatment using CW light. This article is protected by copyright. All rights reserved.

In patients undergoing breast-conserving surgery (BCS), the rate of re-excision procedures to remove residual tumor left behind after initial resection can be high. Projection radiography, and recently, volumetric x-ray imaging are used to assess margin adequacy, but x-ray imaging lacks contrast between healthy, abnormal benign, and malignant fibrous tissues important for surgical decision making. The purpose of this study was to compare micro-CT and optical scatter imagery of surgical breast specimens and to demonstrate enhanced contrast-to intra-tumoral morphologies and tumor boundary features revealed by optical scatter imaging. A total of 57 breast tumor slices from 57 patients were imaged ex vivo by spatially co-registered micro-CT and optical scatter scanning. Optical scatter exhibited greater similarity with micro-CT in 89% (51/57) of specimens versus diffuse white light (DWL) luminance using mutual information (mean ± standard deviation of 0.48 ± 0.21 versus 0.24 ± 0.12; p p p

Active line scan imaging (LSI) with spatial gating is explored for wide-field, sub-diffuse sensing of scattering microtextures. Spatial gating involves masking detector pixels far from the laser line of incidence on a turbid target. The active line scan provides broadband spatial frequency modulation, and the spatial gating effectively high pass filters the reflectance. The major benefits of LSI are that of high dynamic range signal preservation and high contrast-to-noise when imaging at high spatial frequencies. As such, LSI presents as an inherently high sensitivity and high dynamic range way to image microscopic scattering features on the surfaces of turbid media.

Fluorescence imaging for surgical guidance is a proven modality that allows for visualization of fluorescent markers in numerous biological imaging applications. As the field continues to develop there is an urgent need for fluorescence-imaging standards and targets that enable system characterization, performance monitoring, and the development of analytical algorithms. 3D-printing technology has shown promise in providing biomimicking phantoms that allow simulation of realistic clinical conditions. Here, we present a comprehensive method for 3D printing fluorescent and tissue-equivalent material using photo-curable resins. We show the ability to print Indocyanine-green (ICG) equivalent material in complex shapes that would enable the evaluation of ICG-specific clinical systems. The method presented allows tuning of both the reduced scattering and absorption coefficients at multiple wavelengths, allowing for application-specific manufacturing of 3D-printed phantoms.

A new low-cost imaging system has been developed for MRI-guided Near-Infrared Spectral Tomography (MRI-NIRST) for breast cancer detection. In this new system, 8 flexible sensing strips with 4 source fibers and 4 photodetectors will be applied directly to the breast, from nipple towards chest-wall, to cover the entire breast. For each source illumination, data will be collected at 32 detector locations. The feasibility of this new imaging system was demonstrated using 3D simulation breast phantoms that created from real breast MR images. Our results indicated that total hemoglobin in a tumor smaller than 1 mm could be recovered with this system.

A new photo-detector (PD) and source fiber based wearable MRI-guide near infrared spectroscopic tomographic imaging (MRg-NIRST) system has been designed and is underdevelopment. In this new system, eight flex circuit strips, each with 6 PDs and 6 side-firing fibers will be applied to the breast, prior to coil/biopsy positioning. The intensity of the diffused light will be acquired at 48x68 source-detector positions at 12 wavelengths. The 3D optical image reconstruction will be guided through the structural information obtained from T2 MRI and the scattering properties associated with MRI defined breast density.

Total Skin Electron Therapy (TSET) utilizes high-energy electrons to treat cancers on the entire body surface. The otherwise invisible radiation beam can be observed via the optical Cherenkov photons emitted from interaction between the high-energy electron beam and tissue. Cherenkov emission can be used to evaluate the dose uniformity on the surface of the patient in real-time using a time-gated intensified camera system. Each patient was monitored during TSET by in-vivo detectors (IVD) as well as Scintillators. Patients undergoing TSET in various conditions (whole body and half body) were imaged and analyzed. A rigorous methodology for converting Cherenkov intensity to surface dose as products of correction factors, including camera vignette correction factor, incident radiation correction factor, and tissue optical properties correction factor. A comprehensive study has been carried out by inspecting various positions on the patients such as vertex, chest, perineum, shins, and foot relative to the umbilicus point (the prescription point).

Significance: Deep-tissue penetration by x-rays to induce optical responses of specific molecular reporters is a new way to sense and image features of tissue function in vivo. Advances in this field are emerging, as biocompatible probes are invented along with innovations in how to optimally utilize x-ray sources. Aim: A comprehensive review is provided of the many tools and techniques developed for x-ray-induced optical molecular sensing, covering topics ranging from foundations of x-ray fluorescence imaging and x-ray tomography to the adaptation of these methods for sensing and imaging in vivo. Approach: The ways in which x-rays can interact with molecules and lead to their optical luminescence are reviewed, including temporal methods based on gated acquisition and multipoint scanning for improved lateral or axial resolution. Results: While some known probes can generate light upon x-ray scintillation, there has been an emergent recognition that excitation of molecular probes by x-ray-induced Cherenkov light is also possible. Emission of Cherenkov radiation requires a threshold energy of x-rays in the high kV or MV range, but has the advantage of being able to excite a broad range of optical molecular probes. In comparison, most scintillating agents are more readily activated by lower keV x-ray energies but are composed of crystalline inorganic constituents, although some organic biocompatible agents have been designed as well. Methods to create high-resolution structured x-ray-optical images are now available, based upon unique scanning approaches and/or a priori knowledge of the scanned x-ray beam geometry. Further improvements in spatial resolution can be achieved by careful system design and algorithm optimization. Current applications of these hybrid x-ray-optical approaches include imaging of tissue oxygenation and pH as well as of certain fluorescent proteins. Conclusions: Discovery of x-ray-excited reporters combined with optimized x-ray scan sequences can improve imaging resolution and sensitivity.

We examine the value of an active line scan with spatial gating for imaging sub-diffuse, wide-field reflectance microtexture. Line scanning combined with spatial gating and linear translation can be used for localized detection of features in the surface layer of a turbid target. The line scan provides broadband spatial frequency modulation, and the spatial gating effectively high-pass filters the reflectance. The major benefit of this approach is that of high dynamic range (70%–90%) signal preservation and high contrast to noise when imaging at high spatial frequencies. Alternative approaches, such as spatial frequency domain imaging, are degraded by low dynamic range in demodulated images, making it nearly impossible to image over a wide field of view at frequencies over 1.5 m m − 1 using commercial technology. As such, active line scanning with spatial gating presents as an inherently high sensitivity and high dynamic range method of imaging microscopic scattering features in only the surface layer of a turbid medium.

Significance: The necessity to use exogenous probes for optical oxygen measurements in radiotherapy poses challenges for clinical applications. Options for implantable probe biotechnology need to be improved to alleviate toxicity concerns in human use and facilitate translation to clinical trial use. Aim: To develop an implantable oxygen sensor containing a phosphorescent oxygen probe such that the overall administered dose of the probe would be below the Federal Drug Administration (FDA)-prescribed microdose level, and the sensor would provide local high-intensity signal for longitudinal measurements of tissue pO2. Approach: PtG4, an oxygen quenched dendritic molecule, was mixed into an agarose matrix at 100 μM concentration, allowing for local injection into tumors at the total dose of 10 nmol per animal, forming a gel at the site of injection. Cherenkov-excited luminescence imaging (CELI) was used to acquire the phosphorescence and provide intratumoral pO2. Results: Although PtG4 does not form covalent bonds with agarose and gradually leaches out into the surrounding tissue, its retention time within the gel was sufficiently long to demonstrate the capability to measure intratumoral pO2 with the implantable gel sensors. The sensor’s performance was first evaluated in vitro in tissue simulation phantoms, and then the sensor was used to measure changes in oxygen in MDA-MB-231 tumors during hypofractionated radiotherapy. Conclusions: Our study demonstrates that implantable oxygen sensors in combination with CELI present a promising approach for quantifying oxygen changes during the course of radiation therapy and thus for evaluating the tumor response to radiation. By improving the design of the gel–probe composition in order to prevent leaching of the probe into the tissue, biosensors can be created that should allow longitudinal oxygen measurements in tumors by means of CELI while using FDA-compliant microdose levels of the probe and thus lowering toxicity concerns.

Concurrent chemoradiotherapy is used for advanced cancers, but the chemotherapy is dose limited by normal tissue toxicity. Localized X-ray activation of chemotherapy could overcome this, as studied here, with release from self-assembled nanomicelles (NMs) created from copolymers loaded with doxorubicin (DOX) having a photocleavable o-nitrobenzyl ester (o-Ne) group. The micelles demonstrated release of DOX from X-ray-induced Cherenkov light and conversion from a caged hydrophobic form to hydrophilic DOX, which achieves nuclear localization. Folate on the exterior of the NMs directed them for effective intracellular uptake prior to irradiation. Irradiation with 8 Gy released the DOX, which then entered the cell nucleus, providing near-complete in vivo tumor eradication and negligible off-target organ damage. Micelles were assembled from molecular component materials that are commonly in human use. This study realizes triple targeting in chemoradiation with potential for cell-receptor-mediated uptake, localized radiotherapy activation, and nuclear relocalization, all leading to limited off-target toxicity.

Fluorescence imaging is severely limited by the background and autofluorescence of tissues for in vivo detection of circulating tumor cells (CTCs). Time-gated luminescence (TGL) imaging, in combination with luminescent probes that possess hundreds of microsecond emission lifetimes, can be used to effectively suppress this background, which has predominantly nanosecond lifetimes. This Letter demonstrates the feasibility of TGL imaging using luminescent probes for the in vivo real time imaging and tracking of single CTCs circulating freely in the blood vessels with higher accuracy given by substantially higher signal-to-noise ratio. The luminescent probe used in this Letter was a commercial E u 3 + chelate (EuC) nanosphere with a super-long lifetime of near 800 µs, which enabled TGL imaging to achieve background-free detection with ∼ 5 times higher SNR versus steady state. Phantom and in vivo mouse studies indicated that EuC labeled tumor cells moving in medium or bloodstream at the speed of 1–2 mm/s could be captured in real time.

Purpose: The goal of this work was to create a technique that could measure all possible spatial and temporal delivery rates used in pencil-beam scanning (PBS) proton therapy. The proposed system used a fast scintillation screen for full-field imaging to resolve temporal and spatial patterns as it was delivered. Methods and Materials: A fast intensified CMOS camera imaged used continuous mode 10 ms temporal frame rate and 1x1 mm^2 spatial resolution, imaging a scintillation screen during clinical proton PBS delivery. PBS plans with varying dose, dose rate, energy, field size, and spot-spacing were generated, delivered and imaged. The captured images were post processed to provide dose and dose rate values after background subtraction, perspective transformation, uniformity correction for the camera and the scintillation screen, and calibration into dose. Results: The linearity in scintillation response with respect to varying dose rate, dose, and field size was within 2%. The quenching corrected response with varying energy was also within 2%. Large spatio-temporal variations in dose rate were observed, even for plans delivered with similar dose distributions. Dose and dose rate histograms and maximum dose rate maps were generated for quantitative evaluations. With the fastest PBS delivery on a clinical system, dose rates up to 26.0 Gy/s were resolved. Conclusions: The scintillation imaging technique was able to quantify proton PBS dose rate profiles with spot weight as low as 2 MU, with spot-spacing of 2.5 mm, having an ultimate 1x1 mm^2 spatial resolution. These dose rate temporal profiles, spatial maps, and cumulative dose rate histograms provide useful metrics for the potential evaluation and optimization of dose rate in treatment plans.

Many tumors for which fluorescence guided surgery (FGS) has been developed are surface tumors, where direct visualization by the surgeon is straightforward. On the other hand, cancers such as soft-tissue sarcomas, are present at a subsurface level. Resection of these sub-surface tumors is performed using 'wide local excision' where a single, complete mass is removed with an intact zone of normal tissue (~ 1 cm 'margin'). We used a phantom model for sarcoma with near infrared fluorophore IRDye800 CW that defined different tissue properties. We compare the detection sensitivity of two commercially available near infrared (NIR) surgical imaging systems, Solaris (Perkin Elmer) and SPY PHI (Novadaq) using the phantom models of sarcoma. We also determine targeted fluorescence signal on both systems for blinded surgical phantom dissection by a surgeon. The fluorescence intensities are higher for Solaris than for SPY-PHI. On average, the fluorescence increased with an increase in intralipid concentration and decreased with an increase in blood concentration. The depth of imaging was higher for Solaris than for SPY PHI. Using the target values, the surgeon successfully dissected all phantoms using Solaris. Using fat phantoms for SPY PHI, the surgeon cut through four out of the total. Further improvement in FGS will improve cancer recurrence and morbidity.

Purpose Tattoo fiducials are commonly used in radiotherapy patient alignment, and recent studies have examined the use of UV-excited luminescent tattoo ink as a cosmetic substitute to make these visible under UV illumination. The goal of this study was to show how luminescent tattoo inks could be excited with MV radiation and imaged during beam delivery for direct visualization of field position. Methods A survey of nine UV-sensitive tattoo inks with various emission spectra were investigated using both UV and MV excitation. Images of liquid solutions were collected under MV excitation using an intensified-CMOS imager. Solid skin-simulating phantoms were imaged with both surface-painted ink and in situ tattooing during dose delivery by both a clinical linear accelerator and cobalt-60 source. Results The UV inks have peak fluorescence emission ranging from approximately 440 to 600 nm with lifetimes near 11-16 μs. The luminescence intensity is approximately 6x higher during the x-ray pulse than after the pulse, however, the signal-to-noise is only approximately twice as large. Spatial resolution for imaging was achieved at 1.6 mm accuracy in a skin test phantom. Optical filtering allows for continuous imaging using a cobalt source and provides a mechanism to discriminate ink colors using a monochromatic image sensor. Conclusions This study demonstrates how low-cost inks can be used as fiducial markers and imaged both using time-gated and continuous modes during MV dose delivery. Phantom studies demonstrate the potential application of real-time field verification. Further studies are required to understand if this technique could be used as a tool for radiation dosimetry.

Significance: Photodynamic therapy (PDT) involves complex light-drug-pathophysiology interactions that can be affected by multiple parameters and often leads to large variations in treatment outcome from patient to patient. Direct PDT dosimetry technologies have been sought to optimize the control variables (e.g., light dose, drug administration, tissue oxygenation, and patient conditioning) for best patient outcomes. In comparison, singlet oxygen (O21) dosimetry has been tested in various forms to provide an accurate and perhaps comprehensive prediction of the treatment efficacy. Aim: We discuss an advanced version of this approach provided by a noninvasive, continuous wave dosimeter that can measure near-infrared spectrally resolved luminescence of both photosensitizer (PS) and O21 generated during PDT cancer treatment. Approach: This dosimetry technology uses an amplified, high quantum efficiency InGaAs detector with spectroscopic decomposition during the light treatment to continuously extract the maximum signal of O21 phosphorescence while suppressing the strong PS luminescence background by spectrally fitting the data points across nine narrow band wavelengths. O21 and PS luminescence signals were measured in vivo in FaDu xenograft tumors grown in mice during PDT treatment using Verteporfin as the PS and a continuous laser treatment at 690 nm wavelength. Results: A cohort of 19 mice was used and observations indicate that the tumor growth rate inhibition showed a stronger correlation with O21 than with just the PS signal. Conclusions: These results suggest that O21 measurement may be a more direct dosimeter of PDT damage, and it has potential value as a definitive diagnostic for PDT treatment, especially with spectral separation of the background luminescence and online estimation of the PS concentration.

Biomedical imaging techniques are often limited by the loss of resolution with depth due to light scattering in biological tissue. Beyond a few millimeters in depth, diffuse transport dominates and makes high resolution imaging impossible using conventional techniques. In this work, light sheet imaging using x-ray photons was developed with a keV x-ray source. This partially overcomes this scattering by generating light within tissue at depth. The light excites fluorescent probes that can be used for tumor tracking based upon molecular targeting. Most of the fluorescent probes have a lifetime in the nanosecond range. In this study, the use of a portable linear accelerator delivering 30-ns x-ray pulses was explored. Using x-ray excitable fluorophores, light was generated within a tissue phantom. Image stacks were acquired using an intensified camera (PiMAX4 – Princeton Instrument – USA) placed perpendicularly to the slicing direction of the sample. A solid-state silicon photomultiplier was used to gate acquisition. Although this delayed acquisition slightly, it improved the fluorescence signal-to-noise ratio (SNR). A deconvolution algorithm counteracted the blurring effects of tissue, and image stacks were converted to 3D reconstructions. In summary, nanosecond x-ray pulses can be used to excite fluorophores through radioluminescence phenomenon. Combined with slice imaging, this approach shows promise for time-resolved x-ray luminescence.

PURPOSE The purpose of this study was to identify the optimal treatment geometry for total skin electron therapy (TSET) using a new optimization metric from Cherenkov image analysis, and to investigate the sensitivity of the Cherenkov imaging method to floor scatter effects in this unique treatment setup. METHODS Cherenkov imaging using an intensified charge coupled device (ICCD) was employed to measure the relative surface dose distribution as a 2D image in the total skin electron treatment plane. A 1.2 m × 2.2 m × 1 cm white polyethylene sheet was placed vertically at a source to surface distance (SSD) of 300 cm, and irradiated with 6 MeV high dose rate TSET beams. The linear accelerator coordinate system used stipulates 0° is the bottom of the gantry arc, and progresses counterclockwise so that gantry angle 270° produces a horizontal beam orthogonal to the treatment plane. First, all unique pairs of treatment beams were analyzed to determine the performance of the currently recommended symmetric treatment angles (±20° from the horizontal), compared to treatment geometries unconstrained to upholding gantry angle symmetry. This was performed on two medical linear accelerators (linacs). Second, the extent of the floor scatter contributions to measured surface dose at the extended SSD required for TSET were imaged using three gantry angles of incidence: 270° (horizontal), 253° (-17°), and 240° (-30°). Images of the surface dose profile at each angle were compared to the standard concrete floor when steel plates, polyvinyl chloride (PVC), and solid water were placed on the ground at the base of the treatment plane. Postprocessing of these images allowed for comparison of floor material-based scatter profiles with previously published simulation results. RESULTS Analysis of the symmetric treatment geometry (270 ± 20°) and the identified optimal treatment geometry (270 + 23° and 270 - 17°) showed a 16% increase in the 90% isodose area for the latter field pair on the first linac. The optimal asymmetric pair for the second linac (270 + 25° and 270 - 17°) provided a 52% increase in the 90% isodose area when compared to the symmetric geometry. Difference images between Cherenkov images captured with test materials (steel, PVC, and solid water) and the control (concrete floor) demonstrated relative changes in the two-dimensional (2D) dose profile over a 1 × 1.9 m region of interest (ROI) that were consistent with published simulation data. Qualitative observation of the residual images demonstrates localized increases and decreases with respect to the change in floor material and gantry angle. The most significant changes occurred when the beam was most directly impinging the floor (gantry angle 240°, horizontal -30°), where the PVC floor material decreased scatter dose by 1-3% in 7.2% of the total ROI area, and the steel plate increased scatter dose by 1-3% in 7.0% of the total ROI area. CONCLUSIONS An updated Cherenkov imaging method identified asymmetric, machine-dependent TSET field angle pairs that provided much larger 90% isodose areas than the commonly adopted symmetric geometry suggested by Task Group 30 Report 23. A novel demonstration of scatter dose Cherenkov imaging in the TSET field was established.

Low signal-to-noise ratios and limited imaging depths restrict the ability of optical-imaging modalities to detect and accurately quantify molecular emissions from tissue. Here, by using a scanning external X-ray beam from a clinical linear accelerator to induce Cherenkov excitation of luminescence in tissue, we demonstrate in vivo mapping of the oxygenation of tumours at depths of several millimetres, with submillimetre resolution and nanomolar sensitivity. This was achieved by scanning thin sheets of the X-ray beam orthogonally to the emission-detection plane, and by detecting the signal via a time-gated CCD camera synchronized to the radiation pulse. We also show with experiments using phantoms and with simulations that the performance of Cherenkov-excited luminescence scanned imaging (CELSI) is limited by beam size, scan geometry, probe concentration, radiation dose and tissue depth. CELSI might provide the highest sensitivity and resolution in the optical imaging of molecular tracers in vivo.

In this work, Cherenkov-excited molecular sensing was used to assess the potential for simultaneous quantitative sensing of two NIR fluorophores within tissue-simulating phantoms through spectral separation of signals. Cherenkov emissions induced by external beam gamma photon radiation treatment to tissues/tissue-simulating phantoms were detectable over the 500-900-nm wavelength range. The presence of blood was demonstrated to reduce the integrated intensity of detected Cherenkov emissions by nearly 50%, predominantly at wavelengths below 620 nm. The molecular dyes, IRDye 680RD and IRDye 800CW, have excitation and emission spectra at longer wavelengths than the strongest blood absorption peaks, and also where the intensity of Cherenkov light is at its lowest, so that the emission signal relative to background signal is maximized. Tissue phantoms composed of 1% intralipid and 1% blood were used to simulate human breast tissue, and vials containing fluorophore were embedded in the media, and irradiated with gamma photons for Cherenkov excitation. We observed that fluorescence emissions excited by the Cherenkov signal produced within the phantom could be detected at 5-mm depth into the media within a 0.1-25 μ M fluorophore concentration range. The detected fluorescence signals from these dyes showed linear relationships with radiation doses down to the cGy level. In vivo tests were successful only within the range near a μ M, suggesting that these could be used for metabolic probes in vivo where the local concentrations are near this range.

Integration of magnetic resonance imaging (MRI) and near-infrared spectral tomography (NIRST) has yielded promising diagnostic performance for breast imaging in the past. This study focused on whether MRI-guided NIRST can quantify hemoglobin concentration using only continuous wave (CW) measurements. Patients were classified into four breast density groups based on their MRIs. Optical scattering properties were assigned based on average values obtained from these density groups, and MRI-guided NIRST images were reconstructed from calibrated CW data. Total hemoglobin (HbT) contrast between suspected lesions and surrounding normal tissue was used as an indicator of the malignancy. Results obtained from simulations and twenty-four patient cases indicate that the diagnostic power when using only CW data to differentiate malignant from benign abnormalities is similar to that obtained from combined frequency domain (FD) and CW data. These findings suggest that eliminating FD detection to reduce the cost and complexity of MRI-guided NIRST is possible.

We realized subsurface fluorescence LiDAR with a large format single-photon avalanche diode array capable of localizing and quantifying concentration of fluorescent molecules in heavily scattering media, such as tissue, with submillimeter depth accuracy. (C) 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

PURPOSE: Treatment planning systems (TPS) for MR-linacs must employ Monte Carlo based simulations of dose deposition to model the effects of the primary magnetic field on dose. However, the accuracy of these simulations, especially for areas of tissue-air interfaces where the electron return effect (ERE) is expected, are difficult to validate due to physical constraints and magnetic field compatibility of available detectors. This study employs a novel dosimetric method based on remotely captured, real-time optical Cherenkov and scintillation imaging to visualize and quantify the ERE. METHODS: An intensified CMOS camera was used to image two phantoms with designed ERE cavities. Phantom A was a 40cm × 10cm × 10cm clear acrylic block drilled with 5 holes of increasing diameters (0.5cm, 1cm, 2cm, 3cm, 4cm). Phantom B was a clear acrylic block with 3 cavities of increasing diameter (3cm, 2cm, 1cm) split into two halves in the transverse plane to accommodate radiochromic film. Both phantoms were imaged while being irradiated by 6MV flattening filter free (FFF) beams within a MRIdian Viewray (Viewray, Cleveland, OH) MR-linac (0.34T primary field). Phantom A was imaged while being irradiated by 6MV FFF beams on a conventional linac (TrueBeam, Varian Medical Systems, San Jose, CA) to serve as a control. Images were post processed in Matlab (Mathworks Inc., Natick, MA) and compared to treatment planning system (TPS) dose volumes. RESULTS: Control imaging of Phantom A without the presence of a magnetic field supports the validity of the optical image data to a depth of 6cm. In the presence of the magnetic field, the optical data shows deviations from the commissioned TPS dose in both intensity and localization. The largest air cavity examined (3cm) indicated the largest dose differences, which were above 20% at some locations. Experiments with Phantom B illustrated similar agreement between optical and film dosimetry comparisons with TPS data in areas not affected by ERE. CONCLUSION: There are some appreciable differences in dose intensity and spatial dose distribution observed between the novel experimental data set and the dose models produced by the current clinically implemented MR-IGRT TPS. Further validation of the proposed experimental methods, as well as the magnetic field effect models within the TPS, is recommended.

Cherenkov excitation of molecular probes in tissue can be used for photodynamic effect or diagnostic luminescence imaging. Excitation of dendritic platinum-based metalloporphyrn molecule complexes as an oxygen sensor (PtG4) has been shown to allow oxygen measurement in vivo with spatial resolution defined by the exciting x-ray beam. By sweeping the beamlet shapes and geometries, and detecting the emitted luminscence lifetime, a highly accurate pO2 distribution can be reconstructed of the tissue volume. This has been shown in tumors as well as in normal tissues, and now for implanted microtubes. Additional imaging of luminescent agents based upon Cherenkov or direct radio-excitation could be used for molecular sensing. The combination of depth, concentration, and radiation dose have been analyzed for the tradeoffs in signal to noise, and the signal to background optimization has been completed. The use of this approach can be applied to any clinical radiation therapy linac.

Purpose The purpose of this study was to demonstrate high resolution optical luminescence sensing, referred to as Cherenkov excited luminescence scanning imaging (CELSI), could be achieved during a standard dynamic treatment plan for a whole breast radiotherapy geometry. Methods The treatment plan beams induce Cherenkov light within tissue, and this excitation projects through the beam trajectory across the medium, inducing luminescence where there can be molecular reporter. Broad beams generally produce higher signal but low spatial resolution, yet for dynamic plans the scanning of the multileaf collimator allows for a beam-narrowing strategy by recursively temporal differencing each of the Cherenkov images and associated luminescence images. Then reconstruction from each of these size-reduced beamlets defined by the differenced Cherenkov images provides a well-conditioned matrix inversion, where the spatial frequencies are limited by the higher signal-to-noise ratio beamlets. A built-in stepwise convergence relies on stepwise beam size reduction, which is associated with a widening of the bandwidth of Cherenkov spatial frequency and resultant increase in spatial resolution. For the phantom experiments, europium nanoparticles were used as luminescent probes and embedded at depths ranging from 3 to 8 mm. An intensity modulated radiotherapy (IMRT) plan was used to test this. Results The Cherenkov images spatially guided where the luminescence was measured from, providing high lateral resolution, and iterative reconstruction convergence showed that optimization of the initial and stopping beamlet widths could be achieved with 15 and 4.5 mm, respectively, using a luminescence imaging frame rate of 5/s. With the IMRT breast plan, the original lateral resolution was improved 2X, that is, 0.08-0.24 mm for target depths of 3-8 mm. In comparison, a dynamic wedge (DW) plan showed an inferior image fidelity, with relative contrast recovery decreasing from 0.86 to 0.79. The methodology was applied to a three-dimensional dataset to reconstruct Cherenkov excited luminescence intensity distributions showing volumetric recovery of a 0.5 mm diameter object composed of 0.5 μM luminescent microbeads. Conclusions High resolution CELSI was achieved with a clinical breast external beam radiotherapy (EBRT) plan. It is anticipated that this method can allow visualization and localization for luminescence/fluorescence tagged vasculature, lymph nodes, or superficial tagged regions with most dynamic treatment plans.

Surface dosimetry is required for ensuring effective administration of total skin electron therapy (TSET); however, its use is often reduced due to the time consuming and complex nature of acquisition. A new surface dose imaging technique was characterized in this study and found to provide accurate, rapid and remote measurement of surface doses without the need for post-exposure processing. Disc-shaped plastic scintillators (1 mm thick × 15 mm ) were chosen as optimal-sized samples and designed to attach to a flat-faced phantom for irradiation using electron beams. Scintillator dosimeter response to radiation damage, dose rate, and temperature were studied. The effect of varying scintillator diameter and thickness on light output was evaluated. Furthermore, the scintillator emission spectra and impact of dosimeter thickness on surface dose were also quantified. Since the scintillators were custom-machined, dosimeter-to-dosimeter variation was tested. Scintillator surface dose measurements were compared to those obtained by optically stimulated luminescence dosimeters (OSLD). Light output from scintillator dosimeters evaluated in this study was insensitive to radiation damage, temperature, and dose rate. Maximum wavelength of emission was found to be 422 nm. Dose reported by scintillators was linearly related to that from OSLDs. Build-up from placement of scintillators and OSLDs had a similar effect on surface dose (4.9% increase). Variation among scintillator dosimeters was found to be 0.3 ± 0.2%. Scintillator light output increased linearly with dosimeter thickness (~1.9 × /mm). All dosimeter diameters tested were able to accurately measure surface dose. Scintillator dosimeters can potentially improve surface dosimetry-associated workflow for TSET in the radiation oncology clinic. Since scintillator data output can be automatically recorded to a patient medical record, the chances of human error in reading out and recording surface dose are minimized.

Previous work has shown that capturing optical emission from plastic discs attached directly to the skin can be a viable means to accurately measure surface dose during total skin electron therapy. This method can provide accurate dosimetric information rapidly and remotely without the need for postprocessing. The objective of this study was to: (1) improve the robustness and usability of the scintillators and (2) enhance sensitivity of the optical imaging system to improve scintillator emission detection as related to tissue surface dose. Baseline measurements of scintillator optical output were obtained by attaching the plastic discs to a flat tissue phantom and simultaneously irradiating and imaging them. Impact on underlying surface dose was evaluated by placing the discs on-top of the active element of an ionization chamber. A protective coating and adhesive backing were added to allow easier logistical use, and they were also subjected to disinfection procedures, while verifying that these changes did not affect the linearity of response with dose. The camera was modified such that the peak of detector quantum efficiency better overlapped with the emission spectra of the scintillating discs. Patient imaging was carried out and surface dose measurements were captured by the updated camera and compared to those produced by optically stimulated luminescence detectors (OSLD). The updated camera was able to measure surface dose with3 % difference compared to OSLD–Cherenkov emission from the patient was suppressed and scintillation detection was enhanced by 25 × and 7 × , respectively. Improved scintillators increase underlying surface dose on average by 5.2 ± 0.1 % and light output decreased by 2.6 ± 0.3 % . Disinfection had0.02 % change on scintillator light output. The enhanced sensitivity of the imaging system to scintillator optical emission spectrum can now enable a reduction in physical dimensions of the dosimeters without loss in ability to detect light output.

Cherenkov-excited luminescence scanned imaging (CELSI) is achieved with a clinical linear accelerator during External Beam Radiotherapy, to map out molecular luminescence intensity or lifetime in tissue. In order to realize a deeper imaging depth with a reasonable spatial resolution in CELSI, we optimized the original scanning procedure to complete this in a similar way to x-ray computed tomography and with image reconstruction from Maximum-likelihood expectation maximization and multi-pinhole irradiation for parallelization. Resolution phantom studies showed that a 0.3 mm diameter capillary tube containing 0.01 nM luminescent nanospheres could be recognized at a depth of 21 mm into tissue-like media. Small animal imaging with a 1 mm diameter cylindrical target demonstrated that fast 3D data acquisition can be achieved by this multi-pinhole collimator approach to image high resolution luminescence through a whole animal.

In recent years, numerous publications have documented the growing consensus among dermatologists for daylight-photodynamic therapy (dPDT) treatment of Actinic Kerasotis (AK), with additional evidence supporting treatment of certain non-melanoma skin cancers (NMSC). While these publications aim to address the minimum effective surface-irradiance required for successful clearance, our current work investigates how the tissue optical properties influence the fluence rate within tissue. While it is known red and blue light will have drastically different attenuation profiles in tissue, it is harder to quantify this for broad-spectrum light sources. Our model aims to expand the current PpIX-weighted irradiance metric by incorporating a clinically relevant depth distribution factor. Using a 7-layer skin model, Monte Carlo simulations of optical photons ranging from 350nm – 900nm provide insight into the potential depth of activation of the photosensitizer. Additionally, these models can be applied to known light spectra for both narrow-band conventional treatments (415nm, 633nm), as well as for the Sun and other white light sources (CFL, Halogen). Using this model, we show even when the effective surface-irradiance of the Sun is 4x a halogen light source, the effective fluence within the top 3mm of tissue is generally equivalent, due to the higher proportion of UV-blue light in Sun spectrum which is highly attenuated within the first 50m. We plan to use this model to inform which light source or light combinations would be most appropriate for specific lesion morphologies.

Cherenkov emission, which is generated during radiation therapy, can be utilized for imaging that is synergistic with radiation therapy. Cherenkov light can be utilized to excite phosphors, which then can be imaged utilizing Cherenkov excited luminescence scanned imaging (CELSI). Europium chelate microspheres, which exhibit bright luminescence with long luminescent lifetime, were appended with multiple copies of cetuximab. This will allow for selective imaging of EGFR overexpressing tumors during the course of radiation therapy via CELSI. We have characterized the functionality of the cetuximab loaded microspheres in vitro via ELISA, as well as via fluorescence microscopy in EGFR overexpressing A431 cells. These microspheres were intravenously injected into athymic nude mice bearing A431 flank tumors and allowed to incubate for a series of time points. They were then imaged first via standard fluorescence imaging to determine the ideal time point for visualizing tumors via CELSI. After demonstrating selective accumulation in tumors, imaging was then undertaken in vivo via CELSI. These antibody conjugated europium microspheres provide promise to image tumors selectively with CELSI. Future studies involve conjugating other antibodies to the europium microspheres to utilize in CELSI.

Total Skin Electron Therapy (TSET) utilizes high-energy electrons to treat cancers on the entire body surface. The otherwise invisible radiation beam can be observed via the optical Cherenkov photons emitted from interaction between the high-energy electron beam and tissue. Cherenkov emission is used to evaluate the dose uniformity on the surface of the patient in real-time. We have utilized a structured light sensor to determine the surface contour of each patient. Each patient was also monitored during TSET via in-vivo detectors (IVD) and/or scintillating discs in nine locations. The Cherenkov image is converted to dose distribution after a two-dimensional perspective geometry correction and the IVD measured dose at umbilicus. Cumulative dose on patient surface is obtained by projecting the two-dimensional dose distribution onto a cylindrical geometry representing the patient anatomic geometry. Patients undergoing TSET in various conditions (whole body and half body) were imaged and analysed, and the cumulative dose based on Cherenkov imaging was evaluated on various patients.

Photodynamic Therapy (PDT) is a promising modality for cancer treatment. Typically, a laser is used to photo-excite a photosensitizer (PS) that subsequently collides with oxygen molecules promoting them to the metastable singlet delta state O2(1Δ). Singlet oxygen molecules are believed to be the species that destroys cancerous cells during PDT. In this paper we describe a novel 2D imaging sensor for photosensitizer fluorescence and singlet oxygen luminescence. We describe our instrument and initial results from both in-vitro and in-vivo studies that indicate that this system may be a valuable dosimeter for both PDT researchers and eventually for clinical application.

Purpose.In this study, spatio-temporal beam profiling for electron ultra-high dose rate (UHDR; >40 Gy s-1) radiation via Cherenkov emission and radioluminescence imaging was investigated using intensified complementary metal-oxide-semiconductor cameras.Methods.The cameras, gated to FLASH optimized linear accelerator pulses, imaged radioluminescence and Cherenkov emission incited by single pulses of a UHDR (>40 Gy s-1) 10 MeV electron beam delivered to the isocenter. Surface dosimetry was investigated via imaging Cherenkov emission or scintillation from a solid water phantom or Gd2O2S:Tb screen positioned on top of the phantom, respectively. Projected depth-dose profiles were imaged from a tank filled with water (Cherenkov emission) and a 1 g l-1quinine sulfate solution (scintillation). These optical results were compared with projected lateral dose profiles measured by Gafchromic film at different depths, including the surface.Results.The per-pulse beam output from Cherenkov imaging agreed with the photomultiplier tube Cherenkov output to within 3% after about the first five to seven ramp-up pulses. Cherenkov emission and scintillation were linear with dose (R2 = 0.987 and 0.995, respectively) and independent of dose rate from ∼50 to 300 Gy s-1(0.18-0.91 Gy/pulse). The surface dose distribution from film agreed better with scintillation than with Cherenkov emission imaging (3%/3 mm gamma pass rates of 98.9% and 88.8%, respectively). Using a 450 nm bandpass filter, the quinine sulfate-based water imaging of the projected depth optical profiles agreed with the projected film dose to within 5%.Conclusion.The agreement of surface dosimetry using scintillation screen imaging and Gafchromic film suggests it can verify the consistency of daily beam quality assurance parameters with an accuracy of around 2% or 2 mm. Cherenkov-based surface dosimetry was affected by the target's optical properties, prompting additional calibration. In projected depth-dose profiling, scintillation imaging via spectral suppression of Cherenkov emission provided the best match to film. Both camera-based imaging modalities resolved dose from single UHDR beam pulses of up to 60 Hz repetition rate and 1 mm spatial resolution.

Significance: The effects of varying the indocyanine green injection dose, injection rate, physiologic dispersion of dye, and intravenous tubing volume propagate into the shape and magnitude of the arterial input function (AIF) during intraoperative fluorescence perfusion assessment, thereby altering the observed kinetics of the fluorescence images in vivo. Aim: Numerical simulations are used to demonstrate the effect of AIF on metrics derived from tissue concentration curves such as peak fluorescence, time-to-peak (TTP), and egress slope. Approach: Forward models of tissue concentration were produced by convolving simulated AIFs with the adiabatic approximation to the tissue homogeneity model using input parameters representing six different tissue examples (normal brain, glioma, normal skin, ischemic skin, normal bone, and osteonecrosis). Results: The results show that AIF perturbations result in variations in estimates of total intensity of up to 80% and TTP error of up to 200%, with the errors more dominant in brain, less in skin, and less in bone. Interestingly, error in ingress slope was as high as 60% across all tissue types. These are key observable parameters used in fluorescence imaging either implicitly by viewing the image or explicitly through intensity fitting algorithms. Correcting by deconvolving the image with a measured subject-specific AIF provides an intuitive means of visualizing the data while also removing the source of variance and allowing intra- and intersubject comparisons. Conclusions: These results suggest that intraoperative fluorescence perfusion assessment should be corrected by patient-specific AIFs measured by pulse dye densitometry.

Cherenkov emission induced by external beam radiation therapy from a clinical linear accelerator (LINAC) can be used to excite phosphors deep in biological tissues. As with all luminescence imaging, there is a desire to minimize the spectral overlap between the excitation light and emission wavelengths, here between the Cherenkov and the phosphor. Cherenkov excited short-wavelength infrared (SWIR, 1000 to 1700 nm) fluorescence imaging has been demonstrated for the first time, using long Stokes-shift fluorophore PdSe quantum dots (QD) with nanosecond lifetime and an optimized SWIR detection. The 1/λ2 intensity spectrum characteristic of Cherenkov emission leads to low overlap of this into the fluorescence spectrum of PdSe QDs in the SWIR range. Additionally, using a SWIR camera itself inherently ignores the stronger Cherenkov emission wavelengths dominant across the visible spectrum. The SWIR luminescence was shown to extend the depth sensitivity of Cherenkov imaging, which could be used for applications in radiotherapy sensing and imaging in human tissue with targeted molecular probes.

Cherenkov-excited luminescence scanned imaging (CELSI) is achieved with external beam radiotherapy to map out molecular luminescence intensity or lifetime in tissue. Just as in fluorescence microscopy, the choice of excitation geometry can affect the imaging time, spatial resolution and contrast recovered. In this study, the use of spatially patterned illumination was systematically studied comparing scan shapes, starting with line scan and block patterns and increasing from single beams to multiple parallel beams and then to clinically used treatment plans for radiation therapy. The image recovery was improved by a spatial-temporal modulation-demodulation method, which used the ability to capture simultaneous images of the excitation Cherenkov beam shape to deconvolve the CELSI images. Experimental studies used the multi-leaf collimator on a clinical linear accelerator (LINAC) to create the scanning patterns, and image resolution and contrast recovery were tested at different depths of tissue phantom material. As hypothesized, the smallest illumination squares achieved optimal resolution, but at the cost of lower signal and slower imaging time. Having larger excitation blocks provided superior signal but at the cost of increased radiation dose and lower resolution. Increasing the scan beams to multiple block patterns improved the performance in terms of image fidelity, lower radiation dose and faster acquisition. The spatial resolution was mostly dependent upon pixel area with an optimized side length near 38mm and a beam scan pitch of P = 0.33, and the achievable imaging depth was increased from 14mm to 18mm with sufficient resolving power for 1mm sized test objects. As a proof-of-concept, in-vivo tumor mouse imaging was performed to show 3D rendering and quantification of tissue pO2 with values of 5.6mmHg in a tumor and 77mmHg in normal tissue.