Cherenkov imaging provides a valuable tool for the quality assurance of dose homogeneity in total skin electron therapy (TSET), in which patients were treated in six different postures using the Stanford standing technique. The emitted Cherenkov signals can be captured by three Cherenkov cameras, converted to 2D dose maps after certain corrections, and projected onto the patient specific 3D model to evaluate the cumulative total skin dose distribution. This study aims to improve the accuracy and reliability of Cherenkov converted dose obtained from the combination of multi-view Cherenkov images. The extra correction factors are investigated by conducting manikin phantom experiments as well as GAMOS based Monte Carlo simulations, and validated with in vivo dosimetry (IVD). We have also calibrated the side cameras and achieved a straightforward conversion of all Cherenkov images into doses using only the front camera. We have improved the Cherenkov-to-dose correction and conversion method, significantly reducing the deviation between the Cherenkov-converted dose and IVD measurements.
The delivery of genetic payloads to cells using genetic medicines is challenging to predict and the existing tools to assess delivery in animal models, although critical for discovering and advancing these drugs to the clinic, do not readily facilitate assessments of functional delivery throughout the entire body. To complement these existing techniques, our lab has used whole-body 3-D hyperspectral fluorescence cryo-imaging for the evaluation of functional delivery in whole animal specimens at high resolution. This instrument acquires hyperspectral fluorescence images of whole animal specimen while they are sectioned at micron-level resolution. In this study, mice were administered AAV9, a common adeno-associated viral delivery vehicle to express and image a fluorescent reporter. As the specimen is sectioned the acquisition can be paused to collect whole-body tissue samples which are then stained for immunofluorescence (IF) analysis. Herein, we describe a technique to reconstruct IF images into a single whole-body tissue specimen to be assessed alongside the co-registered cryo-images.
Intraoperative photodynamic therapy (PDT) has proven effective in treating malignant pleural mesothelioma. Achieving uniform light dose delivery is vital for its efficiency. Currently, eight light detectors are placed inside the pleural cavity to monitor light distribution. To enhance this process, an updated navigation system, combined with a novel scanning system, has been developed to provide real-time guidance to physicians during pleural PDT, thereby improving light delivery. The scanning system incorporates two handheld three-dimensional (3D) scanners, enabling rapid and precise capture of the pleural cavity's surface topography before PDT. This allows for identification of the target surface for real-time calculation of light fluence distribution during treatment. An algorithm has been devised to further process the scanned volume, facilitating continuous tracking of the light source position within the pleural cavity throughout the treatment process. During PDT, real-time 3D and 2D visualizations of the light source position, scanned pleural cavity, and light fluence distribution across the entire cavity's surface are displayed, providing physicians with invaluable guidance to enhance overall treatment efficiency. To validate the system, phantom studies were conducted using three newly 3D-printed lung phantoms of varying volumes based on individual CT scans. A set of liquid tissue-simulating phantoms with different combinations of optical properties (μa, μs') was utilized for improved clinical simulation. These lung phantoms, designed to mimic surgical conditions, feature side openings similar to the actual surgery and are treated with eight isotropic detectors fixed on the inner surface at positions predetermined by the physician.
The application of Photodynamic (PDT) for malignant pleural mesothelioma (MSM) is complex due to post-surgery alteration. To address this issue of calculating dose for complex anatomical geometries, our study utilized advanced digital technologies to construct and analyze 3D models of the human lung pleural cavity. This study aims to support a translational model that will eventually enhance PDT dose delivery while minimizing effects on surrounding healthy tissues. The pleural cavity geometries were digitally extracted from medical CT scans, transformed into stereolithography (STL) files, and 3D-printed using acrylic resins. Three volumes corresponding to small, medium, and large patient sizes were prepared. Each model was asymmetrically coated with non-reflective adhesive paper to mimic realistic conditions. Data capture utilized a single handheld scanning device operating at distances to accommodate generalized and refined detail capture. This approach enabled the precise capture of internalized cavity shapes and critical asymmetries. Medical CT validated all 3D printed models. The results confirmed the precise generation and capture of multiple asymmetric 3D human cavity models in actual size. The proposed workflow shows potential for clinicians to accurately map post-surgical pleural cavity changes, thereby improving PDT light dose delivery. This could enhance treatment efficacy and patient outcomes, underlining the potential of digital technology in advancing precision oncology and integration into future clinical practice.
Imaging Cherenkov photons emitted during radiation therapy can provide real-time information of the external beam field. It is well established that Cherenkov emission is correlative to dose deposited; however, differential photon energies and tissue attenuation properties, along with complicated camera geometries, entangle this relationship and introduce variability in the Cherenkov emission-to-dose ratio from patient-to-patient. This study aims to better understand the effects of optical properties, skin color, and patient-specific geometries (i.e. angle of camera incidence and curvature) on the Cherenkov emission-to-dose relationship. To do so, a series of phantom experiments were conducted with tissuesimulating optical phantoms and an andromorphic breast phantom in which optical properties, curvature, and camera angle of incidence were all examined as a function of normalized Cherenkov emission-to-dose. To acquire clinical Cherenkov data along with patient skin color, Cherenkov images and OSLD measurements for the ground-truth surface dose were collected weekly on 13 whole-breast radiotherapy patients, alongside high-resolution 3D color and texture scans. Phantom results suggest there to be a moderately strong correlation between dose percent error and patient curvature (R2 = 0.57), as well as angle of camera incidence (R2 = 0.56). Initial patient results suggest there to be a correlation between the redness of a patient’s skin, and the Cherenkov emission-to-dose ratio, with higher amounts of redness correlating to lower Cherenkov signal. By better characterizing these trends, we are potentially able to find generalizable optics-based corrections that improve the accuracy in mapping Cherenkov emission to real-time skin dose.
Neurosurgical fluorescence guidance relies on contrast agents to identify tumor regions to aid in increasing the extent of resection. Existing contrast agents for this indication each have their own limitation: unpredictable contrast from tumor heterogeneity, significant extravasation into the background brain and long incubation times. An ideal contrast agent should have high and rapid contrast that persists well into the surgical procedure. By using a whole animal hyperspectral cryo-imaging system several CAs were screened for these favorable properties and compared to the gold standard of gadolinium enhanced MR. Herein, we briefly report on the leading candidate Rd-PEG1k, which shows high contrast within minutes of administration that persists for at least 90 minutes.
Timely assessment of bone perfusion in orthopaedic trauma surgery plays an important role in successful treatment outcome. For guiding accurate debridement of bones with impaired blood supply, fluorescence-guided surgery (FGS) technique have gained increasingly popularity. Compared to other imaging modalities like computed tomography and nuclear magnetic resonance imaging that are time consuming and less practical during surgery, fluorescence imaging can be performed intraoperatively and is able to visualize the bone blood flow in real time. In order to link the blood flow fluorescence imaging to quantitative bone perfusion numbers, in this study we are using a modified fluorescent microsphere (FM) approach called microsphere quantification using imaging cryomacrotome (mQUIC). Bone perfusion is assessed by identifying the density of deposited microspheres in reconstructed imaging volumes, which are proportional to the regional blood flow. In the rabbit model presented here, cryoimaging was used to scan femurs injected with three colors of microspheres corresponding to three conditions: baseline, post-osteotomy and post-periosteal stripping. Image processing, such as top-hat transform and object-based colocalization, was used to enable accurate counting of FMs to produce their 3D-localization within the bones. FM density volumes were converted to bone perfusion units (mL/min/100g) using the reference organ technique. This study provides a groundwork for direct comparison with our DCE-FI technique for measuring bone perfusion in orthopaedic trauma surgery models.
In orthopedic trauma surgery, timely assessment of bone tissue perfusion plays a vital role in the successful treatment outcome. Fluorescence-guidance is gaining increased surgical interest, especially with respect to hemodynamic assessment of bone. Intraoperative dynamic contrast-enhanced fluorescence imaging (DCE-FI) not only enables visualization of the perfused areas of the injured bone, but with subsequent analysis using kinetic models, may also provide a valuable quantitative bone blood flow information to a surgeon. In this study, we are validating this quantitative approach with a modified fluorescent microsphere (FM) technique using a custom-built four-channel imaging cryomacrotome. We demonstrate that FMs of four different colors can be accurately detected in controlled phantoms and evaluate their detection accuracy in real blood samples. In a rabbit model of orthopaedic trauma, we show that blood flow measurements using the DCE-FI technique can be compared with the FM technique. This feasibility pilot study provides the groundwork for investigation of the correlation between bone perfusion measurements using DCE-FI and using fluorescent microspheres, in units of ml/min/100g.
As rapidly accelerating technology, fluorescence guided surgery (FGS) has the potential to place molecular information directly into the surgeon’s field of view by imaging administered fluorescent contrast agents in real time, circumnavigating pre-operative MR registration challenges with brain deformation. The most successful implementation of FGS is 5-ALAPpIX guided glioma resection which has been linked to improved patient outcomes. While FGS may offer direct in-field guidance, fluorescent contrast agent distributions are not as familiar to the surgical community as Gd-MRI uptake, and may provide discordant information from previous Gd-MRI guidance. Thus, a method to assess and validate consistency between fluorescence-labeled tumor regions and Gd-enhanced tumor regions could aid in understanding the correlation between optical agent fluorescence and Gd-enhancement. Herein, we present an approach for comparing whole-brain fluorescence biodistributions with Gd-enhancement patterns on a voxel-by-voxel basis using co-registered fluorescent cryo-volumes and Gd-MRI volumes. In this initial study, a porcine-human glioma xenograft model was administered 5-ALA-PpIX, imaged with MRI, and euthanized 22 hours following 5-ALA administration. Following euthanization, the extracted brain was imaged with the cryo-macrotome system. After image processing steps and non-rigid, point-based registration, the fluorescence cryo-volume and Gd-MRI volume were compared for similarity metrics including: image similarity, tumor shape similarity, and classification similarity. This study serves as a proof-of-principle in validating our screening approach for quantitatively comparing 3D biodistributions between optical agents and Gd-based agents.
Fluorescent contrast agents targeted to cancer biomarkers are increasingly being explored for cancer detection, surgical guidance, and response monitoring. Efforts have been underway to topically apply such biomarker-targeted agents to freshly excised specimen for detecting cancer cell receptors on the surface as a method for intraoperative surgical margin assessment, including dual-probe staining methods introduce a second ‘non-specific’ optical agent as a control to help compensate for heterogeneous uptake and normalize the imaging field. Still, such specimen staining protocols introduce multifaceted complexity with unknown variables, such as tissue-specific diffusion, cell-specific binding and disassociation rates, and other factors, affecting the interpreted cancer-biomarker distribution across the specimen surface. The ability to recover three-dimensional dual-probe biodistributions throughout whole-specimens could offer a ground-truth validation method for examining topical staining uptake behaviors. Herein, we report on a novel method for characterizing dual-probe accumulation with 3D depth-profiles observed from a dual-probe fresh-specimen staining experiment.
Characterizing an administered drug’s pathway from initial systemic uptake, to targeted tissue accumulation, and the eventual excretion route is an important component of clinical translation. For mapping such pharmacokinetic behaviors in a biologically-relevant system, fluorescently-tagged drugs are commonly administered and examined in preclinical animal models. Broadband fluorescence cryo-imaging offers a high-resolution, whole-animal technique for recovering such fluorescently-tagged biodistributions, although agent-specificity remains a challenge due to unknown levels of heterogeneous tissue autofluorescence. Herein, we report on a new hyperspectral multichannel fluorescence cryo-imaging system and demonstrate higher agent-specificity and signal-sensitivity compared to conventional broadband fluorescence.
MRI images of gadolinium-based contrast agents (GBCA’s) acquired before surgery are often registered to patients and used to guide surgical resection of intracranial tumors. Yet, the accuracy of these MR images in describing the surgical field degrades as surgery progresses; a well-recognized problem which has prompted efforts to develop new techniques that provide updated guidance information on residual tumor location. These efforts span a wide array of technologies, including image updating with deformation models, intraoperative MRI, and fluorescence guided surgery, among others. However, introduction of a straightforward technique that provides surgeons with a current view of GBCA distribution in real time remains an important goal. In this context, development of a fluorescent agent that recapitulates the kinetic behavior of GBCA’s could provide familiar information directly in the surgical field in real time. To advance this strategy, we have begun identifying fluorescent contrast agents that show similar kinetic behavior to GBCA’s. Using a novel hyperspectral whole body cryo-imaging system, we acquired highresolution 3-D volumes of the distribution of multiple candidate fluorophores in whole heads bearing orthotopic brain tumors. Preliminary results reveal significant differences in the distribution of candidate optical agents, some of which show strong similarity to the GBCA uptake. Identification and eventual translation of a reliable GBCAoptical analog could improve and simplify surgical resection of brain tumors.
Short-wave infrared (SWIR/NIR-II) fluorescence imaging has received increased attention for use in fluorescence-guided surgery (FGS) due to the potential for higher resolution imaging of subsurface structures and reduced autofluorescence compared to conventional NIR-I imaging. As with any fluorescence imaging modality introduced in the operating room, an appropriate accounting of contaminating background signal from other light sources in the operating room is an important step. Herein, we report the background signals in the SWIR and NIR-I emitted from commonly-used equipment in the OR, such as ambient and operating lights, LCD screens and surgical guidance systems. These results can guide implementation of protocols to reduce background signal.
The ability to directly measure whole-body fluorescence can enable tracking of labeled cells, metastatic spread, and drug bio-distribution. We describe the development of a new hyperspectral imaging whole body cryomacrotome designed to acquire 3-D fluorescence volumes in large specimens (whole animals) at high resolution. The use of hyperspectral acquisition provides full spectra at every voxel, enabling spectral decoupling of multiple fluorohpores and autofluorescence. We present examples of tissue spectra and spectral fitting in a rodent glioma xenograft.
The potential to image subsurface fluorescent contrast agents at high spatial resolution has facilitated growing interest in short-wave infrared (SWIR) imaging for biomedical applications. The early but growing literature showing improvements in resolution in small animal models suggests this is indeed the case, yet to date, images from larger animal models that more closely recapitulate humans have not been reported. We report the first imaging of SWIR fluorescence in a large animal model. Specifically, we imaged the vascular kinetics of an indocyanine green (ICG) bolus injection during open craniotomy of a mini-pig using a custom SWIR imaging instrument and a clinical-grade surgical microscope that images ICG in the near-infrared-I (NIR-I) window. Fluorescence images in the SWIR were observed to have higher spatial and contrast resolutions throughout the dynamic sequence, particularly in the smallest vessels. Additionally, vessels beneath a surface pool of blood were readily visualized in the SWIR images yet were obscured in the NIR-I channel. These first-in-large-animal observations represent an important translational step and suggest that SWIR imaging may provide higher spatial and contrast resolution images that are robust to the influence of blood.
The observed behavior of short-wave infrared (SWIR) light in tissue, characterized by relatively low scatter and subdiffuse photon transport, has generated considerable interest for the potential of SWIR imaging to produce high-resolution, subsurface images of fluorescence activity in vivo. These properties have important implications for fluorescence-guided surgery and preclinical biomedical research. Until recently, translational efforts have been impeded by the conventional understanding that fluorescence molecular imaging in the SWIR regime requires custom molecular probes that do not yet have proven safety profiles in humans. However, recent studies have shown that two readily available near-infrared (NIR-I) fluorophores produce measurable SWIR fluorescence, implying that other conventional fluorophores produce detectable fluorescence in the SWIR window. Using SWIR spectroscopy and wide-field SWIR imaging with tissue-simulating phantoms, we characterize and compare the SWIR emission properties of eight commercially available red/NIR-I fluorophores commonly used in preclinical and clinical research, in addition to a SWIR-specific fluorophore. All fluorophores produce measurable fluorescence emission in the SWIR, including shorter wavelength dyes such as Alexa Fluor 633 and methylene blue. This study is the first to report SWIR fluorescence from six of the eight conventional fluorophores and establishes an important comparative reference for developing and evaluating SWIR imaging strategies for biomedical applications.
Short-wave infrared imaging in tissue in the 1000-2000 nm range is characterized by reduced photon scatter and comparable or higher absorption compared to the NIR-I regime. These characteristics have implications for the performance of fluorescence molecular tomography (FMT) techniques, potentially improving the resolution of subsurface structure, possibly at the expense of depth sensitivity. To examine these questions, we have developed a SWIR small animal fluorescence tomography system. This instrument acquires multi-angle SWIR projection images of a stationary platform through a rotating gantry technique. These images are then processed for tomographic reconstruction of the SWIR fluorescence activity. Herein, we describe the development of this system and show multi-angle images from a mouse carcass containing a SWIR-specific fluorophore inclusion.
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