Validation of Monte Carlo 131 I radiopharmaceutical dosimetry workflow using a 3D-printed anthropomorphic head and neck phantom.

PURPOSE: Approximately 50% of head and neck cancer (HNC) patients will experience loco-regional disease recurrence following initial courses of therapy. Retreatment with external beam radiotherapy (EBRT) is technically challenging and may be associated with a significant risk of irreversible damage to normal tissues. Radiopharmaceutical therapy (RPT) is a potential method to treat recurrent HNC in conjunction with EBRT. Phantoms are used to calibrate and add quantification to nuclear medicine images, and anthropomorphic phantoms can account for both the geometrical and material composition of the head and neck. In this study, we present the creation of an anthropomorphic, head and neck, nuclear medicine phantom, and its characterization for the validation of a Monte Carlo, SPECT image-based, 131 I RPT dosimetry workflow. METHODS: 3D-printing techniques were used to create the anthropomorphic phantom from a patient CT dataset. Three 131 I SPECT/CT imaging studies were performed using a homogeneous, Jaszczak, and an anthropomorphic phantom to quantify the SPECT images using a GE Optima NM/CT 640 with a high energy general purpose collimator. The impact of collimator detector response (CDR) modeling and volume-based partial volume corrections (PVCs) upon the absorbed dose was calculated using an image-based, Geant4 Monte Carlo RPT dosimetry workflow and compared against a ground truth scenario. Finally, uncertainties were quantified in accordance with recent EANM guidelines. RESULTS: The 3D-printed anthropomorphic phantom was an accurate re-creation of patient anatomy including bone. The extrapolated Jaszczak recovery coefficients were greater than that of the 3D-printed insert (∼22.8 ml) for both the CDR and non-CDR cases (with CDR: 0.536 vs. 0.493, non-CDR: 0.445 vs. 0.426, respectively). Utilizing Jaszczak phantom PVCs, the absorbed dose was underpredicted by 0.7% and 4.9% without and with CDR, respectively. Utilizing anthropomorphic phantom recovery coefficient overpredicted the absorbed dose by 3% both with and without CDR. All dosimetry scenarios that incorporated PVC were within the calculated uncertainty of the activity. The uncertainties in the cumulative activity ranged from 23.6% to 106.4% for Jaszczak spheres ranging in volume from 0.5 to 16 ml. CONCLUSION: The accuracy of Monte Carlo-based dosimetry for 131 I RPT in HNC was validated with an anthropomorphic phantom. In this study, it was found that Jaszczak-based PVCs were sufficient. Future applications of the phantom could involve 3D printing and characterizing patient-specific volumes for more personalized RPT dosimetry estimates.

A device for the controlled cooling and freezing of excised plant specimens during magnetic resonance imaging.

BACKGROUND: Investigating plant mechanisms to tolerate freezing temperatures is critical to developing crops with superior cold hardiness. However, the lack of imaging methods that allow the visualization of freezing events in complex plant tissues remains a key limitation. Magnetic resonance imaging (MRI) has been successfully used to study many different plant models, including the study of in vivo changes during freezing. However, despite its benefits and past successes, the use of MRI in plant sciences remains low, likely due to limited access, high costs, and associated engineering challenges, such as keeping samples frozen for cold hardiness studies. To address this latter need, a novel device for keeping plant specimens at freezing temperatures during MRI is described. RESULTS: The device consists of commercial and custom parts. All custom parts were 3D printed and made available as open source to increase accessibility to research groups who wish to reproduce or iterate on this work. Calibration tests documented that, upon temperature equilibration for a given experimental temperature, conditions between the circulating coolant bath and inside the device seated within the bore of the magnet varied by less than 0.1 °C. The device was tested on plant material by imaging buds from Vaccinium macrocarpon in a small animal MRI system, at four temperatures, 20 °C, - 7 °C, - 14 °C, and - 21 °C. Results were compared to those obtained by independent controlled freezing test (CFT) evaluations. Non-damaging freezing events in inner bud structures were detected from the imaging data collected using this device, phenomena that are undetectable using CFT. CONCLUSIONS: The use of this novel cooling and freezing device in conjunction with MRI facilitated the detection of freezing events in intact plant tissues through the observation of the presence and absence of water in liquid state. The device represents an important addition to plant imaging tools currently available to researchers. Furthermore, its open-source and customizable design ensures that it will be accessible to a wide range of researchers and applications.

Hyperpolarized 13C Magnetic Resonance Spectroscopic Imaging of Pyruvate Metabolism in Murine Breast Cancer Models of Different Metastatic Potential.

This study uses dynamic hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging (MRSI) to estimate differences in glycolytic metabolism between highly metastatic (4T1, n = 7) and metastatically dormant (4T07, n = 7) murine breast cancer models. The apparent conversion rate of pyruvate-to-lactate (kPL) and lactate-to-pyruvate area-under-the-curve ratio (AUCL/P) were estimated from the metabolite images and compared with biochemical metabolic measures and immunohistochemistry (IHC). A non-significant trend of increasing kPL (p = 0.17) and AUCL/P (p = 0.11) from 4T07 to 4T1 tumors was observed. No significant differences in tumor IHC lactate dehydrogenase-A (LDHA), monocarboxylate transporter-1 (MCT1), cluster of differentiation 31 (CD31), and hypoxia inducible factor-α (HIF-1α), tumor lactate-dehydrogenase (LDH) activity, or blood lactate or glucose levels were found between the two tumor lines. However, AUCL/P was significantly correlated with tumor LDH activity (ρspearman = 0.621, p = 0.027) and blood glucose levels (ρspearman = -0.474, p = 0.042). kPL displayed a similar, non-significant trend for LDH activity (ρspearman = 0.480, p = 0.114) and blood glucose levels (ρspearman = -0.414, p = 0.088). Neither kPL nor AUCL/P were significantly correlated with blood lactate levels or tumor LDHA or MCT1. The significant positive correlation between AUCL/P and tumor LDH activity indicates the potential of AUCL/P as a biomarker of glycolytic metabolism in breast cancer models. However, the lack of a significant difference between in vivo tumor metabolism for the two models suggest similar pyruvate-to-lactate conversion despite differing metastatic potential.

Evaluating the effectiveness of a lower extremity venous phantom on developing ultrasound examination skills and confidence.

INTRODUCTION: The aims of this study were: (1) Determine the effect on student ultrasound scanning skills using a lower extremity venous ultrasound phantom in addition to standard teaching methods of didactic lecture and scanning live volunteers and (2) Determine the effect of using a lower extremity venous ultrasound phantom in addition to standard teaching methods of didactic lecture and scanning live volunteers on student confidence levels in performing the lower extremity venous ultrasound examination. METHODS: Participants were first year diagnostic medical sonography students with minimal scanning experience (n = 11), which were randomized into two groups. Group 1 (n = 5) received the standard didactic lecture and attended a scan lab assessment where they performed a lower extremity venous examination on a human volunteer. Group 2 (n = 6) received the standard didactic lecture, performed three scheduled scanning sessions on an anatomic lower extremity venous phantom with flow and then attended the same scan lab assessment as Group 1, where they performed a lower extremity venous examination on a human volunteer. RESULTS: Scan lab assessments on day 4 of the study demonstrated a significant difference in scanning performance (p = 0.019) between the two groups. Post scan lab assessment confidence scores also demonstrated a significant difference between how participants in each group scored their confidence levels (p = 0.0260), especially in the ability to image calf veins. CONCLUSIONS: This study suggests anatomical phantoms can be used to develop scanning skills and build confidence in ultrasound imaging of the lower extremity venous structures.

Modeling early thermal injury using an ex vivo human skin model of contact burns.

BACKGROUND: Early mechanisms underlying the progressive tissue death and the regenerative capability of burn wounds are understudied in human skin. A clinically relevant, reproducible model for human burn wound healing is needed to elucidate the early changes in the human burn wound environment. This study reports a reproducible contact burn model on human skin that explores the extent of tissue injury and healing over time, and defines the inter-individual variability in human skin to enable use in mechanistic studies on burn wound progression and healing. METHODS: Using a customized burn device, contact burns of various depths were created on human skin by two operators and were evaluated for histologic depth by three raters to determine reproducibility. Early burn wound progression and wound healing were also evaluated histologically after the thermally injured human skin was cultured ex vivo for up to 14 days. RESULTS: Burn depths were reproducibly generated on human skin in a temperature- or time-dependent manner. No significant difference in operator-created or rater-determined depth was observed within each patient sample. However, significant inter-individual variation was identified in burn depth in ten patient samples. Burn-injured ex vivo human skin placed into culture demonstrated differential progression of cell death and collagen denaturation for high and low temperature contact burns, while re-epithelialization was observed in superficial burn wounds over a period of 14 days. CONCLUSION: This model represents an invaluable tool to evaluate the inter-individual variability in early burn wound progression and wound healing to complement current animal models and enhance the translation of preclinical research to improvements in patient care.

3-D-Printed Registration Phantom for Combined Ultrasound and Optical Imaging of Biological Tissues.

Efforts to develop quantitative ultrasound biomarkers would benefit from comparisons between ultrasound data and higher-resolution images of the tissue microstructure, such as from optical microscopy. However, only a few studies have used these methods for multiscale imaging because it is difficult to register low-resolution (>100 µm) ultrasound images to high-resolution microscopy images. To address this need, we have designed a 3-D-printed registration phantom that is made of a hard fluorescent resin, fits into a glass-bottom dish and can be used to calculate a coordinate system transform between ultrasound and optical microscopy. We report the phantom design, a registration protocol and an example registration using 18.5-MHz ultrasound and second harmonic generation microscopy. We evaluate the registration precision, achieving standard deviations smaller than the ultrasound resolution across all axes, and illustrate on a mouse mammary gland that this method yields results superior to those of manual landmark registration.

A syringe adapter for reduced muscular strain and fatigue.

Workers in hospitals, clinics, and contract research organizations who repetitively use syringes have an increased risk for musculoskeletal disorders. This study developed and tested a novel syringe adapter designed to reduce muscle strain associated with repetitive fluid draws. Three syringe plunger extension methods (ring-finger, middle-finger, and syringe adapter) were studied across twenty participants. Electromyogram signals for the flexor digitorum superficialis and extensor digitorum muscles were recorded. The syringe adapter required 31% of the 90th percentile flexor muscle activity as compared to the ring-finger syringe extension method, and 45% the 90th percentile flexor muscle activity as compared to the middle-finger method (p < 0.001). The greatest differences were observed when the syringe was near full extension. Although the syringe adapter took more time than the other syringe extension methods (1.5 times greater), it greatly helped reduce physical stress associated with repetitive, awkward syringe procedures.

Improved reconstruction stability for chemical shift encoded hyperpolarized 13 C magnetic resonance spectroscopic imaging using k-t spiral acquisitions.

PURPOSE: A multiecho, field of view (FOV)-oversampled k-t spiral acquisition and direct iterative decomposition of water and fat with echo asymmetry and least-squares estimation reconstruction is demonstrated to improve the stability of hyperpolarized 13 C magnetic resonance spectroscopic imaging (MRSI) in the presence of signal ambiguities attributed to low-SNR (signal-to-noise-ratio) species, local uncertainties in metabolite peaks, and echo-to-echo signal inconsistencies. THEORY: k-t spiral acquisitions redistribute readout points to be more densely spaced radially in k-space by acquiring an FOV and matrix that are oversampled by η. These more densely spaced spiral turns constitute effective intraspiral echoes and can supplement conventional interspiral echoes to improve spectral separation and reduce spectral cross-talk to better resolve 13 C-labeled species for spectroscopic imaging. METHODS: Digital simulations and imaging phantom experiments were performed for a range of interspiral echo spacings and η using multiecho, k-t spiral acquisitions. Image spectral cross-talk artifacts were evaluated both qualitatively and quantitatively as the percent error in measured metabolite ratios. In vivo murine experiments evaluated the feasibility of multiecho, k-t spiral [1-13 C]pyruvate MRSI to reduce spectral cross-talk for 3 scenarios of different expected reconstruction stability. RESULTS: Digital simulations and imaging phantom experiments both demonstrated reduced or comparable image spectral cross-talk and percent errors in measured metabolite ratios with increasing η and better choices of echo spacings. In vivo images displayed markedly reduced spectral cross-talk in lactate images acquired with η = 7 versus η = 1. CONCLUSION: The precision of hyperpolarized 13 C metabolic imaging and quantification in the presence of low-SNR species, local uncertainties in metabolite resonances, and echo-to-echo signal inconsistencies can be improved with the use of FOV-oversampled k-t spiral acquisitions.

Micro-injection molded, poly(vinyl alcohol)-calcium salt templates for precise customization of 3D hydrogel internal architecture.

In tissue engineering applications, sacrificial molding of hydrogel monoliths is a versatile technique for creating 3D molds to control tissue morphology. Previous sacrificial templates fabricated by serial processes such as solvent casting and thermal extrusion/fiber drawing can be used to effectively mold internal geometries within rapidly polymerizing, bulk curing hydrogels. However, they display poorer performance in controlling the geometry of diffusion limited, ionically cross-linked hydrogels, such as alginate. Here, we describe the use of poly(vinyl alcohol)-calcium salt templates (PVOH-Ca) fabricated by micro-injection molding, a parallel mass-production process, to conveniently cast internal geometries within both bulk curing hydrogels and ionically cross-linked alginate hydrogels. Calcium salt solubility was discovered to be a critical factor in optimizing the polymer composite's manufacturability, mechanical properties, and the quantity of calcium released upon template dissolution. Metrological and computed tomography (CT) analysis showed that the template's calcium release enables precise casting of microscale channel geometries within alginate hydrogels (6.4 ±â€¯7.2% average error). Assembly of modular PVOH-Ca templates to mold 3D channel networks within alginate hydrogels is presented to demonstrate engineering scalability. Moreover, the platform is used to create hydrogel molds for engineering human embryonic stem cell (hESC)-derived neuroepithelial organoids of a microscale, biomimetic cylindrical morphology. Thus, injection molded PVOH-Ca templates facilitate customization of hydrogel sacrificial molding, which can be used to generate 3D hydrogels with complex internal microscale architecture for diverse tissue engineering applications. STATEMENT OF SIGNIFICANCE: Sacrificial molding of hydrogel monoliths is a versatile technique for creating 3D molds for tissue engineering applications. Previous sacrificial materials fabricated by serial processes have been used to effectively mold internal geometries within rapidly polymerizing, bulk curing hydrogels. However, they display poor performance in molding geometry within diffusion limited, ionically cross-linked hydrogels, e.g. alginate. We describe the use of poly(vinyl alcohol)-calcium salt templates (PVOH-Ca) fabricated by micro-injection molding, an unparalleled mass-production process, to conveniently cast internal geometries within both bulk curing hydrogels and ionically cross-linked alginate hydrogels. Calcium release from the PVOH-Ca templates enables precise sacrificial molding of alginate hydrogels and the process is biocompatible. Moreover, we demonstrate its use to engineer the morphology of hPSC-derived neuroepithelial organoids, and modular PVOH-Ca template designs can be assembled to enable scalable 3D customization of hydrogel internal architecture.

A novel bioreactor for combined magnetic resonance spectroscopy and optical imaging of metabolism in 3D cell cultures.

PURPOSE: Fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorescent metabolites permits the measurement of cellular metabolism in cell, tissue and animal models. In parallel, magnetic resonance spectroscopy (MRS) of dynamic nuclear (hyper)polarized (DNP) 13 C-pyruvate enables measurement of metabolism at larger in vivo scales. Presented here are the design and initial application of a bioreactor that connects these 2 metabolic imaging modalities in vitro, using 3D cell cultures. METHODS: The model fitting for FLIM data analysis and the theory behind a model for the diffusion of pyruvate into a collagen gel are detailed. The device is MRI-compatible, including an optical window, a temperature control system and an injection port for the introduction of contrast agents. Three-dimensional printing, computer numerical control machining and laser cutting were used to fabricate custom parts. RESULTS: Performance of the bioreactor is demonstrated for 4 T1 murine breast cancer cells under glucose deprivation. Mean nicotinamide adenine dinucleotide (NADH) fluorescence lifetimes were 10% longer and hyperpolarized 13 C lactate:pyruvate (Lac:Pyr) ratios were 60% lower for glucose-deprived 4 T1 cells compared to 4 T1 cells in normal medium. Looking at the individual components of the NADH fluorescent lifetime, τ1 (free NADH) showed no significant change, while τ2 (bound NADH) showed a significant increase, suggesting that the increase in mean lifetime was due to a change in bound NADH. CONCLUSION: A novel bioreactor that is compatible with, and can exploit the benefits of, both FLIM and 13 C MRS in 3D cell cultures for studies of cell metabolism has been designed and applied.

3D second harmonic generation imaging tomography by multi-view excitation.

Biological tissues have complex 3D collagen fiber architecture that cannot be fully visualized by conventional second harmonic generation (SHG) microscopy due to electric dipole considerations. We have developed a multi-view SHG imaging platform that successfully visualizes all orientations of collagen fibers. This is achieved by rotating tissues relative to the excitation laser plane of incidence, where the complete fibrillar structure is then visualized following registration and reconstruction. We evaluated high frequency and Gaussian weighted fusion reconstruction algorithms, and found the former approach performs better in terms of the resulting resolution. The new approach is a first step toward SHG tomography.

Fabrication approaches for the creation of physical models from microscopy data.

BACKGROUND: Three-dimensional (3D) printing has become a useful method of fabrication for many clinical applications. It is also a technique that is becoming increasingly accessible, as the price of the necessary tools and supplies decline. One emerging, and unreported, application for 3D printing is to aid in the visualization of 3D imaging data by creating physical models of select structures of interest. METHODS: Presented here are three physical models that were fabricated from three different 3D microscopy datasets. Different methods of fabrication and imaging techniques were used in each case. RESULTS: Each model is presented in detail. This includes the imaging modality used to capture the raw data, the software used to create any computer models and the 3D printing tools used to create each model. Despite the differences in their creation, these examples follow a simple common workflow that is also detailed. CONCLUSIONS: Following these approaches, one can easily make 3D printed models from 3D microscopy datasets utilizing off the shelf commercially available software and hardware.

Development of a novel linearly-filled Derenzo microPET phantom.

Positron emission tomography (PET) phantoms are used to calibrate PET scanners so that inter-scanner and inter-isotope comparison can be made between PET datasets. Hot rod style phantoms have a hole pattern, which is filled with a positron-emitting isotope and typically involves using two radioisotope reservoirs with the pattern created with channels in between. However, this configuration is difficult to fill and requires an excess of activity and volume. Here we present an alternative design, a phantom that is linearly filled-one channel at a time. The process of fabrication of prototypes of the design is described and PET images of the prototyped phantom are also shown for a variety of commonly used radioisotopes ((52)Mn, (64)Cu, (76)Br, (124)I). This design allows for a large reduction in isotope volume and required filling time making a quality assurance (QA) protocol safer, more efficient and less costly.

Multi-view second-harmonic generation imaging of mouse tail tendon via reflective micro-prisms.

Here we experimentally show that second-harmonic generation (SHG) imaging is not sensitive to collagen fibers oriented parallel to the direction of laser propagation and, as a consequence, can potentially miss important structural information. As an alternative approach, we demonstrate the use of reflective micro-prisms to enable multi-view SHG imaging of mouse tail tendon by redirecting the focused excitation and collection of subsequent emission. Our approach data corroborates the theoretical treatment on vanishing and nonvanishing orientations, where fibers along the laser direction are largely transparent by SHG. In strong contrast, the two-photon excited fluorescence of dye-labeled collagen fibers is isotropic and is not subject to this constraint. We utilized Pearson correlation to quantify differences in fluorescent and backward detected SHG images of the tendon fiber structure, where the SHG and TPEF were highly statistically correlated (0.6-0.8) for perpendicular excitation but were uncorrelated for excitation parallel to the fiber axis. The results suggest that improved imaging of 3D collagen structure is possible with multi-view SHG microscopy.

Strategies from UW-Madison for rescuing biomedical research in the US.

A cross-campus, cross-career stage and cross-disciplinary series of discussions at a large public university has produced a series of recommendations for addressing the problems confronting the biomedical research community in the US.

Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination.

The recombination activity of Escherichia coli (E. coli) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A recA gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis. An iterative selection process generated at least 18 recA gene variants capable of producing a higher yield of transconjugants. Three of the variant proteins, RecA I102L, RecA V79L and RecA E86G/C90G were characterized based on their prominence. Relative to wild type RecA, the selected RecA variants exhibited faster rates of ATP hydrolysis, more rapid displacement of SSB, decreased inhibition by the RecX regulator protein, and in general displayed a greater persistence on DNA. The enhancement in conjugational function comes at the price of a measurable RecA-mediated cellular growth deficiency. Persistent DNA binding represents a barrier to other processes of DNA metabolism in vivo. The growth deficiency is alleviated by expression of the functionally robust RecX protein from Neisseria gonorrhoeae. RecA filaments can be a barrier to processes like replication and transcription. RecA regulation by RecX protein is important in maintaining an optimal balance between recombination and other aspects of DNA metabolism.

The sweet spot: FDG and other 2-carbon glucose analogs for multi-modal metabolic imaging of tumor metabolism.

Multi-modal imaging approaches of tumor metabolism that provide improved specificity, physiological relevance and spatial resolution would improve diagnosing of tumors and evaluation of tumor progression. Currently, the molecular probe FDG, glucose fluorinated with (18)F at the 2-carbon, is the primary metabolic approach for clinical diagnostics with PET imaging. However, PET lacks the resolution necessary to yield intratumoral distributions of deoxyglucose, on the cellular level. Multi-modal imaging could elucidate this problem, but requires the development of new glucose analogs that are better suited for other imaging modalities. Several such analogs have been created and are reviewed here. Also reviewed are several multi-modal imaging studies that have been performed that attempt to shed light on the cellular distribution of glucose analogs within tumors. Some of these studies are performed in vitro, while others are performed in vivo, in an animal model. The results from these studies introduce a visualization gap between the in vitro and in vivo studies that, if solved, could enable the early detection of tumors, the high resolution monitoring of tumors during treatment, and the greater accuracy in assessment of different imaging agents.

3D collagen alignment limits protrusions to enhance breast cancer cell persistence.

Patients with mammographically dense breast tissue have a greatly increased risk of developing breast cancer. Dense breast tissue contains more stromal collagen, which contributes to increased matrix stiffness and alters normal cellular responses. Stromal collagen within and surrounding mammary tumors is frequently aligned and reoriented perpendicular to the tumor boundary. We have shown that aligned collagen predicts poor outcome in breast cancer patients, and postulate this is because it facilitates invasion by providing tracks on which cells migrate out of the tumor. However, the mechanisms by which alignment may promote migration are not understood. Here, we investigated the contribution of matrix stiffness and alignment to cell migration speed and persistence. Mechanical measurements of the stiffness of collagen matrices with varying density and alignment were compared with the results of a 3D microchannel alignment assay to quantify cell migration. We further interpreted the experimental results using a computational model of cell migration. We find that collagen alignment confers an increase in stiffness, but does not increase the speed of migrating cells. Instead, alignment enhances the efficiency of migration by increasing directional persistence and restricting protrusions along aligned fibers, resulting in a greater distance traveled. These results suggest that matrix topography, rather than stiffness, is the dominant feature by which an aligned matrix can enhance invasion through 3D collagen matrices.