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Ensemble representations have been considered as one of the strategies that the visual system adopts to cope with its limited capacity. Thus, they include various statistical summaries such as mean, variance, and distributional properties and are formed over many stages of visual processing. The current study proposes a population coding model of ensemble perception to provide a theoretical and computational framework for these various facets of ensemble perception. The proposed model consists of a simple feature layer and a pooling layer. We assumed ensemble representations as population responses in the pooling layer and decoded various statistical properties from population responses. Our model successfully predicted averaging performance in orientation, size, color, and motion direction across different tasks. Furthermore, it predicted variance discrimination performance and the priming effects of feature distributions. Finally, it explained the well-known variance and set size effects and has a potential for explaining the adaptation and clustering effects.

Contextual cueing refers to finding a target more efficiently in repeated displays than in novel displays. However, conflicting results have been reported regarding whether target absent judgments can also be efficient in repeated displays. To resolve this controversy, we first tested 3 factors that might influence the strength of distractor-distractor associations and then investigated how such associations produced faster responses on repeated target absent trials by measuring the patterns of eye movements. The factors were the number of distractors, number of repeated configurations, and diversity of the distractors' properties. In Experiments 1 and 2, we found that the diversity of the distractors was the only factor producing contextual cueing without a target. In Experiment 3, we recorded eye movements during a search task and found that the contextual cueing effect in the target absent condition was due to the lower number of fixations and larger mean saccadic amplitudes. Overall, these results suggest that the distractor-distractor associations, strengthened by the diversity of the distractors' properties, widened the attentional window. This enlarged window in turn helps people to reject more distractors at once and enables them to terminate a visual search faster in repeated target absent trials. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Cryopreservation represents one if not the only long-term option for tissue and perhaps future organ banking. In one particular approach, cryopreservation is achieved by completely avoiding ice formation (or crystallization) through a process called vitrification. This "ice-free" approach to tissue banking requires a combination of high-concentration cryoprotective additives such as M22 (9.4 M), VS55 (8.4 M), or DP6 (6 M) and sufficiently fast rates of cooling and warming to avoid crystallization. In this article, we report the temperature-dependent specific heat capacity of the above-mentioned cryoprotective additives in small volumes (10 mg sample pans) at rates of 5°C/min and 10°C/min using a commercially available differential scanning calorimetry (TA Instruments Q1000), in the temperature range of -150°C to 30°C. This data can be utilized in heat-transfer models to predict thermal histories in a cryopreservation protocol. More specifically, the effects of temperature dependence of specific heat due to the presence of three different phases (liquid, ice, and vitreous phase) can dramatically impact the thermal history and therefore the outcome of the cryopreservation procedure. The crystallization potential of these cryoprotectants was also investigated by studying cases of maximal and minimal crystallization in VS55 and DP6, where M22 did not crystallize under any rates tested. To further reduce crystallization in VS55 and DP6, a stabilizing sugar (sucrose) was added in varying concentrations (0.15 M and 0.6 M) and was shown to further reduce crystallization, particularly in VS55, at modest rates of cooling (1°C/min, 5°C/min, and 10°C/min).

Atrial fibrillation (AF) currently affects millions of people in the U.S. alone. Focal therapy is an increasingly attractive treatment for AF that avoids the debilitating effects of drugs for disease control. Perhaps the most widely used focal therapy for AF is heat-based radiofrequency (heating), although cryotherapy (cryo) is rapidly replacing it due to a reduction in side effects and positive clinical outcomes. A third focal therapy, irreversible electroporation (IRE), is also being considered in some settings. This study was designed to help guide treatment thresholds and compare mechanism of action across heating, cryo, and IRE. Testing was undertaken on HL-1 cells, a well-established cardiomyocyte cell line, to assess injury thresholds for each treatment method. Cell viability, as assessed by Hoechst and propidium iodide (PI) staining, was found to be minimal after exposure to temperatures ≤−40 °C (cryo), ≥60 °C (heating), and when field strengths ≥1500 V/cm (IRE) were used. Viability was then correlated to protein denaturation fraction (PDF) as assessed by Fourier transform infrared (FTIR) spectroscopy, and protein loss fraction (PLF) as assessed by bicinchoninic acid (BCA) assay after the three treatments. These protein changes were assessed both in the supernatant and the pellet of cell suspensions post-treatment. We found that dramatic viability loss (≥50%) correlated strongly with ≥12% protein change (PLF, PDF or a combination of the two) in every focal treatment. These studies help in defining both cellular thresholds and protein-based mechanisms of action that can be used to improve focal therapy application for AF.

The use of thermal based therapies for treatment of atrial fibrillation is increasing. While numerous reports are available in the literature regarding the efficacy of cryotherapy on pulmonary vein survival, there are no reports specifically at the cellular level that establish thermal thresholds and mechanisms of cellular destruction. The current article reports on the response of HL-1 cardiomyocytes to cooling rates and end temperatures during cryothermal exposure. The focus is on establishment of in vitro thresholds while also establishing mechanisms of action due to biophysical events (i.e. intracellular ice formation and water transport).

While vitrified cryopreservation holds great promise, practical application has been limited to smaller systems (cells and thin tissues) due to diffusive heat and mass transfer limitations, which are typically manifested as devitrification and cracking failures during thaw. Here then we describe a new approach for rapidly and uniformly heating cryopreserved biospecimens with radiofrequency (RF) excited magnetic nanoparticles (mNPs). Importantly, heating rates can be increased several fold over conventional boundary heating techniques and are independent of sample size. Initial differential scanning calorimetry studies indicate that the addition of the mNPs has minimal impact on the freeze-thaw behavior of the cryoprotectant systems themselves. Then proof-of-principle experiments in aqueous and cryoprotectant solutions demonstrate the ability to heat at rates high enough to mitigate or eliminate devitrification (hundreds of °C/min) and scaled heat transfer modeling is used to illustrate the potential of this innovative approach. Finally, X-ray micro-computed-tomography (micro-CT) is investigated as a planning and quality control tool, where the density-based measurements are able to quantify changes in cryoprotectant concentration, mNP concentration, and the frozen state (i.e. crystallized versus vitrified).

The outcome of systemic and local therapies (e.g. chemotherapy, radiotherapy, surgery, focal ablation) for prostate cancer can be significantly improved by using tumor-specific adjuvants prior to treatment ("preconditioning"). We propose to use dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to monitor the in vivo response of a mouse model of prostate cancer treated with a vascular disruptive agent, TNF-α, delivered on a gold nanoparticle (NP-TNF). Six male nude mice bearing 4-5 week old LNCaP tumors were scanned at 9.4 T. DCE-MRI was performed two days before and 4-5 h after treatment with NP-TNF. An intraperitoneal (i.p.) bolus of gadolinium-DTPA (Gd) was administered and contrast enhancement was measured for 90 min. Concentration-time curves of Gd were calculated and the area under the Gd curve (AUGC) was determined pre- and post-treatment. NP-TNF treatment caused an increase in contrast uptake in tumors. Interestingly, the early concentration (10 min post Gd bolus i.p.) was similar in both untreated and treated conditions; however, 90 min after injection, [Gd] was 3.4 times higher than before treatment. AUGC doubled from (11 ± 6) [Gd] × min before treatment to (22 ± 9) [Gd] × min after treatment. An increase in signal enhancement was also observed in the muscle but to a lesser degree. We also evaluated the kinetics of intravenous Gd administration in mice bearing a jugular vein catheter to mimic the delivery method used in clinical trials. The overall treatment effects were independent of the delivery pathway of the contrast agent. In conclusion, we show that DCE-MRI is suitable to detect changes associated with a vascular disruptive agent in a mouse model of prostate cancer. The ability to characterize the effects of nanoparticle therapy in vivo with non-destructive methods is important, as such compounds, in combination with treatment strategies, are progressing towards clinical trials.

The current study presents a new and novel analysis of heat release signatures measured by a differential scanning calorimeter (DSC) associated with water transport (WT), intracellular ice formation (IIF) and extracellular ice formation (EIF). Correlative cryomicroscopy experiments were also performed to validate the DSC data. The DSC and cryomicroscopy experiments were performed on human dermal fibroblast cells (HDFs) at various cytocrit values (0-0.8) at various cooling rates (0.5-250 °C/min). A comparison of the cryomicroscopy experiments with the DSC analysis show reasonable agreement in the water transport (cellular dehydration) and IIF characteristics between both the techniques with the caveat that IIF measured by DSC lagged that measured by cryomicroscopy. This was ascribed to differences in the techniques (i.e. cell vs. bulk measurement) and the possibility that not all IIF is associated with visual darkening. High and low rates of 0.5 °C/min and 250 °C/min were chosen as HDFs did not exhibit significant IIF or WT at each of these extremes respectively. Analysis of post-thaw viability data suggested that 10 °C/min was the presumptive optimal cooling rate for HDFs and was independent of the cytocrit value. The ratio of measured heat values associated with IIF (q(IIF)) to the total heat released from both IIF and water transport or from the total cell water content in the sample (q(CW)) was also found to increase as the cooling rate was increased from 10 to 250 °C/min and was independent of the sample cytocrit value. Taken together, these observations suggest that the proposed analysis is capable of deconvolving water transport and IIF data from the measured DSC latent heat thermograms in cell suspensions during freezing.

The mechanisms of cold injury to biosystems are investigated at the cellular, tissue, and systemic levels, with details concerning the related experimental methods. Cellular level studies have shown a direct relationship between biophysical changes versus freeze-thaw survival. Advances in experimental and analytical methods have resulted in qualitatively similar results for native and artificial tissue systems, with several important caveats relating to cell-cell, cell-ECM effects. While these biophysical events have informed a better understanding of immediate injury after freezing at the cell and tissue level, further understanding of delayed injury effects after cryosurgery including at the cellular (i.e. apoptosis) and host mediated (i.e. vascular and immunological) events remain important areas of research and an opportunity to improve the technique.

Solid tumors such as hepatocellular carcinoma are very often not amenable to chemotherapy and radiotherapy. Local ablation methods, including chemical ablation with absolute ethanol, are therefore an option for treatment but lack of information about the mechanism of devitalization leading to cell death is a hindrance to further adoption. Systemic toxicity also has limited the amount of ethanol that can be used in a single treatment session. Therefore we evaluated the mechanism of urea, a denaturant with little or no systemic toxicity, for potential use in chemical ablation. In this study we report on the use of three methods to analyze the effects in cell culture with a view towards eventual clinical application. Human hepatoma HuH-7 cells were analyzed at several time points after treatment using FTIR, DSC, and Raman microspectroscopy based on MTT and PI-exclusion viability assays. Time course fractional denaturation data plotted against viability show that a 50% viability drop occurs after only a 10–20% drop in overall protein denaturation. Other methods of cell death such as apoptosis may also be operative, but this result implies that protein denaturation is one of the major mechanisms of cell death. This is in line with what has been previously suggested for purely thermal methods, and opens the way to mechanism-based improvements in chemical ablation of solid tumors.

While studies on the freezing of cells in suspension have been carried out extensively, corresponding studies with cells in the attached state and in tissue or tissue-equivalents are less developed. As attachment is a hallmark of the tissue state it is important to understand its impact on biophysics and viability to better apply freezing towards tissue preservation. The current study reports on observed biophysical response changes observed during freezing human dermal fibroblasts in suspension, attached cell, and fibrin tissue-equivalent models. Specifically, intracellular ice formation is shown to increase and dehydration is inferred to increase from suspension to attached systems. Biophysical model parameters fit to these experimental observations reflect the higher kinetics in the attached state. Post-thaw viability values from fast cooling rates were higher for suspension systems, and correlated well with the amount of IIF observed. On the other hand, viability values from slow cooling rates were higher for attached systems, although the degree of dehydration was predicted to be comparable to suspension cells. This disconnect between biophysics and viability predictions at slow rates clearly requires further investigation as it runs counter to our current understanding of dehydration injury in cells. This may suggest a possible protective effect of the attachment state on cell systems.

It is well accepted in cryobiology that the temperature history and cooling rates experienced in biomaterials during freezing procedures correlate strongly with biological outcome. Therefore, heat transfer measurement and prediction in the cryogenic regime is central to the field. Although direct measurement of temperature history (i.e. heat transfer) can be performed, accuracy is usually achieved only for local measurements within a given system and cannot be readily generalized to another system without the aid of predictive models. The accuracy of these models rely upon thermal properties which are known to be highly dependent on temperature, and in the case of significant cryoprotectant loading, also on crystallized fraction. In this work, we review the available thermal properties of biomaterials in the cryogenic regime. The review shows a lack of properties for many biomaterials in the subzero temperature domain, and especially for systems with cryoprotective agents. Unfortunately, use of values from the limited data available (usually only down to -40 degrees C) lead to an underestimation of thermal property change (i.e. conductivity rise and specific heat drop due to ice crystallization) with lower temperatures. Conversely, use of surrogate values based solely on ice thermal properties lead to an overestimation of thermal property change for most biomaterials. Additionally, recent work extending the range of available thermal properties to -150 degrees C has shown that the thermal conductivity will drop in both PBS and tissue (liver) due to amorphous/glassy phases (versus crystalline) of biomaterials with the addition of cryoprotective additives such as glycerol. Thus, we investigated the implications of using approximated or constant property values versus measured temperature-dependent values for predicting temperature history during freezing in PBS (phosphate-buffered saline) and porcine liver with and without cryoprotectants (glycerol). Using measured property values (thermal conductivity, specific heat, and latent heat of phase change) of porcine liver, a standard was created which showed that values based on surrogate ice properties under-predicted cooling times, while constant properties (i.e. based on limited data reported near the freezing point) over-predicted cooling times. Additionally, a new iterative numerical method that accommodates non-equilibrium cooling effects as a function of time and position (i.e. crystallization versus amorphous phase) was used to predict temperature history during freezing in glycerol loaded systems. Results indicate that in addition to the increase in cooling times due to the lowering of thermal diffusivity with more glycerol, non-equilibrium effects such as the prevention of maximal crystallization (i.e. amorphous phases) will further increase required cooling times. It was also found that the amplified effect of non-equilibrium cooling and crystallization with system size prevents the thermal history to be described with non-dimensional lengths, such as was possible under equilibrium cooling. These results affirm the need to use accurate thermal properties that incorporate temperature dependence and crystallized fraction. Further studies are needed to extract thermal properties of other important biomaterials in the subzero temperature domain and to develop accurate numerical methods which take into account non-equilibrium cooling events encountered in cryobiology when partial or total vitrification occurs.

Though cryopreservation of mouse sperm yields good survival and motility after thawing, cryopreservation of rat sperm remains a challenge. This study was designed to evaluate the biophysics (membrane permeability) of rat in comparison to mouse to better understand the cooling rate response that contributes to cryopreservation success or failure in these two sperm types. In order to extract subzero membrane hydraulic permeability in the presence of ice, a differential scanning calorimeter (DSC) method was used. By analyzing rat and mouse sperm frozen at 5 degrees C/min and 20 degrees C/min, heat release signatures characteristic of each sperm type were obtained and correlated to cellular dehydration. The dehydration response was then fit to a model of cellular water transport (dehydration) by adjusting cell-specific biophysical (membrane hydraulic permeability) parameters L(pg) and E(Lp). A "combined fit" (to 5 degrees C/min and 20 degrees C/min data) for rat sperm in Biggers-Whitten-Whittingham media yielded L(pg) = 0.007 microm min(-1) atm(-1) and E(Lp) = 17.8 kcal/mol, and in egg yolk cryopreservation media yielded L(pg) = 0.005 microm min(-1) atm(-1) and E(Lp) = 14.3 kcal/mol. These parameters, especially the activation energy, were found to be lower than previously published parameters for mouse sperm. In addition, the biophysical responses in mouse and rat sperm were shown to depend on the constituents of the cryopreservation media, in particular egg yolk and glycerol. Using these parameters, optimal cooling rates for cryopreservation were predicted for each sperm based on a criteria of 5%-15% normalized cell water at -30 degrees C during freezing in cryopreservation media. These predicted rates range from 53 degrees C/min to 70 degrees C/min and from 28 degrees C/min to 36 degrees C/min in rat and mouse, respectively. These predictions were validated by comparison to experimentally determined cryopreservation outcomes, in this case based on motility. Maximum motility was obtained with freezing rates between 50 degrees C/min and 80 degrees C/min for rat and at 20 degrees C/min with a sharp drop at 50 degrees C/min for mouse. In summary, DSC experiments on mouse and rat sperm yielded a difference in membrane permeability parameters in the two sperm types that, when implemented in a biophysical model of water transport, reasonably predict different optimal cooling rate outcomes for each sperm after cryopreservation.

Rigid polyurethane foams (PU) are widely used as thermal insulators in various applications. The thermal conductivity of the foam is the key parameter that governs the efficiency of thermal insulation provided by the foam. The usual technique employed to measure thermal conductivity is based on the rate of steady state heat transfer across a known thickness, induced by two different known temperatures at two opposite surfaces of the foam. We introduce a technique based on the transient measurement of heat transfer measured by an embedded needle probe. This technique is not only rapid but the instrumentation required for such a measurement is simple and the cost is only a fraction of the steady state counterpart. The values of thermal conductivity obtained by both methods are compared and found to agree within 4% over the range of 0.02—0.03 W/mK, which is the usual range of thermal conductivity for commercial rigid PU foams. The sensitivity of the needle probe technique is demonstrated by measuring the thermal conductivity values of foams made with various concentrations of chemical blowing agent (water). The present technique is also shown to be effective for measuring the thermal conductivity of small samples, especially, free rise cup foams for which the steady state technique can not be used.

This study reports the first comprehensive data on the effects of glycerol, a widely used cryoprotective agent (CPA), on the thermal properties of Phosphate Buffered Saline at subzero and cryogenic (⩽−40 °C) temperatures. A Differential Scanning Calorimeter was used to measure specific and latent heats. Thermal conductivity was determined using a thermistor probe technique. Results differed significantly from known water (ice) or glycerol values, and a simple weight averaging method between water and glycerol was found to over-predict thermal conductivity below −100 °C due to glass formation. This indicates the need for these newly measured thermal properties of CPA solutions particularly below −100 °C.

The latent heat during phase change of water-NaCl binary mixture was measured using a differential scanning calorimeter, and the magnitude for two distinct phase change events, water/ice and eutectic phase change, were analyzed considering the phase change characteristics of a binary mixture. During the analysis, the latent heat associated with each event was calculated by normalizing the amount of each endothermic peak with only the amount of sample participating in each event estimated from the lever rule for the phase diagram. The resulting latent heat of each phase change measured is 303.7 +/- 2.5 J/g for water/ice phase change, and 233.0 +/- 1.6 J/g for eutectic phase change, respectively regardless of the initial concentration of mixture. Although the latent heats of water/ice phase change in water-NaCl mixtures are closely correlated, further study is warranted to investigate the reason for smaller latent heat of water/ice phase change than that in pure water (335 J/g). The analysis using the lever rule was extended to estimate the latent heat of dihydrate as 115 J/g with the measured eutectic and water/ice latent heat values. This new analysis based on the lever rule will be useful to estimate the latent heat of water-NaCl mixtures at various concentrations, and may become a framework for more general analysis of latent heat of various biological solutions.

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The outcome of systemic and local therapies (e.g., chemotherapy, radiotherapy, surgery, focal ablation) for prostate cancer can be significantly improved by using tumor-specific adjuvants prior to treatment (“preconditioning”). We propose to use dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to monitor the in vivo response of a mouse model of prostate cancer treated with a vascular disruptive agent, TNF-α, delivered on a gold nanoparticle (NP-TNF). Six male nude mice bearing 4–5 weeks old LNCaP tumors were scanned at 9.4T. DCE-MRI was performed two days before and 4–5 hours after treatment with NP-TNF. An intraperitoneal bolus of Gadolinium-DTPA (Gd) was administered and contrast enhancement was measured for 90 minutes. Concentration time curves of Gd were calculated and the area under the Gd curve (AUGC) was determined pre and post treatment. NP-TNF treatment caused an increase in contrast uptake in tumors. Interestingly, the early concentration (10 minutes post Gd bolus i.p.) was similar in both untreated and treated conditions; however, 90 minutes after injection, [Gd] was 3.4 times higher after treatment compared to before. AUGC doubled from 11 ± 6 [Gd] × min before treatment to 22 ± 9 [Gd] × min after treatment. An increase in signal enhancement was also observed in the muscle but to a lesser degree. We also evaluated the kinetics of intravenous gadolinium administration in mice bearing a jugular vein catheter to mimic the delivery method used in clinical trials. The overall treatment effects were independent of the delivery pathway of the contrast agent. In conclusion, we show that DCE-MRI is suitable to detect changes associated with a vascular disruptive agent in a mouse model of prostate cancer. The ability to characterize the effects of nanoparticle therapy in vivo with non-destructive methods is important as such compounds, in combination with treatment strategies, progress towards clinical trials.

Tumor hypoxia is an important factor in assessment of both cancer progression and cancer treatment efficacy. This has driven a substantial effort toward development of imaging modalities that can directly measure oxygen distribution and therefore hypoxia in tissue. Although several approaches to measure hypoxia exist, direct measurement of tissue oxygen through an imaging approach is still an unmet need. To address this, we present a new approach based on in vivo application of photoacoustic lifetime imaging (PALI) to map the distribution of oxygen partial pressure (pO2) in tissue. This method utilizes methylene blue, a dye widely used in clinical applications, as an oxygen-sensitive imaging agent. PALI measurement of oxygen relies upon pO2-dependent excitation lifetime of the dye. A multimodal imaging system was designed and built to achieve ultrasound (US), photoacoustic, and PALI imaging within the same system. Nude mice bearing LNCaP xenograft hindlimb tumors were used as the target tissue. Hypoxic regions were identified within the tumor in a combined US/PALI image. Finally, the statistical distributions of pO2 in tumor, normal, and control tissues were compared with measurements by a needle-mounted oxygen probe. A statistically significant drop in mean pO2 was consistently detected by both methods in tumors.

Biothermal engineering applications impose thermal excursions on tissues with an ensuing biological outcome (i.e., life or death) that is tied to the molecular state of water and protein in the system. The accuracy of heat transfer models used to predict these important processes in turn depends on the kinetics and energy absorption of molecular transitions for both water and protein and the underlying temperature dependence of the tissue thermal properties. Unfortunately, a lack of tissue thermal property data in the literature results in an overreliance on property estimates. This work addresses these thermal property limitations in liver, a platform tissue upon which hyperthermic engineering applications are routinely performed and a test bed that will allow extension to further tissue property measurement in the future. Specifically, we report on the thermal properties of cadaveric human and porcine liver in the suprazero range between 25 °C to 80 °C for thermal conductivity and 25 °C to 85 °C for apparent specific heat. Denaturation and water vaporization are shown to reduce thermal conductivity and apparent specific heat within the samples by up to 20% during heating. These changes in thermal properties significantly altered thermal histories during heating compared to conditions when properties were assumed to remain constant. These differences are expected to alter the biological outcome from heating as well.

Microbubble ultrasound contrast agents (UCAs) have been extensively used in medical ultrasound for enhancing the echo components from blood vessels. However, their ultimate promise of enhancing the echoes from the microvasculature with high specificity remains unfulfilled using existing methods. We have previously shown that the Volterra filter can be used to enhance UCA echo components from flow channels with dimensions similar to peripheral vessels, e.g. the carotid artery. In this paper, we investigate a new receiver architecture based on an adaptive third-order Volterra Filter (VF) in conjunction with beamformed echo data from imaging tumor microvasculature in vivo. It is shown that the cubic and quadratic components of the VF provide significant enhancement of the UCA echoes from the tumor compared to the echoes from the same tissue regions in the absence of the UCA. We describe an adaptive recursive least squares (RLS) implementation of the VF and give examples of the contrast enhancement. Further enhancement of the UCA contrast is achieved by applying a dynamic statistical decision rule that produces a parameter we refer to as the temporal perfusion index (TPI). The TPI rewards transient echo oscillations while rejecting tissue motion and noise variation. These in vivo results demonstrate the potential for advanced signal processing in increasing the sensitivity and specificity of medical ultrasound in imaging tissue perfusion, a form of functional imaging.

Surgery, radiation and chemotherapy remain the mainstay of current cancer therapy. However, treatment failure persists due to the inability to achieve complete local control of the tumor and curtail metastatic spread. Vascular disrupting agents (VDAs) are a class of promising systemic agents that are known to synergistically enhance radiation, chemotherapy or thermal treatments of solid tumors. Unfortunately, there is still an unmet need for VDAs with more favorable safety profiles and fewer side effects. Recent work has demonstrated that conjugating VDAs to other molecules (polyethylene glycol, CNGRCG peptide) or nanoparticles (liposomes, gold) can reduce toxicity of one prominent VDA (tumor necrosis factor alpha, TNF-α). In this report, we show the potential of a gold conjugated TNF-α nanoparticle (NP-TNF) to improve multimodal cancer therapies with VDAs. In a dorsal skin fold and hindlimb murine xenograft model of prostate cancer, we found that NP-TNF disrupts endothelial barrier function and induces a significant increase in vascular permeability within the first 1–2 hours followed by a dramatic 80% drop in perfusion 2–6 hours after systemic administration. We also demonstrate that the tumor response to the nanoparticle can be verified using dynamic contrast-enhanced magnetic resonance imaging (MRI), a technique in clinical use. Additionally, multimodal treatment with thermal therapies at the perfusion nadir in the sub- and supra- physiological temperature regimes increases tumor volumetric destruction by over 60% and leads to significant tumor growth delays compared to thermal therapy alone. Lastly, NP-TNF was found to enhance thermal therapy in the absence of neutrophil recruitment, suggesting that immune/inflammatory regulation is not central to its power as part of a multimodal approach. Our data demonstrate the potential of nanoparticle-conjugated VDAs to significantly improve cancer therapy by preconditioning tumor vasculature to a secondary insult in a targeted manner. We anticipate our work to direct investigations into more potent tumor vasculature specific combinations of VDAs and nanoparticles with the goal of transitioning optimal regimens into clinical trials.

Oxygen plays a key role in the energy metabolism of living organisms. Any imbalance in the oxygen levels will affect the metabolic homeostasis and lead to pathophysiological diseases. Hypoxia, a status of low tissue oxygen, is a key factor in tumor biology as it is highly prominent in tumor tissues. However, clinical tools for assessing tissue oxygenation are limited. The gold standard is polarographic needle electrode which is invasive and not capable of mapping (imaging) the oxygen content in tissue. We applied the method of photoacoustic lifetime imaging (PALI) of oxygen-sensitive dye to small animal tissue hypoxia research. PALI is new technology for direct, non-invasive imaging of oxygen. The technique is based on mapping the oxygen-dependent transient optical absorption of Methylene Blue (MB) by pump-probe photoacoustic imaging. Our studies show the feasibility of imaging of dissolved oxygen distribution in phantoms. In vivo experiments demonstrate that the hypoxia region is consistent with the site of subcutaneously xenografted prostate tumor in mice with adequate spatial resolution and penetration depth.

While cryosurgery has proven capable in treating of a variety of conditions, it has met with some resistance among physicians, in part due to shortcomings in the ability to predict treatment outcomes. Here we attempt to address several key issues related to predictive modeling by demonstrating methods for accurately characterizing heat transfer from cryoprobes, report temperature dependent thermal properties for ultrasound gel (a convenient tissue phantom) down to cryogenic temperatures, and demonstrate the ability of convective exchange heat transfer boundary conditions to accurately describe freezing in the case of single and multiple interacting cryoprobe(s). Temperature dependent changes in the specific heat and thermal conductivity for ultrasound gel are reported down to −150 °C for the first time here and these data were used to accurately describe freezing in ultrasound gel in subsequent modeling. Freezing around a single and two interacting cryoprobe(s) was characterized in the ultrasound gel phantom by mapping the temperature in and around the “iceball” with carefully placed thermocouple arrays. These experimental data were fit with finite-element modeling in COMSOL Multiphysics, which was used to investigate the sensitivity and effectiveness of convective boundary conditions in describing heat transfer from the cryoprobes. Heat transfer at the probe tip was described in terms of a convective coefficient and the cryogen temperature. While model accuracy depended strongly on spatial (i.e., along the exchange surface) variation in the convective coefficient, it was much less sensitive to spatial and transient variations in the cryogen temperature parameter. The optimized fit, convective exchange conditions for the single-probe case also provided close agreement with the experimental data for the case of two interacting cryoprobes, suggesting that this basic characterization and modeling approach can be extended to accurately describe more complicated, multiprobe freezing geometries. Accurately characterizing cryoprobe behavior in phantoms requires detailed knowledge of the freezing medium's properties throughout the range of expected temperatures and an appropriate description of the heat transfer across the probe's exchange surfaces. Here we demonstrate that convective exchange boundary conditions provide an accurate and versatile description of heat transfer from cryoprobes, offering potential advantages over the traditional constant surface heat flux and constant surface temperature descriptions. In addition, although this study was conducted on Joule–Thomson type cryoprobes, the general methodologies should extend to any probe that is based on convective exchange with a cryogenic fluid.

Accurate knowledge of the thermal conductivities of biological tissues is important for thermal bioengineering, including applications in cryopreservation, cryosurgery, and other thermal therapies. The thermal conductivity of biomaterials is traditionally measured with macroscale techniques such as the steady longitudinal heat flow method or embedded thermistor method. These techniques typically require relatively large, centimeter-scale samples, limiting their applicability to finer biological structures. They are also vulnerable to errors caused by thermal contact resistances and parasitic heat losses. In contrast, the thermal conductivity of inorganic solids such as semiconductor wafers and thin films is commonly measured using the “3 omega method” [1–3]. This frequency domain technique is robust against thermal contact resistances and parasitic heat losses. It routinely has sub-millimeter spatial resolution, with theoretical limits down to tens of microns. Here we adapt the 3 omega method for measurements of biological tissues. Thermal conductivity measurements are made on both frozen and un-frozen samples including agar gel, water, and mouse liver, including samples with sub-millimeter thicknesses. The measurement results compare favorably with literature values and span the range from around 0.5 to 2.5 W/m-K. This study demonstrates the promise that this technique holds for thermal measurements of bulk tissues as well as fine sub-millimeter samples.Copyright © 2012 by ASME

Cryosurgery has demonstrated significant capabilities as a minimally invasive technique for treating cancer and other conditions [1], but has been limited by the ability to accurately predict treatment outcome and damage to surrounding tissues. Contributing to this problem has been the accuracy of predictive modeling and translating this to effective pretreatment planning. The two key factors in accurately modeling cryosurgical freezing are the temperature-dependent thermal properties of the freezing medium and cryoprobe boundary conditions. Both of these issues are considered in this study.

Solid tumors in the liver such as hepatocellular carcinoma, often are not amenable to chemotherapy or surgical therapies. Local “ablation” methods including thermal and chemical ablation are therefore options for treatment. Hyperthermic ablation methods (RF, microwave, HIFU and laser) can potentially accomplish treatment in a single session but are considerably more costly than chemicals. It was therefore of interest to investigate if a known protein denaturant such as urea would destroy liver tumors. Further, we wished to assess whether this destruction occurs by protein denaturation mechanisms similar to hyperthermic destruction. Specifically, Lepock showed that hyperthermic cell destruction occurs for many cells when overall protein denaturation is greater than 5% (i.e. survival drops from 95% to 5% beyond this range). In this study we report on the cytotoxicity of urea on human hepatoma HuH-7. We then quantify the amount of cellular protein denaturation associated with cytotoxicity and show that protein denaturation in excess of 5% of total protein must occur in order for significant cell killing.Copyright © 2011 by ASME

A large number of studies in cryobiology have focused on understanding the underlying biophysics at the cellular level to help predict survival outcome after cryopreservation or cryosurgery. While this behavior is increasingly well studied and understood in cells gaps remain in our understanding of how cells in tissues behave which can hamper freezing applications in tissues. This study compares freezing behavior in cells in suspension vs. attached (a model tissue) state to investigate any differences in cellular behavior in these two states.Copyright © 2011 by ASME

In cryopreservation protocols maximum survivability is achieved when cooling occurs slowly enough to avoid Intracellular ice formation (IIF) yet fast enough to avoid solute effects injury (1). IIF plays a significant role in cell damage during cryopreservation. IIF has been extensively studied using cryomicroscopy. This technique is a useful tool to understand the dynamic processes during cooling, i.e. volume change of cells and IIF occurrence associated with temperature. However it has some limitations in being applied to biological systems. The central assumption in cryomicroscopy is that the projected two-dimensional area of the cell can be extrapolated to a spherical three-dimensional volume. While reasonable for spherical cell systems, this assumption is inappropriate for obtaining quantitative volumetric information in nonspherical cell systems. Differential scanning calorimetry (DSC), however, can be applied to nonspherical cell systems. Thus, DSC exotherms during freezing needed to be compared with cryomicroscopy observations in simple spherical cell systems. Several studies related to IIF using DSC havebeen reported (2)–(5). Most of them, however, discussed only IIF peaks and/or Extracellular ice formation (EIF). In order to predict the optimum cooling rate from DSC results, it is important to quantify not only IIF and EIF but also water transport (WT) during cooling at various cooling rates.Copyright © 2011 by ASME

Cryopreservation is a convenient method for long-term preservation of natural and engineered tissues in regenerative medicine. Homogeneous loading of tissues with CPAs, however, forms one of the major hurdles in tissue cryopreservation. In this study, computer tomography (CT) as a non-invasive imaging method was used to determine the effective diffusion of Me2SO in tissue-engineered collagen scaffolds. The dimensions of the scaffolds were 30 x 30 x 10 mm3 with a homogeneous pore size of 100 microm and a porosity of 98%. CT images were acquired after equilibrating the scaffolds in phosphate buffered saline (PBS) and transferring them directly in 10% (v/v)Me2SO. The Me2SO loading process of the scaffold could thus be measured and visualized in real time. The experimental data were fitted using a diffusion equation. The calculated effective diffusion constant for Me2SO in the PBS loaded scaffold was determined from experimental diffusion studies to be 2.4 x 10(-6) cm2/s at 20 degrees C.

Atrial fibrillation (AF) is the irregular beating of the heart affecting an estimated 2.7 million Americans and responsible for 26 billion dollars of the nation’s annual healthcare cost. Cryoablation is a clinically approved technique for the treatment of paroxysmal AF in which an ablation catheter rapidly cools the cardiac tissue. Through freezing, discrete lesions are created in the region where the pulmonary veins join with the atria in order to terminate the conduction of abnormal electrical signals responsible for AF. While the efficacy of AF cryoablation is well known, the thermal thresholds and mechanisms of cryoinjury that determine efficacy are not known thereby hindering development of the next generation of AF cryoablation systems. This study begins to address this by improving understanding of the mechanisms and thermal thresholds of cryodestruction in a cardiac muscle cell line (HL-1). Specifically, we determine biophysical parameters governing known mechanisms of injury (membrane permeability and likelihood of intracellular ice formation, IIF). Additionally we correlate these biophysics and thermal history (cooling rate and end = temperature) to post-thaw viability in vitro in HL-1s guide optimal lesion formation and thus cryoablation device design. HL-1 cells were cultured using previously published methods. Cells were harvested using trypsinization and kept as a suspension on ice until needed. A temperature stage was used in conjunction with a light microscope to observe freezing of the samples. The controlled thermal history included 3 variables including: cooling rate (0.5 to 130 °C/min), end temperature (−5 to −60 °C), and hold time (0–5 min). These were varied in an experimental matrix to determine the importance of each. Before cooling, samples were pre-nucleated by applying a chilled needle on the outer edge of the sample. Change in water content within cells (cellular dehydration) was measured based on observed changes to projected cell area during freezing. IIF events were measured based on observing a sudden change in opacity (darkening) of the cell interior. Measured data were applied to water transport and nucleation models to determine membrane permeability and IIF nucleation parameters. Additionally, post-thaw viability of samples were determined after a rapid thaw and 15 min incubation period using a fluorescence membrane dye exclusion assay. For cases when the end temperature was set as −20 °C the highest viability of around 15% was observed at a cooling rate of 5 °C/min, while slower or faster cooling rates resulted in reduced viability. Subsequent measurements with cooling rate set as 5 °C/min and varied end temperatures resulted in viability values around 94%, 72%, 15%, 6% and less than 5% at −5 °C, −10 °C, −20 °C, −40 °C, and −60 °C, respectively. Our results suggest a large difference in post-thaw cell viability dependent on cooling rate and end temperature, while difference in hold times (0, 1, and 5 min) appeared less important. The application of this work to pulmonary vein freezing numerical models, and to future work in more translational systems such as attached (monolayer, artificial constructs) cell systems and tissues will be discussed.

While cryopreservation through vitrification holds great promise, practical application has been limited to smaller systems (cells and thin tissues) due to diffusive transfer and phase-change limitations, which are typically manifested as devitrification and cracking failures during thaw. Here we describe a new approach for rapidly and uniformly heating cryopreserved biospecimens with radiofrequency (RF) excited magnetic nanoparticles (mNPs). Importantly, heating rates can be increased several fold over conventional boundary heating and are independent of sample size and dielectric properties, overcoming the inhomogeneity associated with microwave thawing. Proof-of-principle experiments in aqueous and cryoprotectant solutions are presented and then modeling is used to further illustrate the potential of this innovative approach. Three embodiments of mNP suspensions are studied – aqueous, 6 M glycerol in phosphate buffered saline, and 8.4 M VS55 cryoprotectant solution (Mehl, Cryobiology 30 (1993) 509–518). The iron oxide mNPs used are commercially available (Ferrotec, Inc.) and have been previously shown to produce significant heating in aqueous suspensions (Etheridge and Bischof, ABME 41 (2013) 78–88). Nanoparticle concentrations up to 10 mg Fe/ml are shown to have negligible impact on the freezing behavior of these solutions, including the glass transition, critical cooling/warming rates, and specific heat, as measured by differential scanning calorimetry. One milliliter mNP suspensions at 5 and 10 mg Fe/ml in water and cryoprotectant solution were frozen in liquid nitrogen (at sufficient rates to achieve vitrification in the cryoprotectant samples) and then reheated in an insulated, 370 kHz and 20 kA/m RF coil. Sample temperatures were monitored with 40-gauge thermocouples embedded prior to cooling. RF fields can produce interference in metallic thermocouples, but this was characterized and found to be negligible for the fine gauge thermocouples used. Heating rates at the glass transition of approximately 200 ° C/min were achieved in the 10 mg Fe/ml samples, avoiding devitrification in the cryoprotectant solutions. Since RF fields at frequencies Source of funding: This work was supported by the Minnesota Futures Grant Program, NSF/CBET #1066343, and the University of Minnesota Institute for Engineering Seed Grant Program. Conflict of interest: None declared. bischof@umn.edu

There is a lack of information on the effect of cryoprotective agents (CPAs) on the thermal properties of biomaterials at cryobiologically relevant temperatures (i.e.

Accurate knowledge of the thermal conductivity (k) of biological tissues is important for cryopreservation, thermal ablation, and cryosurgery. Here, we adapt the 3ω method-widely used for rigid, inorganic solids-as a reusable sensor to measure k of soft biological samples two orders of magnitude thinner than conventional tissue characterization methods. Analytical and numerical studies quantify the error of the commonly used "boundary mismatch approximation" of the bi-directional 3ω geometry, confirm that the generalized slope method is exact in the low-frequency limit, and bound its error for finite frequencies. The bi-directional 3ω measurement device is validated using control experiments to within ±2% (liquid water, standard deviation) and ±5% (ice). Measurements of mouse liver cover a temperature ranging from -69 °C to +33 °C. The liver results are independent of sample thicknesses from 3 mm down to 100 μm and agree with available literature for non-mouse liver to within the measurement scatter.

The study focuses on evaluating the effects of adding a cryoprotective agent (CPA) on the thermal properties of solutions. Preliminary results for Phosphate Buffered Saline (PBS) with glycerol at subzero temperatures are reported. A Differential Scanning Calorimeter (DSC) was used to measure the specific heat and to observe latent heat release. Cryomicroscopy was performed using a cryostage to control the cooling and heating rate of the sample and visually observe microscopic changes. The specific heat data obtained from the DSC is also used for predicting bulk thermal conductivity by a transient heat transfer model using a thermistor. Thermal properties measurement results show opposing trends prior to and after devitrification and melting events with respect to glycerol concentration.Copyright © 2004 by ASME

Background Cardiac balloon and catheter cryoablations for the treatment of atrial fibrillation have been gaining attention as approaches for treating arrhythmias, in particular for isolation of the pulmonary veins (PV). Despite widespread and growing clinical use of cardiac cryoablation, there are still questions regarding dosing and treatment time, which may affect both efficacy and collateral injury. To date injury thresholds for therapy of cardiac and/or PV tissues are largely unreported in the literature. Methods Slices of ventricular myocardium ( n = 11) and left atrial tissue ( n = 6) from female Yorkshire Cross swine hearts were dissected including the native endocardial surface. Samples were placed in an infrared imaging apparatus consisting of a plastic petri dish with central ablation probe (Galil Medical) and 2 mm of Sylgard polymer formed to the bottom. The tissue slices were impaled on the central cryoprobe and infrared thermography was captured using a Flir A20 looking from the top down. Subsequently, samples were cultured for 24 h in a cell culture incubator (myocardium) at 37 °C for optimal staining with 1% TTC in Trizma buffer for 1 h at 37 °C. Photographs of the resultant TTC staining of each sample were captured and a custom Matlab program was used to normalize staining intensity and correlate the average staining intensity with the average thermal profile at any radial distance from the cryoprobe. The relationship between the staining intensity and end temperature was fit with a sigmoidal curve. Samples with a fit of greater than r 2 > 0.99 were analyzed. The transition from dead to injured tissue was defined as the point on the curve fit where the staining ratio was 10% of the range between the lower and upper asymptotes of the sigmoid (i.e., 10% of the grayscale value change at the lesion margin closest to the lesion). The maximum cooling rate correlated to this ratio and end temperature was then calculated from the thermal profiles. Eight thermal profiles were averaged to obtain a characteristic thermal profile for each sample. This approach has led to the creation of a database of thermal profiles that results in either completely or partially necrotic tissues. Results Initial results suggest that cooling rates of −103 ± 49 and −72 ± 9 °C/min and end temperatures of approximately −21 ± 6 and −17.2 ± 3.2 °C are necessary for complete necrosis of ventricular and left atrial tissue, respectively. Using a two-tailed T -test assuming equal variance the difference in end temperature and cooling rate between ventricular and left atrial tissue were non-significant ( P = .21, P = .20 respectively). Conclusions Experiments are ongoing to determine cryothermal injury thresholds of tissues associated with cardiac ablation, such as: lung and esophagus. The TTC staining protocol is in the process of being validated by comparison to H& E stain read by a blinded pathologist. Cellular viability assays are being performed with dye exclusion assays and results are being compared with tissue assays. Source of funding: GAANN Fellowship, Institute for Engineering in Medicine at the University of Minnesota, Medtronic, Inc. Source of funding: Medtrain, Inc. Conflict of interest: Ryan Goff has an Internship with Medtronic, Inc. goffx073@umn.edu

Surgery, radiation and chemotherapy remain the mainstay of current cancer therapy. However, treatment failure persists due to the inability to achieve complete local control of the tumor and curtail metastatic spread. Vascular disrupting agents (VDAs) are a class of promising systemic agents that are known to synergistically enhance radiation, chemotherapy or thermal treatments of solid tumors. Unfortunately, there is still an unmet need for VDAs with more favorable safety profiles and fewer side effects. Recent work has demonstrated that conjugating VDAs to other molecules (PEG, NGR) or nanoparticles (liposomes, gold) can greatly reduce toxicity of one prominent VDA (tumor necrosis factor alpha, TNF- α ). In this report, we show the potential of a gold conjugated TNF- α nanoparticle (NP-TNF) to improve multimodal cancer therapies with VDAs. In a dorsal skin fold and hindlimb murine xenograft model of prostate cancer, we found that NP-TNF disrupts endothelial barrier function and induces a significant increase in vascular permeability within the first 1–2 h followed by a dramatic 80% drop in perfusion 2–6 h after systemic administration. We also demonstrate that the tumor response to the nanoparticle can be verified using dynamic contrast-enhanced magnetic resonance imaging (MRI), a technique in clinical use. Additionally, multimodal treatment with thermal therapies at the perfusion nadir in the sub- and supra- physiological temperature regimes increases tumor volumetric destruction by over 60% and leads to significant tumor growth delays compared to thermal therapy alone. Lastly, NP-TNF was found to enhance thermal therapy in the absence of neutrophil recruitment, suggesting that immune/inflammatory regulation is not central to its power as part of a multimodal approach. Our data demonstrates the potential of nanoparticle-conjugated VDAs to significantly improve cancer therapy by preconditioning tumor vasculature to a secondary insult in a targeted manner with limited systemic toxicity or inflammatory response. We anticipate our work to direct investigations into more potent tumor vasculature specific combinations of VDAs and nanoparticles with the goal of transitioning optimal regimens into clinical trials.

Cryoablation for atrial fibrillation is a clinically approved technique used to control irregular heart rhythms due to abnormal electrical signals conducted from the pulmonary veins to the atria. The ablation catheter rapidly cools the cardiac tissue to freezing temperatures creating a discrete lesion which terminates these abnormal electrical pulses. Benefits of this approach include decreased risk of thrombus formation, well demarcated lesions, and preservation of extracellular matrix and endothelial integrity. However, possible complications include phrenic nerve injury and pulmonary vein stenosis (although these are not unique to the cryoablative approach). Therefore, further understanding of the mechanisms and thresholds of cryoinjury are required. This study begins to address these issues by improving predictive model accuracy through demonstrating the importance of accurate, temperature-dependent tissue properties. The direct relationship between thermal history and treatment outcome, combined with the difficulties of realtime, clinical thermometry, make heat transfer modeling and numerical predictions important aspects of cryoablation studies. The accuracy of the models depends on numerous factors including the kinetics and energy release during phase change phenomena and knowledge of thermal properties as a function of temperature. However, insufficient data for tissue thermal properties in the subzero domain results in a reliance on property estimations, generally based on water–ice data or weight averaged values from known materials. This study focused on expanding the thermal properties database for biological tissue in the subzero regime for use in cryoablation modeling. Results for porcine myocardium as well as ultrasound gel (a substitute phantom used for cryoprobe characterization) are reported. A differential scanning calorimeter was used to measure the specific heat and to observe latent heat effects. The thermal conductivity was measured using self-heating thermistors, employing both transient pulse decay and quasi-steady constant power methods. Specific heats in porcine myocardium were slightly less than those for ice, ranging from 0.93 J/gK at −150 °C to 2.35 J/gK at −21 °C. Specific heat behavior for ultrasound gel could be bracketed into two ranges, in which the values measured were less than those for ice at temperatures below the glass transition temperature (around −110 °C), and progressively higher than those for ice with temperatures above the glass transition. Thermal conductivities of porcine myocardium rose gradually at lower temperatures, ranging from 1.52 W/mK at −10 °C to 2.31 at −148 °C, and were about 50% less than those values for ice. Thermal conductivities of ultrasound gel were similar to those of porcine myocardium, but with an inflection point at the glass transition temperature after which the rise in thermal conductivity was attenuated. The importance of accurate, temperature-dependent thermal properties is demonstrated by comparing the predicted thermal history versus those estimated from constant property values.