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BACKGROUND A low-cryogen, compact 3T (C3T) MRI scanner with high-performance gradients capable of simultaneously achieving 80 mT/m gradient amplitude and 700 T/m/second slew rate has been in use to study research patients since March 2016 but has not been implemented in the clinical practice. PURPOSE To compare head MRI examinations obtained with the C3T system and a conventional whole-body 3T (WB3T) scanner in seven parameters across five commonly used brain imaging sequences. STUDY TYPE Prospective. SUBJECTS Thirty patients with a clinically indicated head MRI. SEQUENCE 3T; T1 FLAIR, T1 MP-RAGE, 3D T2 FLAIR, T2 FSE, and DWI. ASSESSMENT All patients tolerated the scans well. Three board-certified neuroradiologists scored the comparative quality of C3T and WB3T images in blinded fashion using a five-point Likert scale in terms of: signal-to-noise ratio, lesion conspicuity, motion artifact, gray/white matter contrast, cerebellar folia, susceptibility artifact, and overall quality. STATISTICAL TEST Left-sided, right-sided, and two-sided Wilcoxon signed rank test; Fisher's method. A P value

OBJECTIVE. The Adaptive Image Receive (AIR) radiofrequency coil is an emergent technology that is lightweight and flexible and exhibits electrical characteristics that overcome many of the limitations of traditional rigid coil designs. The purpose of this study was to apply the AIR coil for whole-brain imaging and compare the performance of a prototype AIR coil array with the performance of conventional head coils. SUBJECTS AND METHODS. A phantom and 15 healthy adult participants were imaged. A prototype 16-channel head AIR coil was compared with conventional 8- and 32-channel head coils using clinically available MRI sequences. During consensus review, two board-certified neuroradiologists graded the AIR coil compared with an 8-channel coil and a 32-channel coil on a 5-point ordinal scale in multiple categories. One- and two-sided Wilcoxon signed rank tests were performed. Noise covariance matrices and geometry factor (g-factor) maps were calculated. RESULTS. The signal-to-noise ratio, structural sharpness, and overall image quality scores of the prototype 16-channel AIR coil were better than those of the 8-channel coil but were not as good as those of the 32-channel coil. Noise covariance matrices showed stable performance of the AIR coil across participants. The median g-factors for the 16-channel AIR coil were, overall, less than those of the 8-channel coil but were greater than those of the 32-channel coil. CONCLUSION. On average, the prototype 16-channel head AIR coil outperformed a conventional 8-channel head coil but did not perform as well as a conventional 32-channel head coil. This study shows the feasibility of the novel AIR coil technology for imaging the brain and provides insight for future coil design improvements.

A recently developed compact 3 T (C3T) MRI scanner with high performance gradients [1, 2] has a dedicated radiofrequency (RF) transmit coil that exposes only the head, neck and a small portion of the upper body region during head-first scanning. Due to the unique coil geometry and patient positioning, the established SAR model used for a conventional whole-body scanner cannot be directly translated to the C3T. Here a specific absorption rate (SAR) estimation and validation framework was developed and used to implement a dedicated and accurate SAR prediction model for the C3T. Two different SAR prediction models for the C3T were defined and evaluated: one based on an anatomically derived exposed mass, and one using a fixed anatomical position located caudally to the RF coil to determine the exposed mass. After coil modeling and virtual human body simulation, the designed SAR prediction model was implemented on the C3T and verified with calorimetry and in vivo scan power monitoring. The fixed-demarcation exposed mass model was selected as appropriate exposed mass region to accurately estimate the SAR deposition in the patient on the C3T.

Background Distortion-free, high-resolution diffusion imaging using DIADEM (Distortion-free Imaging: A Double Encoding Method), proposed recently, has great potential for clinical applications. However, it can suffer from prolonged scan times and its reliability for quantitative diffusion imaging has not been evaluated. Purpose To investigate the clinical feasibility of DIADEM-based high-resolution diffusion imaging on a novel compact 3T (C3T) by evaluating the reliability of quantitative diffusion measurements and utilizing both the high-performance gradients (80 mT/m, 700 T/m/s) and the sequence optimization with the navigator acquisition window reduction and simultaneous multislice (multiband) imaging. Study type Prospective feasibility study. Phantom/subjects Diffusion quality control phantom scans to evaluate the reliability of quantitative diffusion measurements; 36 normal control scans for B0 -field mapping; six healthy and two patient subject scans with a brain tumor for comparisons of diffusion and anatomical imaging. Field strength/sequence 3T; the standard single-shot echo-planar-imaging (EPI), multishot DIADEM diffusion, and anatomical (2D-FSE [fast-spin-echo], 2D-FLAIR [fluid-attenuated-inversion-recovery], and 3D-MPRAGE [magnetization prepared rapid acquisition gradient echo]) imaging. Assessment The scan time reduction, the reliability of quantitative diffusion measurements, and the clinical efficacy for high-resolution diffusion imaging in healthy control and brain tumor volunteers. Statistical test Bland-Altman analysis. Results The scan time for high in-plane (0.86 mm2 ) resolution, distortion-free, and whole brain diffusion imaging were reduced from 10 to 5 minutes with the sequence optimizations. All of the mean apparent diffusion coefficient (ADC) values in phantom were within the 95% confidence interval in the Bland-Altman plot. The proposed acquisition with a total off-resonance coverage of 597.2 Hz wider than the expected bandwidth of 500 Hz in human brain could yield a distortion-free image without foldover artifacts. Compared with EPI, therefore, this approach allowed direct image matching with the anatomical images and enabled improved delineation of the tumor boundaries. Data conclusion The proposed high-resolution diffusion imaging approach is clinically feasible on C3T due to a combination of hardware and sequence improvements. Level of evidence 3 TECHNICAL EFFICACY: Stage 1 J. Magn. Reson. Imaging 2020;51:296-310.

ObjectiveThis study investigates a locally low-rank (LLR) denoising algorithm applied to source images from a clinical task-based functional MRI (fMRI) exam before post-processing for improving statistical confidence of task-based activation maps.MethodsTask-based motor and language fMRI was obtained in eleven healthy volunteers under an IRB approved protocol. LLR denoising was then applied to raw complex-valued image data before fMRI processing. Activation maps generated from conventional non-denoised (control) data were compared with maps derived from LLR-denoised image data. Four board-certified neuroradiologists completed consensus assessment of activation maps; region-specific and aggregate motor and language consensus thresholds were then compared with nonparametric statistical tests. Additional evaluation included retrospective truncation of exam data without and with LLR denoising; a ROI-based analysis tracked t-statistics and temporal SNR (tSNR) as scan durations decreased. A test-retest assessment was performed; retest data were matched with initial test data and compared for one subject.ResultsfMRI activation maps generated from LLR-denoised data predominantly exhibited statistically significant ( p = 4.88×10–4to p = 0.042; one p = 0.062) increases in consensus t-statistic thresholds for motor and language activation maps. Following data truncation, LLR data showed task-specific increases in t-statistics and tSNR respectively exceeding 20 and 50% compared to control. LLR denoising enabled truncation of exam durations while preserving cluster volumes at fixed thresholds. Test-retest showed variable activation with LLR data thresholded higher in matching initial test data.ConclusionLLR denoising affords robust increases in t-statistics on fMRI activation maps compared to routine processing, and offers potential for reduced scan duration while preserving map quality.

Improved gradient performance in an MRI system reduces distortion in echo planar imaging (EPI), which has been a key imaging method for functional studies. A lightweight, low-cryogen compact 3T MRI scanner (C3T) is capable of achieving 80 mT m−1 gradient amplitude with 700 T m−1 s−1 slew rate, in comparison with a conventional whole-body 3T MRI scanner (WB3T, 50 mT m−1 with 200 T m−1 s−1). We investigated benefits of the high-performance gradients in a high-spatial-resolution (1.5 mm isotropic) functional MRI study. Reduced echo spacing in the EPI pulse sequence inherently leads to less severe geometric distortion, which provided higher accuracy than with WB3T for registration between EPI and anatomical images. The cortical coverage of C3T datasets was improved by more accurate signal depiction (i.e. less dropout or pile-up). Resting-state functional analysis results showed that greater magnitude and extent in functional connectivity (FC) for the C3T than the WB3T when the selected seed region is susceptible to distortions, while the FC matrix for well-known brain networks showed little difference between the two scanners. This shows that the improved quality in EPI is particularly valuable for studying certain brain regions typically obscured by severe distortion.

Purpose To build and evaluate a small-footprint, lightweight, high-performance 3T MRI scanner for advanced brain imaging with image quality that is equal to or better than conventional whole-body clinical 3T MRI scanners, while achieving substantial reductions in installation costs. Methods A conduction-cooled magnet was developed that uses less than 12 liters of liquid helium in a gas-charged sealed system, and standard NbTi wire, and weighs approximately 2000 kg. A 42-cm inner-diameter gradient coil with asymmetric transverse axes was developed to provide patient access for head and extremity exams, while minimizing magnet-gradient interactions that adversely affect image quality. The gradient coil was designed to achieve simultaneous operation of 80-mT/m peak gradient amplitude at a slew rate of 700 T/m/s on each gradient axis using readily available 1-MVA gradient drivers. Results In a comparison of anatomical imaging in 16 patients using T2 -weighted 3D fluid-attenuated inversion recovery (FLAIR) between the compact 3T and whole-body 3T, image quality was assessed as equivalent to or better across several metrics. The ability to fully use a high slew rate of 700 T/m/s simultaneously with 80-mT/m maximum gradient amplitude resulted in improvements in image quality across EPI, DWI, and anatomical imaging of the brain. Conclusions The compact 3T MRI system has been in continuous operation at the Mayo Clinic since March 2016. To date, over 200 patient studies have been completed, including 96 comparison studies with a clinical 3T whole-body MRI. The increased gradient performance has reliably resulted in consistently improved image quality.

Purpose To investigate the effect of the asymmetric gradient concomitant fields (CF) with zeroth and first-order spatial dependence on fast/turbo spin-echo acquisitions, and to demonstrate the effectiveness of their real-time compensation. Methods After briefly reviewing the CF produced by asymmetric gradients, the effects of the additional zeroth and first-order CFs on these systems are investigated using extended-phase graph simulations. Phantom and in vivo experiments are performed to corroborate the simulation. Experiments are performed before and after the real-time compensations using frequency tracking and gradient pre-emphasis to demonstrate their effectiveness in correcting the additional CFs. The interaction between the CFs and prescan-based correction to compensate for eddy currents is also investigated. Results It is demonstrated that, unlike the second-order CFs on conventional gradients, the additional zeroth/first-order CFs on asymmetric gradients cause substantial signal loss and dark banding in fast spin-echo acquisitions within a typical brain-scan field of view. They can confound the prescan correction for eddy currents and degrade image quality. Performing real-time compensation successfully eliminates the artifacts. Conclusions We demonstrate that the zeroth/first-order CFs specific to asymmetric gradients can cause substantial artifacts, including signal loss and dark bands for brain imaging. These effects can be corrected using real-time compensation. Magn Reson Med, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

Echo planar imaging is widely-used clinical settings, for its speed. EPI acquisitions typically employ complex sampling protocols, and correspondingly require advanced reconstruction plans both to account for non-uniform data spacing and for managing system imperfections such as B0-field inhomogeneity, gradient non-linearity, and eddy currents, as well as subject-induced non-idealities like susceptibility variation. Such non-idealities are typically managed after the k-space to image domain transformation, which generates geometrical inaccuracies and blurring in images. In this work, we develop and investigate a comprehensive model-based iterative reconstruction (MBIR) framework that prospectively accounts for multiple non-idealities in accelerated single-shot EPI. When necessary, we also employ nonlinear regularization to mitigate noise amplification.

Purpose: To derive a noniterative gridding-type reconstruction framework for nonCartesian magnetic resonance imaging(MRI) that prospectively accounts for gradient nonlinearity (GNL)-induced image geometrical distortion during MR image reconstruction, as opposed to the standard, image-domain based GNL correction that is applied after reconstruction; to demonstrate that such framework is able to reduce the image blurring introduced by the conventional GNL correction, while still offering effective correction of GNL-induced geometrical distortion and compatibility with off-resonance correction. Methods: After introducing the nonCartesian MRI signal model that explicitly accounts for the effects of GNL and off-resonance, a noniterative gridding-type reconstruction framework with integrated GNL correction based on the type-III nonuniform fast Fourier transform (NUFFT) is derived. A novel type-III NUFFT implementation is then proposed as a numerically efficient solution to the proposed framework. The incorporation of simultaneous B0 off-resonance correction to the proposed framework is then discussed. Several phantom and in vivo data acquired via various 2D and 3D nonCartesian acquisitions, including 2D Archimedean spiral, 3D shells with integrated radial and spiral, and 3D radial sampling, are used to compare the results of the proposed and the standard GNL correction methods. Results: Various phantom and in vivo data demonstrate that both the proposed and the standard GNL correction methods are able to correct the coarse-scale geometric distortion and blurring induced by GNL and off-resonance. However, the standard GNL correction method also introduces blurring effects to corrected images, causing blurring of resolution inserts in the phantom images and loss of small vessel clarity in the angiography examples. On the other hand, the results after the proposed GNL correction show better depiction of resolution inserts and higher clarity of small vessel. Conclusions: The proposed GNL-integrated nonCartesian reconstruction method can mitigate the resolution loss that occurs during standard image-domain GNL correction, while still providing effective correction of coarse-scale geometric distortion and blurring induced by GNL and off-resonance.

Purpose To describe how integrated gradient nonlinearity (GNL) correction can be used within noniterative partial Fourier (homodyne) and parallel (SENSE and GRAPPA) MR image reconstruction strategies, and demonstrate that performing GNL correction during, rather than after, these routines mitigates the image blurring and resolution loss caused by postreconstruction image domain based GNL correction. Methods Starting from partial Fourier and parallel magnetic resonance imaging signal models that explicitly account for GNL, noniterative image reconstruction strategies for each accelerated acquisition technique are derived under the same core mathematical assumptions as their standard counterparts. A series of phantom and in vivo experiments on retrospectively undersampled data were performed to investigate the spatial resolution benefit of integrated GNL correction over conventional postreconstruction correction. Results Phantom and in vivo results demonstrate that the integrated GNL correction reduces the image blurring introduced by the conventional GNL correction, while still correcting GNL-induced coarse-scale geometrical distortion. Images generated from undersampled data using the proposed integrated GNL strategies offer superior depiction of fine image detail, for example, phantom resolution inserts and anatomical tissue boundaries. Conclusion Noniterative partial Fourier and parallel imaging reconstruction methods with integrated GNL correction reduce the resolution loss that occurs during conventional postreconstruction GNL correction while preserving the computational efficiency of standard reconstruction techniques. Magn Reson Med, 2015. © 2015 Wiley Periodicals, Inc.

To investigate the effect of the asymmetric gradient concomitant fields (CF) with zeroth and first-order spatial dependence on fast/turbo spin-echo acquisitions, and to demonstrate the effectiveness of their real-time compensation.After briefly reviewing the CF produced by asymmetric gradients, the effects of the additional zeroth and first-order CFs on these systems are investigated using extended-phase graph simulations. Phantom and in vivo experiments are performed to corroborate the simulation. Experiments are performed before and after the real-time compensations using frequency tracking and gradient pre-emphasis to demonstrate their effectiveness in correcting the additional CFs. The interaction between the CFs and prescan-based correction to compensate for eddy currents is also investigated.It is demonstrated that, unlike the second-order CFs on conventional gradients, the additional zeroth/first-order CFs on asymmetric gradients cause substantial signal loss and dark banding in fast spin-echo acquisitions within a typical brain-scan field of view. They can confound the prescan correction for eddy currents and degrade image quality. Performing real-time compensation successfully eliminates the artifacts.We demonstrate that the zeroth/first-order CFs specific to asymmetric gradients can cause substantial artifacts, including signal loss and dark bands for brain imaging. These effects can be corrected using real-time compensation. Magn Reson Med 79:1354-1364, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Many cell types undergo a hypoxic response in the presence of low oxygen, which can lead to transcriptional, metabolic, and structural changes within the cell. Many biophysical studies to probe the localization and dynamics of single fluorescently labeled molecules in live cells either require or benefit from low-oxygen conditions. In this study, we examine how low-oxygen conditions alter the mobility of a series of plasma membrane proteins with a range of anchoring motifs in HeLa cells at 37°C. Under high-oxygen conditions, diffusion of all proteins is heterogeneous and confined. When oxygen is reduced with an enzymatic oxygen-scavenging system for ≥15 min, diffusion rates increase by >2-fold, motion becomes unconfined on the timescales and distance scales investigated, and distributions of diffusion coefficients are remarkably consistent with those expected from Brownian motion. More subtle changes in protein mobility are observed in several other laboratory cell lines examined under both high- and low-oxygen conditions. Morphological changes and actin remodeling are observed in HeLa cells placed in a low-oxygen environment for 30 min, but changes are less apparent in the other cell types investigated. This suggests that changes in actin structure are responsible for increased diffusion in hypoxic HeLa cells, although superresolution localization measurements in chemically fixed cells indicate that membrane proteins do not colocalize with F-actin under either experimental condition. These studies emphasize the importance of controls in single-molecule imaging measurements, and indicate that acute response to low oxygen in HeLa cells leads to dramatic changes in plasma membrane structure. It is possible that these changes are either a cause or consequence of phenotypic changes in solid tumor cells associated with increased drug resistance and malignancy.

Diverse molecules induce general anesthesia with potency strongly correlated both with their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma membrane derived vesicles by lowering the critical temperature ($T_c$) for phase separation. Here we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on $T_c$. First we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described `intoxication reversers' raise $T_c$ and counter ethanol's effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that hydrostatic pressure, long known to reverse anesthesia, also raises $T_c$ in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that $\Delta T_c$ predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia., Comment: 20 pages, 6 figures

Giant plasma membrane vesicles (GPMVs) isolated from RBL-2H3 cells appear uniform at physiological temperatures, contain coexisting liquid-ordered and liquid-disordered phases at low temperatures, and experience micron-sized critical fluctuations close to their critical temperature. We observe a broad distribution of critical temperatures in GPMVs isolated from a dish of cells even though individual vesicles have a well-defined critical temperature. Interestingly, we find that densely plated cells yield GPMVs with lower average critical temperatures than GPMVs from less densely plated cells. Since it is known that cellular doubling times are reduced in cells plated at high density due to contact inhibition, we hypothesized that critical temperatures are linked to the cell cycle. To test this hypothesis, we isolated GPMVs from cells at various stages in their cell cycle. We plated cells in low serum media to produce a population of cells with arrested growth, and found low critical temperatures in GPMVs isolated from these cells (∼10°C). Critical temperatures recovered to typical values (∼20°C) after incubating the growth-arrested cells in serum rich media for 24h. Populations of cells are synchronized at S, G2, M, and G1 stages using a double Thymidine block that arrests cells at the border between G1 and S phases. Critical temperatures are elevated in GPMVs from populations of cells in cell cycle phases that immediately precede cell division (G2 and M) compared to the other stages (G1 and S). These results suggest that the magnitude of plasma membrane heterogeneity may be dependent on the cell cycle. We are currently investigating how position in the cell cycle influences the organization of plasma membrane proteins using quantitative super-resolution microscopy with multiple colors. We are also investigating cell cycle position's impact on outcomes of functional processes known to depend on lipid heterogeneity.

Giant plasma membrane vesicles (GPMVs) isolated from RBL-2H3 cells appear uniform at physiological temperatures, contain coexisting liquid-ordered and liquid-disordered phases at low temperatures, and experience micron-sized critical fluctuations close to their critical temperature. Individual vesicles have well-defined critical temperatures yet there is significant vesicle to vesicle variation even when GPMVs are isolated from a plate of seemingly identical cells. In this study, we explore if heterogeneity in critical temperatures arise, at least in part, from cells being at different stages of the cell cycle. Populations of cells were synchronized at S, G2, M, and G1 stages using a double Thymidine block that arrests cells at the border between G1 and S phases. Critical temperatures were elevated in GPMVs isolated from cells in cell cycle phases that precede cell division (G2 and M) compared to other stages (G1 and S). In unsynchronized cells, critical temperatures were found to be inversely proportional to cell density, suggesting that contact inhibition and associated arrest of cell growth results in lower plasma membrane critical temperatures. Lower critical temperatures were also observed when growth was arrested through overnight serum starvation, and elevated critical temperatures were restored 24h after the addition of serum containing medium. Transition temperatures are also lowered in GPMVs prepared from cells undergoing apoptosis through the application of TRAIL, and vesicles contain a more rigid liquid-ordered or gel phase at low temperature. These results are in agreement with past studies that have indicated that plasma membrane composition varies within the cell cycle. Since GPMV critical temperatures are hypothesized to reflect on the magnitude of lipid-mediated heterogeneity in intact cells, these results suggest that membrane heterogeneity is greatest in cells undergoing rapid growth and cell division and is suppressed in cells under low growth conditions.

Giant plasma membrane vesicle (GPMV) isolated from a flask of RBL-2H3 cells appear uniform at physiological temperatures and contain coexisting liquid-ordered and liquid-disordered phases at low temperatures. While a single GPMV transitions between these two states at a well-defined temperature, there is significant vesicle-to-vesicle heterogeneity in a single preparation of cells, and average transition temperatures can vary significantly between preparations. In this study, we explore how GPMV transition temperatures depend on growth conditions, and find that average transition temperatures are negatively correlated with average cell density over 15°C in transition temperature and nearly three orders of magnitude in average surface density. In addition, average transition temperatures are reduced by close to 10°C when GPMVs are isolated from cells starved of serum overnight, and elevated transition temperatures are restored when serum-starved cells are incubated in serum-containing media for 12 h. We also investigated variation in transition temperature of GPMVs isolated from cells synchronized at the G1/S border through a double Thymidine block and find that average transition temperatures are systematically higher in GPMVs produced from G1 or M phase cells than in GPMVs prepared from S or G1 phase cells. Reduced miscibility transition temperatures are also observed in GPMVs prepared from cells treated with TRAIL to induce apoptosis or sphingomyelinase, and in some cases a gel phase is observed at temperatures above the miscibility transition in these vesicles. We conclude that at least some variability in GPMV transition temperature arises from variation in the local density of cells and asynchrony of the cell cycle. It is hypothesized that GPMV transition temperatures are a proxy for the magnitude of lipid-mediated membrane heterogeneity in intact cell plasma membranes at growth temperatures. If so, these results suggest that cells tune their plasma membrane composition in order to control the magnitude of membrane heterogeneity in response to different growth conditions.

Giant plasma membrane vesicles (GPMVs) isolated from RBL-2H3 cells appear uniform at physiological temperatures, contain coexisting liquid-ordered and liquid-disordered phases at low temperatures, and experience micron-sized critical fluctuations close to their critical temperature. We observe a broad distribution of critical temperatures in GPMVs isolated from a dish of seemingly identical cells even though each individual vesicle has a well defined critical temperature. In addition, we observe that the average transition temperature of GPMVs isolated from a population of cells is inversely proportional to the surface density of cells growing in the culture dish. Since it is known that cellular doubling times are reduced in more densely plated cells due to contact inhibition, we hypothesized that critical temperatures are linked to the cell cycle. To test this hypothesis, we isolated GPMVs from populations of cells synchronized using a double Thymidine block that arrests cells at the border between G1 and S phases. After Thymidine is removed from the culture media, cells proceed synchronously through one cell cycle and GPMVs are isolated at time-points corresponding to S, G2, M, and G1 phases. We find that critical temperatures are significantly elevated in the cell cycle phases that immediately preceding cell division (G2 and M) compared to the remainder of the cell cycle phases (G1 and S), consistent with our observations of higher overall transition temperatures in more rapidly dividing cells. We speculate that membrane heterogeneity arising from this critical point may play a role in cytokinesis or other processes vital for cell division. If this hypothesis is correct, it may suggest that biochemical perturbations that lower critical temperatures in GPMVs can be used to inhibit cellular proliferation, possibly providing a novel treatment strategy for some cancers.

Diverse molecules induce general anesthesia with potency strongly correlated with both their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma-membrane-derived vesicles by lowering the critical temperature (Tc) for phase separation. Here, we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on Tc. First, we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol-induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described “intoxication reversers” raise Tc and counter ethanol’s effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that elevated hydrostatic pressure, long known to reverse anesthesia, also raises Tc in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that ΔTc predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.