Automated quality control of ultrasound based on in-air reverberation patterns.

Ultrasound image degradation originates primarily from transducer defects and potentially undermines reliable image interpretation. Systematic quantitative quality control is often neglected due to the limited resources available for this task. We propose a quantitative quality control based on in-air reverberation images. These images serve as an initial indication of image degradation. They are easily generated for any (curvi-)linear transducer independent of the level of expertise of the operator. Automated analysis is presented to extract quality parameters based on the in-air reverberation pattern. Static images acquired by the clinical user are transferred to a server where analysis is performed. The results are available to the sonographer prior to clinical use and transducer status can be remotely monitored with trend analysis over time. The method was evaluated for normal functioning and defect transducers. A pilot study was performed over a period of three weeks to assess reproducibility and practical feasibility. All reverberation images were successfully analysed for different transducer types and vendor-specific image presentation. The proposed quality parameters are sensitive to signal loss and allow differentiation of type and severity of image degradation. The pilot study was well received by the sonographers for the simplicity of the method and the measurements were consistent over time. The proposed automated analysis method of ultrasound quality control can monitor (curvi-)linear transducer status in the entire hospital, overcoming previous limitations for periodic quality control. Implementation of the method can reduce the number of defective transducers routinely used in clinical practice.

Autofluorescence imaging for improved visualization of joint structures during arthroscopic surgery.

BACKGROUND: The purpose of our study is to develop the arthroscopic autofluorescence imaging (AFI) system to improve the visualization during arthroscopic surgery by real-time enhancing the contrast between joint structures with autofluorescence imaging. Its validity was evaluated around the arthroscopic anterior cruciate ligament (ACL) reconstruction, specifically improving the contrast between the femoral insertion site and its background. The feasibility of the AFI system was validated with bovine and human knees. The spectral responses of the femoral insertion site and its surrounding bone and cartilage were measured with a fluorospectrometer. A prototype of the AFI system was developed based on the spectral responses (SR) and test images of the insertion site. The accuracy was validated by evaluating the overlap between manually segmented insertion sites on the white light color images and on the corresponding spectral unmixed autofluorescence images. The final prototype of the AFI system was tested during arthroscopy in cadaveric knees. RESULTS: The results showed that the joint structures have different SRs. Spectral unmixing enabled separation of the SRs and improved the contrast between the joint structures. The agreement between visible light and autofluorescence ligament insertions had a mean Dice coefficient of 0.84 and the mean Dice coefficient of the interobserver variability for visible light imaging was 0.85. CONCLUSIONS: We have shown that the femoral insertion site can be accurately visualized with autofluorescence imaging combined with spectral unmixing. The AFI system demonstrates the feasibility of real-time and subject-specific visualization of the femoral insertion site which can facilitate anatomic ACL reconstruction. In addition, the AFI system can facilitate arthroscopic procedures in other joints and can also be used as a diagnostic tool.

Transmural distribution and connectivity of coronary collaterals within the human heart.

Despite the importance of collateral vessels in human hearts, a detailed analysis of their distribution within the coronary vasculature based on three-dimensional vascular reconstructions is lacking. This study aimed to classify the transmural distribution and connectivity of coronary collaterals in human hearts. One normotrophic human heart and one hypertrophied human heart with fibrosis in the inferior wall from a previous infarction were obtained. After filling the coronary arteries with fluorescent replica material, hearts were frozen and alternately cut and block-face imaged using an imaging cryomicrotome. Transmural distribution, connectivity, and diameter of collaterals were determined. Numerous collateral vessels were found (normotrophic heart: 12.3 collaterals/cm(3); hypertrophied heart: 3.7 collaterals/cm(3)), with 97% and 92%, respectively, of the collaterals located within the perfusion territories (intracoronary collaterals). In the normotrophic heart, intracoronary collaterals {median diameter [interquartile range (IQR)]: 91.4 [73.0-115.7] µm} were most prevalent (74%) within the left anterior descending (LAD) territory. Intercoronary collaterals [median diameter (IQR): 94.3 (79.9-107.4) µm] were almost exclusively (99%) found between the LAD and the left circumflex artery (LCX). In the hypertrophied heart, intracoronary collaterals [median diameter (IQR): 101.1 (84.8-126.0) µm] were located within both the LAD (48%) and LCX (46%) territory. Intercoronary collaterals [median diameter (IQR): 97.8 (89.3-111.2) µm] were most prevalent between the LAD-LCX (68%) and LAD-right coronary artery (28%). This study shows that human hearts have abundant coronary collaterals within all flow territories and layers of the heart. The majority of these collaterals are small intracoronary collaterals, which would have remained undetected by clinical imaging techniques.

Impact of coronary bifurcation morphology on wave propagation.

The branching pattern of the coronary vasculature is a key determinant of its function and plays a crucial role in shaping the pressure and velocity wave forms measured for clinical diagnosis. However, although multiple scaling laws have been proposed to characterize the branching pattern, the implications they have on wave propagation remain unassessed to date. To bridge this gap, we have developed a new theoretical framework by combining the mathematical formulation of scaling laws with the wave propagation theory in the pulsatile flow regime. This framework was then validated in multiple species using high-resolution cryomicrotome images of porcine, canine, and human coronary networks. Results demonstrate that the forward well-matchedness (no reflection for pressure/flow waves traveling from the coronary stem toward the microcirculation) is a salient feature in the coronary vasculature, and this result remains robust under many scenarios of the underlying pulse wave speed distribution assumed in the network. This result also implies a significant damping of the backward traveling waves, especially for smaller vessels (radius, <0.3 mm). Furthermore, the theoretical prediction of increasing area ratios (ratio between the area of the mother and daughter vessels) in more symmetric bifurcations found in the distal circulation was confirmed by experimental measurements. No differences were observed by clustering the vessel segments in terms of transmurality (from epicardium to endocardium) or perfusion territories (left anterior descending, left circumflex, and right coronary artery).

Selective subepicardial localization of monocyte subsets in response to progressive coronary artery constriction.

Following myocardial infarction and atherosclerotic lesion development, monocytes contribute to myocardial protection and repair, while also partaking in myocardial ischemic injury. The balance of proinflammatory and reparative monocyte subsets is crucial in governing these therapeutic and pathological outcomes. Myocardial ischemic damage displays heterogeneity across the myocardium, whereby the subendocardium shows greatest vulnerability to ischemic damage. In this study we examined the transmural distribution of monocyte subsets in response to gradual coronary artery occlusion. CD14(+) monocytes were isolated from peripheral blood of New Zealand White rabbits and divided into two subgroups based on the expression of CD62L. We employed a rabbit model of progressive coronary artery obstruction to induce chronic myocardial ischemia and reinfused fluorescently labeled autologous monocytes. The distribution of fluorescently labeled autologous monocytes was examined with a high-resolution three-dimensional imaging cryomicrotome. The subepicardial layer contained the largest infiltration of both monocyte subgroups, with a significantly greater proportion of CD14(+)CD62L(+) monocytes at the time when the ischemic area was at a maximum. By targeting CD13(+) angiogenic vessels, we confirmed the presence of angiogenesis in epicardial and midmyocardial regions. These myocardial regions demonstrated the highest level of infiltration of both monocyte subsets. Furthermore, CD14(+)CD62L(+) monocytes showed significantly greater migration towards monocyte chemoattractant protein-1, greater adhesive capacity, and higher expression of C-C chemokine receptor type-2 relative to CD14(+)CD62L(-) monocytes. In conclusion, we note selective subepicardial distribution of monocyte subpopulations, with changes in proportion depending on the time after onset of coronary narrowing. Selective homing is supported by divergent migratory properties of each respective monocyte subgroup.

Microsphere skimming in the porcine coronary arteries: Implications for flow quantification.

Particle skimming is a phenomenon where particles suspended in fluid flowing through vessels distribute disproportionately to bulk fluid volume at junctions. Microspheres are considered a gold standard of intra-organ perfusion measurements and are used widely in studies of flow distribution and quantification. It has previously been hypothesised that skimming at arterial junctions is responsible for a systematic over-estimation of myocardial perfusion from microspheres at the subendocardium. Our objective is to integrate coronary arterial structure and microsphere distribution, imaged at high resolution, to test the hypothesis of microsphere skimming in a porcine left coronary arterial (LCA) network. A detailed network was reconstructed from cryomicrotome imaging data and a Poiseuille flow model was used to simulate flow. A statistical approach using Clopper-Pearson confidence intervals was applied to determine the prevalence of skimming at bifurcations in the LCA. Results reveal that microsphere skimming is most prevalent at bifurcations in the larger coronary arteries, namely the epicardial and transmural arteries. Bifurcations at which skimming was identified have significantly more asymmetric branching parameters. This finding suggests that when using thin transmural segments to quantify flow from microspheres, a skimming-related deposition bias may result in underestimation of perfusion in the subepicardium, and overestimation in the subendocardium.

A quantitative high resolution voxel-wise assessment of myocardial blood flow from contrast-enhanced first-pass magnetic resonance perfusion imaging: microsphere validation in a magnetic resonance compatible free beating explanted pig heart model.

AIMS: To assess the feasibility of high-resolution quantitative cardiovascular magnetic resonance (CMR) voxel-wise perfusion imaging using clinical 1.5 and 3 T sequences and to validate it using fluorescently labelled microspheres in combination with a state of the art imaging cryomicrotome in a novel, isolated blood-perfused MR-compatible free beating pig heart model without respiratory motion. METHODS AND RESULTS: MR perfusion imaging was performed in pig hearts at 1.5 (n = 4) and 3 T (n = 4). Images were acquired at physiological flow ('rest'), reduced flow ('ischaemia'), and during adenosine-induced hyperaemia ('stress') in control and coronary occlusion conditions. Fluorescently labelled microspheres and known coronary myocardial blood flow represented the reference standards for quantitative perfusion validation. For the comparison with microspheres, the LV was divided into 48 segments based on a subdivision of the 16 AHA segments into subendocardial, midmyocardial, and subepicardial subsegments. Perfusion quantification of the time-signal intensity curves was performed using a Fermi function deconvolution. High-resolution quantitative voxel-wise perfusion assessment was able to distinguish between occluded and remote myocardium (P < 0.001) and between rest, ischaemia, and stress perfusion conditions at 1.5 T (P < 0.001) and at 3 T (P < 0.001). CMR-MBF estimates correlated well with the microspheres at the AHA segmental level at 1.5 T (r = 0.94, P < 0.001) and at 3 T (r = 0.96, P < 0.001) and at the subendocardial, midmyocardial, and subepicardial level at 1.5 T (r = 0.93, r = 0.9, r = 0.88, P < 0.001, respectively) and at 3 T (r = 0.91, r = 0.95, r = 0.84, P < 0.001, respectively). CONCLUSION: CMR-derived voxel-wise quantitative blood flow assessment is feasible and very accurate compared with microspheres. This technique is suitable for both clinically used field strengths and may provide the tools to assess extent and severity of myocardial ischaemia.

Quantitative assessment of magnetic resonance derived myocardial perfusion measurements using advanced techniques: microsphere validation in an explanted pig heart system.

BACKGROUND: Cardiovascular Magnetic Resonance (CMR) myocardial perfusion imaging has the potential to evolve into a method allowing full quantification of myocardial blood flow (MBF) in clinical routine. Multiple quantification pathways have been proposed. However at present it remains unclear which algorithm is the most accurate. An isolated perfused, magnetic resonance (MR) compatible pig heart model allows very accurate titration of MBF and in combination with high-resolution assessment of fluorescently-labeled microspheres represents a near optimal platform for validation. We sought to investigate which algorithm is most suited to quantify myocardial perfusion by CMR at 1.5 and 3 Tesla using state of the art CMR perfusion techniques and quantification algorithms. METHODS: First-pass perfusion CMR was performed in an MR compatible blood perfused pig heart model. We acquired perfusion images at physiological flow ("rest"), reduced flow ("ischaemia") and during adenosine-induced hyperaemia ("hyperaemia") as well as during coronary occlusion. Perfusion CMR was performed at 1.5 Tesla (n = 4 animals) and at 3 Tesla (n = 4 animals). Fluorescently-labeled microspheres and externally controlled coronary blood flow served as reference standards for comparison of different quantification strategies, namely Fermi function deconvolution (Fermi), autoregressive moving average modelling (ARMA), exponential basis deconvolution (Exponential) and B-spline basis deconvolution (B-spline). RESULTS: All CMR derived MBF estimates significantly correlated with microsphere results. The best correlation was achieved with Fermi function deconvolution both at 1.5 Tesla (r = 0.93, p < 0.001) and at 3 Tesla (r = 0.9, p < 0.001). Fermi correlated significantly better with the microspheres than all other methods at 3 Tesla (p < 0.002). B-spline performed worse than Fermi and Exponential at 1.5 Tesla and showed the weakest correlation to microspheres (r = 0.74, p < 0.001). All other comparisons were not significant. At 3 Tesla exponential deconvolution performed worst (r = 0.49, p < 0.001). CONCLUSIONS: CMR derived quantitative blood flow estimates correlate with true myocardial blood flow in a controlled animal model. Amongst the different techniques, Fermi function deconvolution was the most accurate technique at both field strengths. Perfusion CMR based on Fermi function deconvolution may therefore emerge as a useful clinical tool providing accurate quantitative blood flow assessment.

Detection and quantification methods of monocyte homing in coronary vasculature with an imaging cryomicrotome.

Cellular imaging modalities are important for revealing the behavior and role of monocytes in response to neovascularization progression in coronary artery disease. In this study we aimed to develop methods for high-resolution three-dimensional (3D) imaging and quantification of monocytes relative to the entire coronary artery network using a novel episcopic imaging modality. In a series of ex vivo experiments, human umbilical vein endothelial cells and CD14+ monocytes were labeled with fluorescent live cell tracker probes and infused into the coronary artery network of excised rat hearts by a Langendorff perfusion method. Coronary arteries were subsequently infused with fluorescent vascular cast material and processed with an imaging cryomicrotome, whereby each heart was consecutively cut (5 µm slice thickness) and block face imaged at appropriate excitation and emission wavelengths. The resulting image stacks yielded 3D reconstructions of the vascular network and the location of cells administered. Successful detection and quantification of single cells and cell clusters were achieved relative to the coronary network using customized particle detection software. These methods were then applied to an in vivo rabbit model of chronic myocardial ischemia in which autologous monocytes were isolated from peripheral blood, labeled with a fluorescent live cell tracker probe and re-infused into the host animal. The processed 3D image stacks revealed homing of monocytes to the ischemic myocardial tissue. Monocytes detected in the ischemic tissue were predominantly concentrated in the mid-myocardium. Vessel segmentation identified coronary collateral connections relative to monocyte localization. This study established a novel imaging platform to efficiently determine the localization of monocytes in relation to the coronary microvascular network. These techniques are invaluable for investigating the role of monocyte populations in the progression of coronary neovascularization in animal models of chronic and sub-acute myocardial ischemia.

Innate collateral segments are predominantly present in the subendocardium without preferential connectivity within the left ventricular wall.

Functional collateral vessels often stem from outward remodelling of pre-existing connections between perfusion territories. Knowledge of the distribution and morphology of innate collateral connections may help in identifying myocardial areas with protection against risk for ischaemia. The coronary network of six healthy canine hearts was investigated with an imaging cryomicrotome. Innate collateral connections ranged from 286 to 1015 µm in diameter. Left ventricular collateral density (number per gram of tissue) was about five in the subendocardium vs. 2.5 in the mid-myocardium (P < 0.01) and 1.3 in the epicardium (P < 0.01). Subendocardial collateral connections were oriented parallel to the long axis of the heart. For the major coronary arteries, five times more intracoronary than intercoronary connections were found, while their median diameter and interquartile range were not significantly different, at 96.1 (16.9) vs. 94.7 (18.9) µm. Collateral vessels connecting crowns from sister branches from a stem are denoted intercrown connections and those within crowns intracrown connections. The number of intercrown connections was related to the mean tissue weight of the crowns (y = 0.73x - 0.33, r2 = 0.85, P < 0.0001). This relation was likewise found to describe intercoronary connections. The median collateral diameter and length were independent of the tissue volumes bridged. We conclude that connectivity and morphology of the innate collateral network are distributed with no preference for intra- or intercrown connections, independent of stem diameter, including epicardial arteries. This renders all sites of the myocardium equally protected in case of coronary artery disease. The orientation of subendocardial collateral vessels indicates the longitudinal direction of subendocardial collateral flow.

Myocardial perfusion distribution and coronary arterial pressure and flow signals: clinical relevance in relation to multiscale modeling, a review.

Coronary artery disease, CAD, is associated with both narrowing of the epicardial coronary arteries and microvascular disease, thereby limiting coronary flow and myocardial perfusion. CAD accounts for almost 2 million deaths within the European Union on an annual basis. In this paper, we review the physiological and pathophysiological processes underlying clinical decision making in coronary disease as well as the models for interpretation of the underlying physiological mechanisms. Presently, clinical decision making is based on non-invasive magnetic resonance imaging, MRI, of myocardial perfusion and invasive coronary hemodynamic measurements of coronary pressure and Doppler flow velocity signals obtained during catheterization. Within the euHeart project, several innovations have been developed and applied to improve diagnosis-based understanding of the underlying biophysical processes. Specifically, MRI perfusion data interpretation has been advanced by the gradientogram, a novel graphical representation of the spatiotemporal myocardial perfusion gradient. For hemodynamic data, functional indices of coronary stenosis severity that do not depend on maximal vasodilation are proposed and the Valsalva maneuver for indicating the extravascular resistance component of the coronary circulation has been introduced. Complementary to these advances, model innovation has been directed to the porous elastic model coupled to a one-dimensional model of the epicardial arteries. The importance of model development is related to the integration of information from different modalities, which in isolation often result in conflicting treatment recommendations.

Model-based vasculature extraction from optical fluorescence cryomicrotome images.

The aim of this study was to develop a novel method to reconstruct 3-D coronary vasculature from cryomicrotome images, comprised of two distinct sets of data-fluorescent microsphere beads and coronary vasculature. Fluorescent beads and cast injected into the vasculature were separately imaged with different filter settings to obtain the microsphere and vascular data, respectively. To extract the vascular anatomy, light scattering in the tissue was modelled using a point spread function (PSF). The PSF was parametrized by optical tissue excitation and emission attenuation coefficients, which were estimated by fitting simulated images of microspheres convolved with the PSF model to the experimental microsphere images. These parameters were then applied within a new model-based method for vessel radius estimation. Current state-of-the-art radii estimation methods and the proposed model-based method were applied on vessel phantoms. In this validation study, the full-width half-maximum method of radii estimation, when performed on the raw data without correcting for the optical blurring, resulted in 42.9% error on average for the 170 µm vessel. In comparison, the model-based method resulted in 0.6% error on average for the same phantom. Whole-organ porcine coronary vasculature was automatically reconstructed with the new model-based vascular extraction method.

3D Imaging of vascular networks for biophysical modeling of perfusion distribution within the heart.

One of the main determinants of perfusion distribution within an organ is the structure of its vascular network. Past studies were based on angiography or corrosion casting and lacked quantitative three dimensional, 3D, representation. Based on branching rules and other properties derived from such imaging, 3D vascular tree models were generated which were rather useful for generating and testing hypotheses on perfusion distribution in organs. Progress in advanced computational models for prediction of perfusion distribution has raised the need for more realistic representations of vascular trees with higher resolution. This paper presents an overview of the different methods developed over time for imaging and modeling the structure of vascular networks and perfusion distribution, with a focus on the heart. The strengths and limitations of these different techniques are discussed. Episcopic fluorescent imaging using a cryomicrotome is presently being developed in different laboratories. This technique is discussed in more detail, since it provides high-resolution 3D structural information that is important for the development and validation of biophysical models but also for studying the adaptations of vascular networks to diseases. An added advantage of this method being is the ability to measure local tissue perfusion. Clinically, indices for patient-specific coronary stenosis evaluation derived from vascular networks have been proposed and high-resolution noninvasive methods for perfusion distribution are in development. All these techniques depend on a proper representation of the relevant vascular network structures.

Gradient changes in porcine renal arterial vascular anatomy and blood flow after cryoablation.

PURPOSE: We quantified temporal changes in vascular structure and blood flow after cryosurgery of the porcine kidney in vivo. MATERIALS AND METHODS: We studied 5 groups of 4 kidneys each with a survival time of 20 minutes, 4 hours, 2 days, and 1 and 2 weeks after cryoablation, respectively. Before harvesting the kidneys, fluorescently labeled microspheres were administrated in the descending aorta. After harvest the kidney and its vasculature were casted with fluorescently dyed elastomer, frozen and processed in an imaging cryomicrotome to reveal the 3-dimensional arterial branching structure and microsphere distribution. In regions of interest vessels were segmented by image analysis software and histograms were constructed to reveal the total summed vessel length as a function of diameter. A characteristic diameter of the ablated area was measured. RESULTS: The 20-minute survival group histograms showed a significant shift of the peak to larger diameters (p<0.002), indicating that smaller vessels were destroyed. Microsphere density was decreased to 2% in the ablated region but not in the nonablated border zone, depending on the remaining crater crossing larger vessels. After 2 weeks neither vessels nor microspheres were left in the ablated area, which had shrunk by about 40% in diameter. Study limitations are the lack of histological confirmation and the use of normal rather than cancerous tissue. CONCLUSIONS: Larger vessels remain patent just after ablation and transport blood to the border of the ablation crater but perfusion within the crater is halted instantly. Characteristic crater diameter increases initially but decreases thereafter. Destruction of vessels and tissue is complete 2 weeks after cryoablation.

Disruption of hexokinase II-mitochondrial binding blocks ischemic preconditioning and causes rapid cardiac necrosis.

RATIONALE: Isoforms I and II of the glycolytic enzyme hexokinase (HKI and HKII) are known to associate with mitochondria. It is unknown whether mitochondria-bound hexokinase is mandatory for ischemic preconditioning and normal functioning of the intact, beating heart. OBJECTIVE: We hypothesized that reducing mitochondrial hexokinase would abrogate ischemic preconditioning and disrupt myocardial function. METHODS AND RESULTS: Ex vivo perfused HKII(+/-) hearts exhibited increased cell death after ischemia and reperfusion injury compared with wild-type hearts; however, ischemic preconditioning was unaffected. To investigate acute reductions in mitochondrial HKII levels, wild-type hearts were treated with a TAT control peptide or a TAT-HK peptide that contained the binding motif of HKII to mitochondria, thereby disrupting the mitochondrial HKII association. Mitochondrial hexokinase was determined by HKI and HKII immunogold labeling and electron microscopy analysis. Low-dose (200 nmol/L) TAT-HK treatment significantly decreased mitochondrial HKII levels without affecting baseline cardiac function but dramatically increased ischemia-reperfusion injury and prevented the protective effects of ischemic preconditioning. Treatment for 15 minutes with high-dose (10 µmol/L) TAT-HK resulted in acute mitochondrial depolarization, mitochondrial swelling, profound contractile impairment, and severe cardiac disintegration. The detrimental effects of TAT-HK treatment were mimicked by mitochondrial membrane depolarization after mild mitochondrial uncoupling that did not cause direct mitochondrial permeability transition opening. CONCLUSIONS: Acute low-dose dissociation of HKII from mitochondria in heart prevented ischemic preconditioning, whereas high-dose HKII dissociation caused cessation of cardiac contraction and tissue disruption, likely through an acute mitochondrial membrane depolarization mechanism. The results suggest that the association of HKII with mitochondria is essential for the protective effects of ischemic preconditioning and normal cardiac function through maintenance of mitochondrial potential.

Porcine coronary collateral formation in the absence of a pressure gradient remote of the ischemic border zone.

In the current paradigm on coronary collateral development, it is assumed that these vessels develop consequentially from increased fluid shear stress (FSS) through preexisting collateral arteries. The increased FSS follows from an increase in pressure gradient between the region at risk and well-perfused surroundings. The objective of this study was to test the hypothesis that, in the heart, collateral connections can form in the absence of an increased FFS and consequentially at any depth and region within the ventricular wall. In Yorkshire pigs, gradual left circumflex coronary artery occlusion was obtained over 6 wk by an ameroid constrictor, whereas the control group underwent a sham operation. Hearts were harvested and subsequently processed in an imaging cryomicrotome, resulting in 40-µm voxel resolution three-dimensional reconstructions of the intramural vascular vessels. Dedicated software segmented the intramural vessels and all continuous vascular pathways containing a collateral connection. In the ameroid group, 192 collaterals, 22-1,049 µm in diameter, were detected with 62% within the subendocardium. Sixty percent of collaterals bridged from the left anterior descending artery to left circumflex coronary artery. A novel result is that 25% (n = 48) of smaller-radius collaterals (P = 0.047) connected with both origin and terminus in the nontarget area where perfusion was assumed uncompromised. In the porcine heart, collateral vessels develop not only in ischemic border zones with increased FSS but also away from such border zones where increased FSS is unlikely. The majority of collaterals were located at the subendocardium, corresponding to the region with highest prevalence for ischemia.

Improved detection of fluorescently labeled microspheres and vessel architecture with an imaging cryomicrotome.

Due to spectral overlap, the number of fluorescent labels for imaging cryomicrotome detection was limited to 4. The aim of this study was to increase the separation of fluorescent labels. In the new imaging cryomicrotome, the sample is cut in slices of 40 microm. Six images are taken for each cutting plane. Correction for spectral overlap is based on linear combinations of fluorescent images. Locations of microspheres are determined by using the system point spread function. Five differently colored microspheres were injected in vivo distributed over two major coronaries, the left anterior descending and left circumflex artery. Under absence of collateral flow, microspheres outside of target perfusion territories were not found and the procedure did not generate false positive detection when spectral overlap was relevant. In silico-generated microspheres were used to test the effect of background image, transparency correction, and color separation. The percentage of microspheres undetected was 2.3 +/- 0.8% in the presence and 1.5 +/- 0.4% in the absence of background structures with a density of 900 microspheres per color per cm(3). The image analysis method presented here, allows for an increased number of experimental conditions that can be investigated in studies of regional myocardial perfusion.

Immediate effect of kidney cryoablation on renal arterial structure in a porcine model studied by imaging cryomicrotome.

PURPOSE: Injury to blood microvessels has a crucial role in effective cryoablation for renal masses. We visualized vascular injury induced by a clinically applied cryoablation instrument and established a microvascular diameter threshold for vascular damage. MATERIALS AND METHODS: In 5 anesthetized pigs 1 kidney each was exposed and 3, 17 gauge cryoneedles were inserted in 1 pole. Tissue was exposed to freezing for 2 x 10 minutes with a 10-minute thaw between freezes. After nephrectomy the arteries were injected with fluorescence dyed casting material and the kidney was frozen to -20C and cut in 40 to 60 micron slices in the imaging cryomicrotome, where fluorescent images of the cutting plane of the bulk were obtained. This resulted in a 3-dimensional image of the arterial tree that was segmented, resulting in unbranched vessel segments. Histograms were constructed with the total segment length per diameter bin plotted as function of diameter. RESULTS: The ablated zone was sharply demarcated on fluorescent and normal light images. Mean +/- SD diameter at the peak of the histogram from control areas was 152.4 +/- 5.3 micron. Compared to control areas the peak diameter of ablated areas was shifted to a larger diameter by an average of 25.4 +/- 2.6 micron. CONCLUSIONS: Immediate renal cryoablation injury destroys arteries smaller than 180 micron. Branching structures of larger arteries remain anatomically intact and connected to vascular structures in surrounding tissue.

Organization and collateralization of a subendocardial plexus in end-stage human heart failure.

In the failing myocardium a subendocardial plexus can develop. Detection of the presence or function, however, of such a plexus does not form part of the present diagnostic spectrum for heart failure. This may now change as new methods for high-resolution imaging of myocardial perfusion distribution are being developed. A severely hypertrophic heart was harvested during transplantation and analyzed for morphology of the intramural coronary arterial vasculature. The heart only had one coronary ostium, and the main branches of the coronary artery were cannulated. A fluorescent casting material was infused that was allowed to harden under physiological pressure. The entire heart was frozen and placed in a novel imaging cryomicrotome and sequentially cut in 25-microm slices. High-resolution images of each cutting plane were acquired, allowing a detailed three-dimensional reconstruction of the arterial vasculature. The epicardial layer of the free wall demonstrated a normal vasculature with penetrating branching arteries. The endocardial layer and the septum revealed a highly interconnected vascular plexus with large vessels oriented parallel to the apicobasal axis. An extensive endocardial network with collaterals was detected, forming connections between the main epicardial branches. We conclude that an outward remodeling of transmural vessels did not prevent the generation and growth of subendocardial conduit arteries. The orientation and vascular volume in the plexus provides an opportunity for detection by novel techniques of MRI contrast imaging currently developed. Knowledge of the effect on perfusion studies is required to prevent a misinterpretation of subendocardial perfusion images in heart failure.

Intramural spatial variation of optical tissue properties measured with fluorescence microsphere images of porcine cardiac tissue.

This proceeding studies the optical fluorescence images of a porcine heart filled with microspheres of two colors, carmine and red. A significant difference in the total optical tissue attenuation coefficient was observed between excitation and emission for both carmine (excitation - 13+/-4(1/mm) and emission - 9.4+/-3(1/mm)) and red (excitation -29+/-5(1/mm) and emission - 25+/-5(1/mm)), indicating that optical tissue properties can change significantly for a small change in light wavelength. The above-mentioned large ranges of variation observed in the tissue attenuation coefficient for excitation and emission (both for carmine and red) suggest significant intramural variation of optical properties across the entire organ. Patterns of global spatial variation in optical attenuation properties in tissue across the entire organ were observed. A novel method using fluorescence microsphere images is presented for measurement of the tissue attenuation's intramural variation across an entire organ.