Histological and SEM Assessment of Blood Stasis in Kidney Blood Vessels after Repeated Intra-Arterial Application of Radiographic Contrast Media.

BACKGROUND: After application of iodinated contrast media (CM), a pronounced deterioration of the microcirculation in skin and myocardium was reported. Clinically, the repeated application of CM, especially, led to an increase of the renal resistance index (RRI). With respect to the transiency of the RRI increase, it is reasonable to assume that the deterioration of blood flow could be due to transient blood stasis caused by reversible morphologic cell alterations due to osmotic discrepancies between CM and human blood. Therefore, the hypothesis was investigated whether CM are able to induce in vivo such blood stasis and cell deformations in the renal vasculature of well-hydrated pigs. METHODS: The in vivo study was performed as a prospective randomized examination to compare the effects of two different CM in 16 pigs (German Landrace). Pigs were randomized to receive either Iodixanol (n = 8), or Iopromide (n = 8). Each animal received 10 injections separated by 5-min intervals via the suprarenal aorta at a rate of 10 mL/s according to the usual procedure during a cardiac catheter examination. Finally, the kidneys were explanted and processed for histology (H & E staining and fibrin staining according to Weigert) as well as for scanning electron microscopy (SEM) with regards to morphologic correlates explaining the changes in the microcirculation. RESULTS: In each of the predefined four categories of vascular diameters, blood stasis were found, but clearly more often after application of Iopromide than after application of Iodixanol (p < 0.001). In addition, Iopromide induced more blood stasis in all of the examined kidney regions compared to Iodixanol (p = 0.0001). There were no obstructive events in the middle cortex following the application of Iodixanol. Except for the region around a puncture channel of a placed-in catheter probe, no fibrin was detected in Weigert's fibrin-stained samples, neither around the histologically assessed thrombi nor in vessels with blood stasis. Complementary SEM analyses revealed in a few cases only a slight generation of fibrin and thrombi and deformations, such as echinocyte and "box-like" deformations. CONCLUSIONS: According to previous in vitro studies, pathological erythrocyte deformations, such as echinocyte and box-like formation of erythrocytes, were observed also in vivo. In addition, blood stasis and/or thrombi could be detected in histological samples from explanted kidneys from young pigs after repeated in vivo administration of CM. In only a few cases, mural platelet aggregates within minimal fibrin meshes occurred only after the application of Iopromide.

Substrate-enzyme affinity-based surface modification strategy for endothelial cell-specific binding under shear stress.

Establishing an endothelial cell (EC) monolayer on top of the blood contacting surface of grafts is considered to be a promising approach for creating a hemocompatible surface. Here we utilized the high affinity interactions between the EC plasma membrane expressed enzyme called endothelin converting enzyme-1 (ECE-1) and its corresponding substrate big Endothelin-1 (bigET-1) to engineer an EC-specific binding surface. Since enzymatic cleavage of substrates require physical interaction between the enzyme and its corresponding substrate, it was hypothesized that a surface with chemically immobilized synthetic bigET-1 will preferentially attract ECs over other types of cells found in vascular system such as vascular smooth muscle cells (VSMCs). First, the expression of ECE-1 was significantly higher in ECs, and ECs processed synthetic bigET-1 to produce ET-1 in a cell number-dependent manner. Such interaction between ECs and synthetic bigET-1 was also detectible in blood. Next, vinyl-terminated self-assembled monolayers (SAMs) were established, oxidized and activated on a glass substrate as a model to immobilize synthetic bigET-1 via amide bonds. The ECs cultured on the synthetic bigET-1-immobilized surface processed larger amount of synthetic bigET-1 to produce ET-1 compared to VSMCs (102.9±5.13 vs. 9.75±0.74 pg/ml). The number of ECs bound to the synthetic bigET-1-immobilized surface during 1 h of shearing (5dyne/cm2) was approximately 3-fold higher than that of VSMCs (46.25±12.61 vs. 15.25±3.69 cells/100×HPF). EC-specific binding of synthetic bigET-1-immobilized surface over a surface modified with collagen, a common substance for cell adhesion, was also observed. The present study demonstrated that using the substrate-enzyme affinity (SEA) of cell type-specific enzyme and its corresponding substrate can be an effective method to engineer a surface preferentially binds specific type of cells. This novel strategy might open a new route toward rapid endothelialization under dynamic conditions supporting the long-term patency of cardiovascular implants.

Vascular Endothelial Cell Biology: An Update.

The vascular endothelium, a monolayer of endothelial cells (EC), constitutes the inner cellular lining of arteries, veins and capillaries and therefore is in direct contact with the components and cells of blood. The endothelium is not only a mere barrier between blood and tissues but also an endocrine organ. It actively controls the degree of vascular relaxation and constriction, and the extravasation of solutes, fluid, macromolecules and hormones, as well as that of platelets and blood cells. Through control of vascular tone, EC regulate the regional blood flow. They also direct inflammatory cells to foreign materials, areas in need of repair or defense against infections. In addition, EC are important in controlling blood fluidity, platelet adhesion and aggregation, leukocyte activation, adhesion, and transmigration. They also tightly keep the balance between coagulation and fibrinolysis and play a major role in the regulation of immune responses, inflammation and angiogenesis. To fulfill these different tasks, EC are heterogeneous and perform distinctly in the various organs and along the vascular tree. Important morphological, physiological and phenotypic differences between EC in the different parts of the arterial tree as well as between arteries and veins optimally support their specified functions in these vascular areas. This review updates the current knowledge about the morphology and function of endothelial cells, particularly their differences in different localizations around the body paying attention specifically to their different responses to physical, biochemical and environmental stimuli considering the different origins of the EC.

Effect of lipopolysaccharide on the adherence of human umbilical vein endothelial cells (HUVEC) on a natural substrate.

 Polymers are often contaminated with lipopolysaccharides also known as endotoxins. Even small amounts of endotoxins can have strong effects on endothelial cell function so that the endothelialisation of cardiovascular implants might be hampered. An open question is how endothelial cells seeded on a body foreign substrate respond to shear load after adding Lipid A (LPA), the domain, which is responsible for much of the toxicity of gram-negative bacteria, and whether morphological changes of endothelial cells occur.LPA supplementation to the culture medium in increasing concentrations (5, 25 and 50µg/ml) resulted in progressive reductions of the density of adherent HUVEC after shear load (p < 0.001). 48% of the HUVEC in control cultures (0µg/ml LPA) were still adherent after 2 hours of shearing at 6 dyne/cm2, while 80 minutes after addition of 50µg/ml LPA, 88% of the HUVEC had already detached from the substrate and after 100 minutes no more HUVEC were attached.The results demonstrate that endotoxins are of extreme importance for the behavior of HUVEC and that in vivo pathologies can be increasingly simulated in vitro.

Shear resistance of endothelial cells in a pathological environment.

BACKGROUND: Endothelial cells (EC) in vivo are strongly influenced by changes of the milieu exterieur. Under pathological conditions EC can become activated e.g. in hypoxic areas or during sepsis. In general, the endothelialization of implant materials is evaluated in vitro under physiological conditions. Though, in patients who receive implant materials pathological conditions are often present. An open question is therefore, how ECs seeded on a body foreign substrate behave in a pathologic microenvironment. In this in vitro study a microenvironment was created mimicking the conditions present in septic patients. To simulate this situation in vitro, serum of patients with septic shock was added to the culture medium of EC cultured on glass. The samples were sheared in a cone-plate rheometer (shear rate of 6 dyn/cm2) with subsequent analysis of the morphology, the microfilament organization and the shear resistance and compared to control cultures of EC without shock serum supplementation. Aim of the study was to investigate whether this in vitro model provides information about the functionality of an EC monolayer on a body foreign surface under pathological conditions. RESULTS: Septic conditions induced severe changes of the morphology of the adherent cells: there was a strong induction of stress fibers. In addition, lots of cells or cell groups were detached visible as denuded areas in the EC monolayer. After shear stress exposure only 28.7% of EC seeded in cell medium supplemented with serum of septic patients remained adherent (control cells: 96.8%). CONCLUSION: The study demonstrates that the microenvironment is of extreme importance for the behavior of EC and that in vivo pathologies can be simulated in vitro. This opens the possibility to evaluate new implant materials under physiological but more important also under certain pathological conditions - simulating the implant size and the disease of the host.

Percutaneous left atrial appendage closure with a novel self-modelizing device: a pre-clinical feasibility study.

The aim of the study is to evaluate the feasibility and safety of a new left atrial appendage (LAA) occluder. Twelve pigs were included. In 2 pigs the implantation process failed due to pericardial tamponade in 1 pig and device embolization in the other pig. The placement of the devices was controlled via TEE and fluoroscopy. After 6 weeks of implantation the hearts were explanted. The devices were found to be easy to deploy and showed a very good adaptation to the LAA tissue. Eight out of 10 pigs had full closure of the LAA directly after implantation. After six weeks, due to the self-modelizing properties of the device, all pigs had a full closure of the LAA. The macroscopic evaluation of the explanted hearts showed that all devices were securely integrated in LAA tissues. There was one case of mild pericarditis but no macroscopic signs of inflammation on the device surrounding endocardium. The explantation revealed that device loops had penetrated the LAA tissue in three pigs. However, no signs of bleeding, pericardial effusion, or other damage to the LAA wall could be detected and the pigs were in good condition with normal weight gain and no clinical symptoms. The Occlutech® LAA occluder achieved complete closure of the LAA in all pigs, and remained in the LAA, with benign healing and no evidence of new thrombus or damage to surrounding structures. Moreover, the uncompromised survival of all implanted pigs demonstrates the feasibility and safety of the device.

Effects of radiographic contrast media on the micromorphology of the junctional complex of erythrocytes visualized by immunocytology.

Effects of radiographic contrast media (RCM) application were demonstrated in vitro and in vivo where the injection of RCM into the A. axillaris of patients with coronary artery disease was followed by a significant and RCM-dependent decrease of erythrocyte velocity in downstream skin capillaries. Another study in pigs revealed that the deceleration of erythrocytes coincided with a significant reduction of the oxygen partial pressure in the myocardium--supplied by the left coronary artery--after the administration of RCM into this artery. Further reports showed RCM dependent alterations of erythrocytes like echinocyte formation and exocytosis, sequestration of actin or band 3 and the buckling of endothelial cells coinciding with a formation of interendothelial fenestrations leading to areas devoid of endothelial cells. Key to morphological alterations of erythrocytes is the membrane cytoskeleton, which is linked to the band 3 in the erythrocyte membrane via the junctional complex. Fundamental observations regarding the cell biological and biochemical aspects of the structure and function of the cell membrane and the membrane cytoskeleton of erythrocytes have been reported. This review focuses on recent results gained, e.g., by advanced confocal laser scanning microscopy of different double-stained structural elements of the erythrocyte membrane cytoskeleton.

Effect of radiographic contrast media on the spectrin/band3-network of the membrane skeleton of erythrocytes.

The membrane of red blood cells consists of a phospholipid bilayer with embedded membrane proteins and is associated on the cytoplasmatic side with a network of proteins, the membrane skeleton. Band3 has an important role as centre of the functional complexes e.g. gas exchange complex and as element of attachment for the membrane skeleton maintaining membrane stability and flexibility. Up to now it is unclear if band3 is involved in the morphology change of red blood cells after contact with radiographic contrast media. The study revealed for the first time that Iopromide induced markedly more severe alterations of the membrane skeleton compared to Iodixanol whose effects were similar to erythrocytes suspended in autologous plasma. A remarkable clustering of band3 was found associated with an accumulation of band3 in spicules and also a sequestration of band3 to the extracellular space. This was evidently accompanied by a gross reduction of functional band3 complexes combined with a dissociation of spectrin from band3 leading to a loss of homogeneity of the spectrin network. It could be demonstrated for the first time that RCM not only induced echinocyte formation but also exocytosis of particles at least coated with band3.

Influence of spray-dried hydroxyapatite-5-fluorouracil granules on cell lines derived from tissues of mesenchymal origin.

In our previous work we described the preparation and characterization of spray dried hydroxyapatite micro granules loaded with 5-fluorouracil (5-FU). These loaded particles are used as a model drug delivery system (DDS). In this study we examined the in vitro response of two cell lines derived from different tissues to 5-FU loaded granules (LG). Both cell lines, either L929 cells of a mouse fibroblast lineage or cells originating from a rat osteosarcoma (ROS 17/2.8) showed a dose dependent decrease in cell proliferation in response to 5-FU-, either dissolved in the culture medium or loaded onto particles. The response of the two cell lines to loaded and nonloaded particles was different. The effect of LG and of a corresponding concentration of free 5-FU was practically the same for the ROS 17/2.8 cells indicating that ROS 17/2.8 cells were not affected by the carrier material. In contrast, L929 cells showed a slight decrease in cell proliferation also in the presence of granules not loaded with 5-FU. This is thought to be attributed to the inhibition of mitogenesis by phosphocitrates, already demonstrated in fibroblasts. In summary, we found that the loaded 5-FU kept its effectivity after the spray drying process and that the response towards the granules varied with cell type. This is the first step towards a tissue specific DDS.

Influence of various radiographic contrast media on the buckling of endothelial cells.

The intra-arterial application of radiographic contrast media (RCM) can induce decreases of blood flow velocity in downstream capillaries as well as a decrease in the tissue oxygen tension. It is unclear whether changes in endothelial cell morphology contribute to the observed microcirculatory disorders. Four RCMs (Iodixanol320, Iohexol350, Iopromide370, and Imeron350) were added to the culture medium of human umbilical venous endothelial cells (HUVEC) and used for short-term incubation studies of these cells. Addition of Iohexol (p=0.6377) and Iodixanol (p=0.6309) did not affect the HUVEC height 1.5 min after incubation in the modified cell culture media supplemented with 30% v/v of the respective RCM. Strong buckling and increased endothelial height appeared after incubation in Iopromide-supplemented medium (the cell height increased by 95% compared to cells incubated under control conditions; p=0.0065). Addition of Iomeprol-supplemented medium caused an increase by 61.6% compared to cells incubated under control conditions; p=0.0051. After 5 min of incubation in any of the RCM-supplemented media, there was no difference in HUVEC height in comparison to incubation in control standard culture media (each p value>0.05). The tremendous buckling caused by Iopromide and Iomeprol, coinciding with an echinocyte formation of erythrocytes might be the reason why a bolus injection of Iopromide in vivo into the left coronary artery was followed by a 50% decrease of oxygen partial pressure in the supplied tissue.

Observations on the microvasculature of bone defects filled with biodegradable nanoparticulate hydroxyapatite.

The microvascularization of metaphyseal bone defects filled with nanoparticulate, biodegradable hydroxyapatite biomaterial with and without platelet factors enrichment was investigated in a minipig model. Results from morphological analysis and PECAM-1 immunohistochemistry showed the formation of new blood vessels into the bone defects by sprouting and intussusception of pre-existing ones. However, no significant differences were observed in the microvascularization of the different biomaterials applied (pure versus platelet factors-enriched hydroxyapatite), concerning the number of vessels and their morphological structure at day 20 after operation. The appearance of VEGFR-2 positive endothelial progenitor cells in the connective tissue between hydroxyapatite particles was also found to be independent from platelet factors enrichment of the hydroxyapatite bone substitute. In both groups formation of lymphatic vessels was detected with a podoplanin antibody. No differences were noted between HA/PLF- and HA/PLF+ implants with respect to the podoplanin expression level, the staining pattern or number of lymphatic vessels. In conclusion, the present study demonstrates different mechanisms of blood and lymphatic vessel formation in hydroxyapatite implants in minipigs.

Effects of platelet factors on biodegradation and osteogenesis in metaphyseal defects filled with nanoparticular hydroxyapatite--an experimental study in minipigs.

There are no studies on the cellular activity in the early phase of biodegradation and bone healing of bone substitutes loaded with platelet factors (PLF). The purpose of this study was to evaluate the cellular effects of PLF in combination with nanoparticulate hydroxyapatite (HA) on the biodegradation and bone formation after 20 days. Autogenous PLFs were obtained by centrifugation of miniature pig blood samples and subsequent degranulation of platelets by calcium and thrombin. A cylindrical bone defect with a diameter of 8.9 mm was created in the distal femoral condyle of 20 miniature pigs. Four of the defects were left empty, 8 were filled with HA with loading and 8 with HA loaded with PLF. The distal femur was harvested after 20 days and TRAP-staining, cathepsin-K and CD44 staining and scanning electron microscopy were performed for cellular assessment of biodegradation was done. Histomorphometry of new bone formation and of biodegradation of HA material was performed. PLF loading of HA led to statistically significant more TRAP-positive cells with enhanced biodegradation of the nanoparticulate HA but no statistically enhanced new bone formation compared to unloaded HA. Furthermore, there was a higher number of CD44 and cathepsin-K positive cells by PLF-loading. In summary, PLF led to stimulation of the cellular process of the biodegradation of HA.

The influence of a multifunctional, polymeric biomaterial on the concentration of acute phase proteins in an animal model.

The concentrations of the acute phase proteins alpha1-Acid Glycoprotein (AAG) and haptoglobin were determined in Sprague-Dawley-rats after implantation of a novel biodegradable multifunctional polymeric biomaterial for the reconstruction of a gastric wall defect (polymer group; n=42). For comparison, the concentrations of AAG and haptoglobin were measured as well after primary wound closure of the gastric wall defect without biomaterial implantation (control group; n=21) and in rats without any surgical procedure (baseline group; n=21). The implantation periods were 1 week, 4 weeks and 6 months. The concentrations of AAG and haptoglobin were measured by an ELISA assay. Gastrointestinal complications like fistula, perforation or peritonitis did not occur in any of the animals. No statistically significant differences in the concentrations of AAG and haptoglobin were detected between the polymer and the control group. An adequate mechanical stability of the polymeric biomaterial was detectable under the extreme pathophysiological conditions of the stomach milieu. In further examinations the correlation between the intraperitoneal cytokine levels of the animals and the following systemic inflammatory markers should be analysed. Further investigations are needed to analyse the mechanisms of the tissue integration of a biomaterial as well as the process of the tissue remodeling and the influence of the immune system on these mechanisms. The knowledge of these processes is necessary to adapt the multifunctional biomaterial and prepare it thus for the use and implantation in different body locations and to develop novel therapeutical options in medicine.

[First results of the investigation of the stability and tissue integration of a degradable, elastomeric copolymer in an animal model]. / Erste Ergebnisse zur Untersuchung der Stabilität und Gewebeintegration eines abbaubaren, elastischen Copolymers im Tiermodell.

The stability and tight integration into adjacent tissue of a novel, degradable, elastic copolymer were examined in an animal model. The biomaterial was used for the reconstruction of a gastric wall defect in Sprague-Dawley rats (n=42) to test the polymeric material under the extreme chemical, enzymatical and mechanical conditions of the stomach. In the control group (n=21) the same defect of the gastric wall was primarily closed without biomaterial implantation. In the baseline group (n=21) the animals were kept under standard conditions without any surgical procedure. The implantation periods were 1 week, 4 weeks and 6 months. The animals' weight was determined preoperatively and before explantation. After explantation, air was pumped into the stomach and the pressure was measured by using a pressure-gauge in order to test whether the surgically produced union of the stomach wall and the polymer patch was gas-tight. After 1 week of implantation time a statistically significant increase of the body weight of the animals was found only in the baseline group. Four weeks and 6 months after the abdominal surgical procedure, a statistically significant increase of the animals' weight was found in the implantation group, the control and the baseline group. Gastrointestinal complications like fistula, perforation or peritonitis did not occur in any of the animals. The measurement of the stomach pressure after maximal gas insufflation did not show significant differences between the implantation group, the control and the baseline group in any of the time periods investigated. Despite very high strains of the gastric wall, no gas leakage was detected. There was a tight connection between the polymer and the adjacent stomach wall in all animals investigated. An adequate mechanical stability of the biomaterial was detectable under the extreme pathophysiological conditions of the stomach milieu. A fast and unfavourable degradation of the degradable polymer was not found in any of the animals. Further investigations are needed to analyse the mechanisms of the tissue integration of the biomaterial as well as the degradation kinetic of the polymer and the process of the tissue remodeling. The knowledge of these processes is necessary to adapt the novel biomaterial and thus prepare it for the use and implantation in different body locations and to develop novel therapeutical options in medicine.

Plants grow better if seeds see green.

We report on the response of dry plant seeds to their irradiation with intense green light applied at biostimulatory doses. Red and near-infrared light delivered by lasers or arrays of light emitting diodes applied at such doses have been shown previously by us to have effects on mammalian cells. Effects include cell proliferation and elevation of cell vitality, and have practical applications in various biomedical fields. Growth processes induced by photoreceptor stimulation (phytochromes and cryptochromes) in plant seeds with green light were described so far only for imbibed seeds. In this paper, we show that irradiation of dry cress, radish and carrot seeds with intense green light (laser or arrays of light emitting diodes), applied at biostimulatory doses, resulted in a significant increase in biomass--14, 26, and 71 days after seeding, respectively. In the case of radish and carrot, the irradiation led to important changes in the root-to-foliage surface ratio. Seeds with a potential to grant growth acceleration could be of special interest in agricultural applications, and could even compensate for shorter growth seasons caused by climate change.

Biocompatibility testing of novel multifunctional polymeric biomaterials for tissue engineering applications in head and neck surgery: an overview.

Biomaterial research and tissue engineering are rapidly growing scientific fields that need an interdisciplinary approach where clinicians should be included from the onset. Biocompatibility testing in vitro and in vivo comprise the agarose-overlay test, the MTT test, direct cell seeding tests and the chorioallantoic membrane test for angiogenic effects, among others. Molecular biology techniques such as real-time polymerase chain reaction and microarray technology facilitate the investigation of tissue integration into biomaterials on a cellular and molecular level. The physicochemical characterization of biomaterials is conducted using such methods as X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Excellent biocompatibility and biofunctionality were demonstrated for a series of recently developed multifunctional biodegradable, polymeric biomaterials both in vitro and in vivo. Novel, multifunctional polymeric biomaterials offer a highly specific adjustment to the physiological, anatomical and surgical requirements and can thereby facilitate new therapeutic options in head and neck surgery.

Magnetic resonance-guided large-core breast biopsy inside a 1.5-T magnetic resonance scanner using an automatic system: in vitro experiments and preliminary clinical experience in four patients.

RATIONALE AND OBJECTIVE: The aim of this study was to investigate the feasibility and the precision of magnetic resonance (MR)-guided large-core breast biopsies (LCBB) by using the second prototype of an automatic system (ROBITOM II), which is used to localize lesions while operating at the isocenter of a 1.5-T whole-body scanner. METHODS AND MATERIALS: In comparison to the first prototype, ROBITOM II is equipped with a dedicated double breast coil and a high-speed trocar setting unit. In vitro experiments (n = 25) with grapefruit phantoms, which contained multiple vitamin E capsules (12 x 7 mm in size) as artificial lesions, were performed. Four patients with MR-detectable breast lesions underwent biopsy. A trocar was positioned in front of the lesion and inserted into the breast. Specimens were harvested with a coaxial technique by using a 14-G core needle biopsy gun. RESULTS: In all 25 in vitro experiments, capsule material was detected in the specimen cylinder. In 4 patients, the coaxial needle was detected exactly at the expected position. Between 8 and 16 tissue cylinders were harvested. Histologic evaluation resulted in 1 invasive ductal carcinoma and 1 papilloma, which were confirmed after open surgery. One patient who had a proven breast cancer was biopsied for exclusion of multifocal disease. She showed fibrocystic changes, whereas open surgery revealed 3 small areas of ductal carcinoma in situ (DCIS). Another patient showed fibroadenoma after biopsy. This patient is in the follow-up period, which has lasted between 3 and 4 months up until now. CONCLUSIONS: In this pilot patient study, the feasibility of manipulator-assisted large-core breast biopsy inside a 1.5-T whole-body scanner was demonstrated by using ROBITOM II. The precision of the device was confirmed with in vitro experiments. Although these findings are preliminary and the follow-up period is rather short, they nevertheless represent a successful proof-of-principle of LCBB with ROBITOM II.

Interaction between a perfluorocarbon emulsion and radiographic contrast media.

This study evaluated specially designed perfluorocarbon (PFC) emulsions as blood substitutes in case of induced ischemia of the left heart ventricle in healthy farm pigs. Two hundred ml of perfluorocarbon emulsion were infused while 200 ml of blood were simultaneously drawn. Radiographic contrast media were given to aid placement of balloon catheters in the left coronary artery. Histopathological analysis showed that right heart failure caused the deaths of both pigs. Particles (up to>3 micro) of foreign body materials obstructed capillaries of all organs analyzed (heart, lung, liver, kidneys and spleen). Laboratory investigation showed severe interference between the PFC emulsion and radiographic contrast media, resulting in the deterioration of the PFC emulsion. The strongest interference occurred when PFC emulsion and Accupaque interacted; particle size started at an initial 311 nm and went up to >3 micro within seconds. Great care must be taken when PFC emulsions are used in combination with x-ray contrast media. None of the described radiographic contrast media should be used within 48 hours prior to the use of this PFC emulsion. Also, the use of these contrast media should be avoided for a certain period of time after using PFC emulsion. The mechanisms of elimination of PFC emulsions from the circulation are not completely understood and has yet to be evaluated.

Near-field optical analysis of living cells in vitro.

Near-field optical analysis (NOA) provides morphological nanoscale mappings of living cells in liquid cell culture media and nondestructive insight into cell functionality. Here we show for the first time the performance of NOA in imaging living cells. Unlabeled human endothelial cells attached to polished titanium disks were analyzed with hydrophobically coated optical biosensors mounted to a near-field scanning optical microscope (NSOM). Biosensors and titanium substrates could be simply implemented in standard NSOM and high-throughput NOA.