Striving for atomic resolution in biomolecular topography: The scanning force microscope (SFM)

The invention in 1986 of scanning force microscopy (SFM) provided a new and powerful tool for the investigation of biological structures. SFM yields a three-dimensional view at nanometer resolution of the surface topography associated with biological objects. The potential for imaging either macromolecules or biomolecules and cells under native (physiological) conditions is currently being exploited to obtain functional information at the molecular level. In addition, the forces involved in individual bimolecular interactions are being assessed under static and dynamic conditions. In this report we focus on the imaging capability of the SFM. The rather broad spectrum of applications represented is intended to orient the prospective user of biological SFM.     

Changes in the phospholipid composition of the arterial cell can result in severe atherosclerotic lesions

The oxysterol concentration in the plasma and the phospholipid composition of vascular tissue obtained by coronary artery bypass grafting (CABG) were compared with plasma and vascular tissue from age and sex matched controls. The plasma from CABG patients had a higher concentration of oxysterols than was present in the controls. Human endothelial cells were cultured for 72 hours in a medium containing plasma obtained from CABG patients, from controls or from the same controls to which 5 oxysterols were added to make the total oxysterol level equivalent to that in the CABG plasma and then pulsed with calcium (45Ca(2+)) for one hr. A significantly higher influx of 45Ca(2+) was noted in the endothelial cells cultured in the plasma obtained from CABG patients and from the controls with 5 added oxysterols, but not in those cultured without added oxysterols indicating that oxysterols increased calcium influx into endothelial cells. A phospholipid analysis indicated that the arterial tissue from CABG patients had 48.2% sphingomyelin in its phospholipid fraction compared to 10% in arterial tissue from umbilical cords. The saphenous vein obtained during CABG surgery from the same patient had only 24% sphingomyelin in its phospholipid fraction and unlike the coronary arteries had no atherosclerotic lesions. The higher level of oxysterol in the plasma of patients suffering from severe atherosclerosis could increase the concentration of sphingomyelin in the arterial cell membrane and thereby increase calcium influx required for producing the calcific type VII lesions in the coronary arteries.     

Scanning force microscopy in the applied biological sciences

Fifteen years after its invention, the scanning force microscope (SFM) is rooted deep in the biological sciences. Here we discuss the use of SFM in biotechnology and biomedical research. The spectrum of applications reviewed includes imaging, force spectroscopy and mapping, as well as sensor applications. It is our hope that this review will be useful for researchers considering the use of SFM in their studies but are uncertain about its scope of capabilities. For the benefit of readers unfamiliar with SFM technology, the fundamentals of SFM imaging and force measurement are also briefly introduced.     

Quantitative 3D fluorescence technique for the analysis of en face preparations of arterial walls using quantum dot nanocrystals and two-photon excitation laser scanning microscopy

Traditional imaging with one-photon confocal microscopy and organic fluorophores poses several challenges for the visualization of vascular tissue, including autofluorescence, fluorophore crosstalk, and photobleaching. We studied human coronary arteries (HCAs) and mouse aortas with a modified immunohistochemical (IHC) "en face" method using quantum dot (Qdot) bioconjugates and two-photon excitation laser scanning microscopy (TPELSM). We demonstrated the feasibility of multilabeling intimal structures by exciting multicolored Qdots with only one laser wavelength (750 nm). Detailed cell structures, such as the granular appearance of von Willebrand factor (VWF) and the subcellular distribution of endothelial nitric oxide synthase, were visualized using green dots (525 nm), even when the emission maximum of these Qdots overlapped that of tissue autofluorescence (510-520 nm). In addition, sensitive fluorescence quantification of vascular cell adhesion molecule 1 expression at areas of varying hemodynamics (intercostal branches vs. nonbranching areas) was performed in normal C57Bl/6 mice. Finally, we took advantage of the photostability of Qdots and the inherent three-dimensional (3D) resolution of TPELSM to obtain large z-stack series without photobleaching. This innovative en face method allowed simple multicolor profiling, highly sensitive fluorescence quantitation, and 3D visualization of the vascular endothelium with excellent spatial resolution. This is a promising technique to define the spatial and temporal interactions of endothelial inflammatory markers and quantify the effects of different interventions on the endothelium.     

Cell-surface receptors and proteins on platelet membranes imaged by scanning force microscopy using immunogold contrast enhancement.

High resolution scanning force microscope (SFM) images of fibrinogen-exposed platelet membranes are presented. Using ultrasharp carbon tips, we are able to obtain submolecular scale resolution of membrane surface features. Corroboration of SFM results is achieved using low voltage, high resolution scanning electron microscopy (LVHRSEM) to image the same protein molecule that is seen in the SFM. We obtain accurate height dimensions by SFM complemented by accurate lateral dimensions obtained by LVHRSEM. The use of 14- and 5-nm gold labels to identify specific membrane-bound biomolecules and to provide contrast enhancement with the SFM is explored as a useful adjunct to observation of unlabeled material. It is shown that the labels are useful for locating specific protein molecules on platelet membrane surfaces and for assessing the distribution of these molecules using the SFM. Fourteen nm labels are shown to be visible over the membrane corrugation, whereas 5-nm labels appear difficult to resolve using the present SFM instrumental configuration. When using the 5-nm labels, collateral use of LVHRSEM allows one to examine SFM images at submolecular resolution and associate function with the structures imaged after the SFM experiment is completed.     

Comparative study of cellular and extracellular matrix composition of native and tissue engineered heart valves.

Tissue engineering of heart valves utilizes biodegradable or metabolizable scaffolds for remodeling by seeded autologous cells. The aim of this study was to determine and compare extracellular matrix (ECM) formations, cellular phenotypes and cell location of native and tissue engineered (TE) valve leaflets. Ovine carotid arteries, ovine and porcine hearts were obtained from slaughterhouses. Cells were isolated from carotid arteries and dissected ovine, porcine and TE leaflets. TE constructs were fabricated from decellularized porcine pulmonary valves, seeded ovine arterial cells and subsequent 16 days dynamic in vitro culture using a pulsatile bioreactor. Native and TE valves were studied by histology (hematoxylin-eosin, resorcin-fuchsin, Movat pentachrome), NIR femtosecond multiphoton laser scanning microscopy and scanning electron microscopy (SEM). Cells of native and TE tissues were identified and localized by immunohistochemistry. Arterial, valvular and re-isolated TE-construct cells were processed for immunocytochemistry and Western blotting. ECM analysis and SEM revealed characteristical and comparable structures in native and TE leaflets. Most cells in native leaflets stained strongly positive for vimentin. Cells positive to alpha-smooth muscle actin (alpha-SMA), myosin and calponin were only found at the ventricular (inflow) side of ovine aortic and porcine pulmonary valve leaflets. Cells from TE constructs had a strong expression of vimentin, alpha-SMA, myosin, calponin and h-caldesmon throughout the entire leaflet. Comparable ECM formation and endothelial cell lining of native and TE leaflets could be demonstrated. However, immunostaining revealed significant differences between valvular cell phenotypes of native and TE leaflets. These results may be essential for further cardiovascular tissue engineering efforts.     

Time-Lapse Imaging of Conformational Changes in Supercoiled DNA by Scanning Force Microscopy

Most of the scanning force microscopy (SFM) images of supercoiled DNA on untreated mica thus far reported have not shown tight plectonemic structure seen by electron microscopy, but instead less coiled molecules and sometimes a partly "condensed" state with intimate chain-chain interactions. By observing time-lapse images of conformational changes of DNA induced by decreasing ionic strength of imaging buffer in solution SFM, we could show that the process of water rinsing, an indispensable step for preparation of dried samples, may be responsible for some of the conformational anomalies in the images previously reported. We have studied several protocols to observe supercoiled DNA molecules by SFM and discuss the merits and the demerits. Images obtained following uranyl acetate treatment may be ideal for the detection of DNA damage, as the supercoiled and nicked forms are easily distinguishable.     

Peritruncal Coronary Endothelial Cells Contribute to Proximal Coronary Artery Stems and Their Aortic Orifices in the Mouse Heart

Avian embryo experiments proved an ingrowth model for the coronary artery connections with the aorta. However, whether a similar mechanism applies to the mammalian heart still remains unclear. Here we analyzed how the main coronary arteries and their orifices form during murine heart development. Apelin (Apln) is expressed in coronary vascular endothelial cells including peritruncal endothelial cells. By immunostaining, however, we did not find Apln expression in endothelial cells of the aorta during the period of coronary vessel development (E10.5 to E15.5). As a result of this unique expression difference, Apln^CreERT2/+ genetically labels nascent coronary vessels forming on the heart, but not the aorta endothelium when pulse activated by tamoxifen injection at E10.5. This allowed us to define the temporal contribution of these distinct endothelial cell populations to formation of the murine coronary artery orifice. We found that the peritruncal endothelial cells were recruited to form the coronary artery orifices. These cells penetrate the wall of aorta and take up residence in the aortic sinus of valsalva. In conclusion, main coronary arteries and their orifices form through the recruitment and vascular remodeling of peritruncal endothelial cells in mammalian heart.     

Endothelial cells in culture produce a vasoconstrictor substance

We report that cultured vascular endothelial cells release into the culture medium a vasoconstrictor peptide, a substance we call an endothelium-derived constricting factor (EDCF). Conditioned medium from cultured bovine aortic and pulmonary artery endothelial cells caused sustained, dose-dependent isometric constriction of vascular rings isolated from bovine coronary and pulmonary arteries and rat and guinea pig pulmonary arteries and aortas. The medium also caused vasoconstriction when infused into isolated, perfused rabbit hearts and rat kidneys. Conditioned medium from bovine aortic intimal explants also contained constrictor activity, whereas medium from denuded intimal explants, cultured microvascular endothelial cells, vascular smooth muscle cells, or lung fibroblasts did not. Constrictor activity increased progressively in the culture medium over 2-12 h of incubation. Thrombin stimulated the release of constrictor activity; hypoxia, anoxia and meclofenamate had no effect and the calcium ionophore A23187 inhibited EDCF release. The EDCF caused a characteristic slow-onset and sustained constriction of the vascular rings that relaxed slowly over 60-90 min following removal. The constriction was not affected by inhibitors of arachidonic acid metabolism or by antagonists of serotonergic, histaminergic, alpha-adrenergic, opioid, leukotriene, angiotensin II, or substance P receptors; constriction was reversed partly by verapamil and acetylcholine and completely by nitroprusside and isoproterenol. EDCF was heat stable, not extractable into organic solvents, and completely destroyed by trypsin and neutral protease. Cycloheximide blocked the production of EDCF. These properties and the results of polyacrylamide gel filtration experiments suggested that EDCF was a peptide with a molecular weight of 3,000 daltons. These findings show that endothelial cells in culture produce a vasoconstrictor substance and support the idea that endothelial cell products play a role in mediating vascular tone.     

Imaging of estrogen receptor-α in rat pial arterioles using a digital immunofluorescent microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors (1, 2, 3). Although two sub-types exist (estrogen receptor -α and β),estrogen receptor-α has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy (4). Furthermore, ER-α is located in the nuclei and in the cytoplasm of rat basilar arteries (5). The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments (6). Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-α using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 μm cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.     

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

BACKGROUND: Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. METHODS: We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. RESULTS: With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. CONCLUSIONS: SFM is a valuable method for the evaluation of fluorescently labeled cells.     

Complementary visualization of mitotic barley chromatin by field-emission scanning electron microscopy and scanning force microscopy

The surface structure of mitotic barley chromatin was studied by field-emission scanning electron microscopy (FESEM) and scanning force microscopy (SFM). Different stages of the cell cycle were accessible after a cell suspension was dropped onto a glass surface, chemical fixed, and critically point dried. Imaging was carried out with metal-coated specimen or uncoated specimen (only for SFM). The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data. The experimentally determined volume of the residue chromatin during mitosis was within the range of 65-85 microm(3). A comparison with the theoretically calculated volume indicated a contribution of about 40% of internal cavities. Decondensation of chromosomes by proteinase K led to a drastic decrease in the chromosome volume, and a 3-D netlike architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious. Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing. We imaged intact chromosomes in liquid by SFM without any intermediate drying step. A granular surface was obvious but with an appreciably lower resolution. Under similar imaging conditions proteinase K-treated chromosomes exhibited low topographic contrast but were susceptible to plastic deformations.     

Identification of Caveolae-like Structures on the Surface of Intact Cells Using Scanning Force Microscopy

Caveolae are small, functionally important membrane invaginations found on the surface of many different cell types. Using electron microscopy, caveolae can be unequivocally identified in cell membranes by virtue of their size and the presence of caveolin/VIP22 proteins in the caveolar coat. In this study we have applied for the first time scanning force microscopy (SFM), to visualize caveolae on the surface of living and fixed cells. By scanning the membranes of Chinese hamster ovary cells (CHO), using the tapping mode of the SFM in fluid, we could visualize small membrane pits on the cell membranes of living and fixed cells. Two populations of pits with mean diameters of around 100 nm and 200 nm were present. In addition, the location of many pits visualized with the SFM was coincident with membrane spots fluorescently labeled with a green fluorescent protein-caveolin-1 fusion protein. Scanning force microscopy on cells treated with methyl--cyclodextrin, an agent that sequesters cholesterol and disrupts caveolae, abolished pits with a measured diameter of 100 nm but left pits of around 200 nm diameter intact. Thus, the smallest membrane pits measured with the SFM in CHO cells were indeed very likely to be identical to caveolae. These experiments show for the first time that SFM can be used to visualize caveolae in intact cells.     

Differential proinflammatory and prooxidant effects of bone morphogenetic protein-4 in coronary and pulmonary arterial endothelial cells

There is increasing evidence that TGF-beta family member cytokine bone morphogenetic protein (BMP)-4 plays different pathophysiological roles in the pulmonary and systemic circulation. Upregulation of BMP-4 has been linked to atherosclerosis and hypertension in the systemic circulation, whereas disruption of BMP-4 signaling is associated with the development of pulmonary hypertension. To test the hypothesis that BMP-4 elicits differential effects in the pulmonary and systemic circulation, we compared the prooxidant and proinflammatory effects of BMP-4 in cultured human coronary arterial endothelial cells (CAECs) and pulmonary arterial endothelial cells (PAECs). We found that BMP-4 (from 0.3 to 10 ng/ml) in CAECs increased O(2)(*-) and H(2)O(2) generation, induced NF-kappaB activation, upregulated ICAM-1, and induced monocyte adhesiveness to ECs. In contrast, BMP-4 failed to induce oxidative stress or endothelial activation in PAECs. Also, BMP-4 treatment impaired acetylcholine-induced relaxation and increased O(2)(*-) production in cultured rat carotid arteries, whereas cultured rat pulmonary arteries were protected from these adverse effects of BMP-4. Thus, we propose that BMP-4 exerts prooxidant, prohypertensive, and proinflammatory effects only in the systemic circulation, whereas pulmonary arteries are protected from these adverse effects of BMP-4. The vascular bed-specific endothelial effects of BMP-4 are likely to contribute to its differential pathophysiological role in the systemic and pulmonary circulation.     

Scanning electron microscopy substantiates histology in showing the inadequacy of the existing theories on the development of the proximal coronary arteries and their connections with the arterial trunks

Development of proximal coronary arterial segments and coronary arterial orifices was studied by scanning electron microscopy in 20 rat embryos and by light microscopy in serial sections of 20 human and another 18 rat embryos. Neither by scanning electron microscopy nor by light microscopy did we observe more than two coronary arterial orifices. These coronary orifices were always situated in the sinuses of the aorta that faced the pulmonary artery. In the human embryos the coronary orifices emerged between 37-39 days of gestation (16-19 mm crown-rump length, Streeter horizon XVIII-XIX) and were invariably present beyond 39 days (19 mm crown-rump length, Streeter horizon XIX). In rat embryos, the coronary orifices emerged in both scanning electron microscopy and light microscopy at 15-17 days of gestation (13-17 mm crown-rump length) and were invariably present beyond 17 days (17 mm crown-rump length). In both human and rat embryos, either by scanning electron microscopy and light microscopy, the left coronary orifice was observed significantly earlier. In all the investigated embryos, human as well as rat, septation at arterial orifice level was complete, including the earliest stages studied. Light microscopy showed that at the emerging stages of the coronary orifices, the proximal epicardial segments of the left and right coronary arteries could already be identified in a peritruncal ring of epicardial vasculature, before the coronary orifice was observed. This was the case in human as well as in rat embryos. Thus, a coronary orifice was never seen in the absence of a proximal coronary artery. The present theories on development of the proximal coronary arteries and coronary orifices do not offer an adequate explanation for either these data or the known possible congenital abnormalities of the coronary arteries. Our study supports dual proximal coronary arterial development. These two proximal coronary arteries develop out of a peritruncal ring of vascular structures on to the aorta. The process by which the coronary orifices actually develop remains to be explained.     

Short-term exercise training improves vascular function in hypercholesterolemic rabbit femoral artery

Chronic exercise in healthy or hypercholesteremic animals for at least two months improves their vascular functions. This study is to examine whether short-term exercise training protocols can correct early-stage vascular dysfunction induced by high-cholesterol diet feeding. Male New Zealand White rabbits were fed for 2, 4 or 6 weeks with rabbit chow with or without the addition of 2% (w/w) cholesterol. They were further divided into control and exercise groups. Animals in exercise groups ran on a leveled treadmill for the same time periods as diet intervention. At the end of experiments, femoral arteries were dissected, loaded with fura 2-AM, and mounted in a tissue flow chamber. Phenylephrine-precontracted vessel specimens were exposed to acetylcholine. The endothelial intracellular calcium elevation and vasorelaxation were determined simultaneously under an epifluorescence microscope with ratio imaging capability. En face oil red O staining was used to evaluate fatty streak formation. Our results showed that 1) high-cholesterol diet feeding for > or = 4 weeks caused lipid deposition, reduced the acetylcholine-evoked endothelial calcium signaling, and impaired both endothelium-dependent and endothelium-independent vascular responses in a time-dependent manner; 2) vasorelaxation at given levels of endothelial intracellular calcium elevation decreased in hypercholesterolemia; 3) concomitant exercise program had reverse effects. We conclude that high-cholesterol diet intervention for as short as 4 weeks induces vascular structural changes, impairs endothelial intracellular calcium signaling and vasodilatation in rabbit femoral arteries. Short-term exercise training in parallel completely eliminates these adverse effects so long as the diet intervention is no more than 6 weeks.     

Expression of TRPC homologs in endothelial cells and smooth muscle layers of human arteries

TRPC channels are a group of Ca²⁺-permeable nonselective cation channels that mediate store-operated and/or agonist-stimulated Ca²⁺ influx in a variety of cell types. In this study, we extensively examined the expression patterns of TRPC homologs in human vascular tissues. RT-PCR amplified cDNA fragments of TRPC1 (505 bp), TRPC3 (372 bp), TRPC4 (499 bp), TRPC5 (325 bp), TRPC6 (509 bp), and TRPC7 (187 bp) from RNA isolated from cultured human coronary artery endothelial cells. In situ hybridization yielded strong labeling of TRPC1,3–6 in the endothelial and smooth muscle cells of human coronary and cerebral arteries. TRPC7 labeling was exclusively found in endothelial cells but not in smooth muscle cells. Results from immunohistochemical staining were consistent with those from in situ hybridization. Similar expression patterns of TRPC homologs were also observed in arterioles and vaso vasora. In conclusion, our study indicates that TRPC homologs are widely expressed in human vessels of all calibers, including medium-sized coronary arteries and cerebral arteries, smaller-sized resistance arteries, and vaso vasora. These results suggest a ubiquitous role of TRPC homologs in regulating blood supply to different regions and in controlling arterial blood pressure.     

Resolving the molecular structure of microtubules under physiological conditions with scanning force microscopy

We have imaged microtubules, essential structural elements of the cytoskeleton in eukaryotic cells, in physiological conditions by scanning force microscopy. We have achieved molecular resolution without the use of cross-linking and chemical fixation methods. With tip forces below 0.3 nN, protofilaments with ~6 nm separation could be clearly distinguished. Lattice defects in the microtubule wall were directly visible, including point defects and protofilament separations. Higher tip forces destroyed the top half of the microtubules, revealing the inner surface of the substrate-attached protofilaments. Monomers could be resolved on these inner surfaces.Abbreviations APTS (3-aminopropyl)triethoxysilane - DETA N¹-[3-(trimethoxysilyl)propyl]diethylenetriamine - EM electron microscopy - MT microtubule - SFM scanning force microscopy     

Differential genomic changes caused by cholesterol- and PUFA-rich diets in regenerated porcine coronary endothelial cells

Endothelial regeneration and dyslipidemia impair endothelium-dependent relaxation, while supplementation with fish oil (FO) prevents it. The genomic impact of different diets was compared in primary cultures derived from native and regenerated endothelial cells. Pigs were fed with high-cholesterol (CHL) or FO-rich diet. Partial in vivo removal of endothelium was performed to induce endothelial regeneration. Native and regenerated cells were harvested, cultured, and prepared for genomic (microarray experiments, real-time PCR) and proteomic (Western blotting) analysis. The analysis identified genomic changes induced by chronic CHL diet in native cultures resembling those induced by in vivo regeneration, as well as those that could be prevented by FO diet. At the protein level, the reduced and increased presences of endothelial nitric oxide synthase and F2, respectively, observed after regeneration combined with CHL diet were alleviated by FO. The comparison of the differential changes induced by regeneration in vivo in endothelial cells from both diet groups revealed a limited number of genes as the most likely contributors to reduction in endothelium-dependent relaxations in porcine coronary arteries lined with regenerated endothelium.