Direct observation of epicardial coronary capillary hemodynamics during reactive hyperemia and during adenosine administration by intravital video microscopy

Using high-resolution intravital charge-coupled device video microscopy, we visualized the epicardial capillary network of the beating canine heart in vivo to elucidate its functional role under control conditions, during reactive hyperemia (RH), and during intracoronary adenosine administration. The pencil-lens video-microscope probe was placed over capillaries fed by the left anterior descending artery in atrioventricular-blocked hearts of open-chest, anesthetized dogs paced at 60-90 beats/min (n = 17). In individual capillaries under control conditions, red blood cell flow was predominant during systole or diastole, indicating that the watershed between diastolic arterial and systolic venous flows is located within the capillaries. Capillary flow increased during RH and reached a peak flow velocity (2.1 +/- 0.6 mm/s), twice as high as control (1.2 +/- 0.5 mm/s), with enhancement of intercapillary cross-connection flow and enlargement of diameter (by 17%). With adenosine, capillary flow velocity significantly increased (1.8 +/- 0.7 mm/s). However, the increase in volumetric capillary flow with adenosine estimated from red blood cell velocity and diameter was less than the increase in arterial flow, whereas that during RH was nearly equivalent to the increase in arterial flow. There was a time lag of approximately 1.5 s for refilling of capillaries during RH, indicating their function as capacitance vessels. In conclusion, the coronary capillary network functions as 1) the major watershed between diastolic-dominant arterial and systolic-dominant venous flows, 2) a capacitor, and 3) a significant local flow amplifier and homogenizer of blood supply during RH, but with adenosine the increase in capillary flow velocity was less than the increase in arterial flow.     

Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy

High-speed atomic force microscopy (HS-AFM) allows direct visualization of dynamic structural changes and processes of functioning biological molecules in physiological solutions, at subsecond to sub-100-ms temporal and submolecular spatial resolution. Unlike fluorescence microscopy, wherein the subset of molecular events that you see is dependent on the site where the probe is placed, dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding of how biological molecules function. Here we present protocols for HS-AFM imaging of proteins in action, including preparation of cantilever tips, step-by-step procedures for HS-AFM imaging, and recycling of cantilevers and sample stages, together with precautions and troubleshooting advice for successful imaging. The protocols are adaptable in general for imaging many proteins and protein-nucleic acid complexes, and examples are described for looking at walking myosin, ATP-hydrolyzing rotorless F(1)-ATPase and cellulose-hydrolyzing cellulase. The entire protocol takes 10-15 h, depending mainly on the substrate surface to be used.     

Automated determination of parameters describing power spectra of micrograph images in electron microscopy

The current theory of image formation in electron microscopy has been semi-quantitatively successful in describing data. The theory involves parameters due to the transfer function of the microscope (defocus, spherical aberration constant, and amplitude constant ratio) as well as parameters used to describe the background and attenuation of the signal. We present empirical evidence that at least one of the features of this model has not been well characterized. Namely the spectrum of the noise background is not accurately described by a Gaussian and associated "B-factor;" this becomes apparent when one studies high-quality far-from focus data. In order to have both our analysis and conclusions free from any innate bias, we have approached the questions by developing an automated fitting algorithm. The most important features of this routine, not currently found in the literature, are (i). a process for determining the cutoff for those frequencies below which observations and the currently adopted model are not in accord, (ii). a method for determining the resolution at which no more signal is expected to exist, and (iii). a parameter-with units of spatial frequency-that characterizes which frequencies mainly contribute to the signal. Whereas no general relation is seen to exist between either of these two quantities and the defocus, a simple empirical relationship approximately relates all three.     

Optimum geometric parameters of the dendrite process

Optimal radius of dendritic spine steam is calculated under constant synaptic excitation using three criteria: maximum of excitatory postsynaptic potential (EPSP), maximal rate of EPSP change due to little change of spine steam radius and maximal rate of EPSP change due to little change of synaptic conductance.     

Scanning gel chromatography: determination of transport parameters from dynamic profiles measured at low solute concentration

Direct optical scanning of solute boundaries in large zone gel Chromatography experiments provides an accurate means of determining boundary profile shapes and rates of motion. A method has been developed for correcting such boundaries to a constant time frame, eliminating the distortion which arises from finite column scanning rate Centroids or the corrected profiles can be used to determine the partition cross section for the solute of interest The partition cross section and flow rate determine translational motion within the column. The axial dispersion coefficient, L, which characterizes rate of boundary spreading may also be calculated from the profiles. In order to explore these procedures a study of four noninteracting solutes was conducted. Partition cross sections determined from rates of motion of boundary centroids were found to be in good agreement with those determined by the equilibrium saturation method on the same column.In order to explore the lowest concentration limits of the technique and to illustrate the boundary characteristics for a selfassociating solute, a study of carboxyhemoglobin was conducted over a wide concentration range. From measurements at 220 nm the lowest concentration where useful data could be obtained was 2 micrograms per ml (0.12 πM heme) These results establish validity of the procedures used in analyzing the rates of boundary transport and in studying solute transport over a wide range of conditions.     

Gravitaxis in Chlamydomonas reinhardtii: characterization using video microscopy and computer analysis

We characterized the gravitactic behavior of Chlamydomonas reinhardtii, a unicellular green alga, using a computer-analysis system in order to study directional swimming. The effects of the calcium-channel inhibitors gadolinium and diltiazem on graviorientation and swimming speed were examined. In addition, we studied directional swimming in the ptx1 strain of C. reinhardtii, a flagellar dominance mutant. Results indicate that Chlamydomonas reorients for gravitactic swimming through a mechanism different from the calcium-mediated pathway believed to be involved in gravity transduction in higher plants. We suggest that calcium-mediated gravitaxis originated in an organism that was more evolutionarily advanced than Chlamydomonas.     

Cervical spine motion in the sagittal plane: Kinematic and geometric parameters

The kinematic function of the cervical spine has been examined previously by means of cineradiography or a sequence of lateral X-rays, usually of a flexion-extension range of motion. Interpretation of these studies, however, presents difficulties. One of the major problems is how to extract information from the X-ray images which is not only explicit, quantitative and accurate, but which is also diagnostically useful. Another problem is that as one increases the number of steps of motion between full flexion and full extension to obtain a more detailed examination, one also increases the amount of radiation exposure and the bulk of the data.

Reported here is a technique which uses one lateral-view X-ray for each of five neck positions: full flexion, full extension, and three intermediate positions. From each set of X-rays, various parameters are derived to describe two types of data: kinematic (angles and centers of rotation), and geometric (pattern of curvature). This technique has been shown capable of identifying functional abnormalities in patients with neck pain who have no structural abnormalities detectable by X-ray. Further experience will be useful in better defining various types of functional abnormalities.     

Protein structure classification using geometric invariants and dynamic programming

Classification of newly determined protein structures is important in understanding their function and mechanism of action. Currently available methods employ a global structure alignment strategy and are computationally expensive. We propose a two-step methodology with a quick screen to significantly reduce the number of candidate structures followed by global structure alignment of the query structure with the reduced set. We represent a protein structure as a sequence of local structures, codified in the form of geometric invariants. Geometric invariants are quantities that remain unchanged under transformations such as translation and rotation. Protein structures represented as multi-attribute sequences are aligned via dynamic programming to identify close neighbors of the query structure. The query structure is then compared with this reduced dataset using conventional structure comparison methods to predict its functional class. For a typical protein structure, the screening method was able to reduce the protein data bank to mere 200 proteins while preserving structurally closest neighbor in the reduced set. This has resulted in 30 to 60 fold improvement in the execution time. We present the results of leave-one-out classification experiment on ASTRAL-95 domains and comparison with SCOP classification hierarchy.     

Direct measurements of cell number using computer-aided video microscopy

Quantitative studies in cell culture require accurate measurements of cell density and kinetics. We have developed a direct, rapid, and noninvasive method for measuring cell number in monolayer culture. Using computer-aided video microscopy, cell number was measured without detaching or chemically destroying the cells, thereby allowing sequential measurements in the same cell population. Cell number measured by computer-aided microscopy closely correlated with hemocytometer counts and determinations of total cell protein. For high-density monolayers of mesenchymal cells, however, staining was required for accurate counts. Unlike other techniques for measuring cell density, computer-aided microscopy was especially accurate in medium- to low-density cultures (less than 6000 cells/cm2). In addition, we applied this technique to the construction of separate proliferation curves for glomerular mesangial and vascular endothelial cells in coculture. These measurements by cell type in coculture are impossible using conventional methods for determining cell number.     

Direct detection of immunogold reactions by real-time video microscopy

Summary Video-enhanced microscopy allows the detection and tracking of individual colloidal gold particles. The analysis of immunogold reactions can also be conducted as a function of time and thus allows the study of dynamic events in living cells. The direct visualization in real time is reported of the reaction of immunogold particles with a surface antigen. This time-resolved immunocytochemistry was achieved by continuous observation of living cells infected with a virus (respiratory syncytial virus) following their incubation with colloidal gold (30 nm) coated with antiviral antibodies. The progress of the immunoreaction was visualized as a sequential deposition of individual gold granules on the viral particles until saturation was reached after 60 min. Binding of colloidal gold was an irreversible event as no elution or dislocation of surface-bound granules took place. Comparative imaging of colloidal gold particles by electron microscopy and by video microscopy demonstrated that the video-imaged immunoreactions represented events involving single gold particles; their signal was sometimes clearly enhanced by secondary depositions taking place in close proximity, i.e. at a distance below the lateral resolution of the light microscope. Our experiments demonstrate that video-enhanced microscopy provides a powerful tool for studying antibody-antigen reactions with a high spatial and temporal resolution.     

Comparison of static and dynamic respirometry for the determination of stoichiometric and kinetic parameters of a nitrifying process

Respirometry consists in the measurement of the biological oxygen consumption rate under well-defined conditions and has been used for the characterization of countless biological processes. In the field of biotechnology and applied microbiology, several respirometry methods are commonly used for the determination of process parameters. Dynamic and static respirometry, which are based on oxygen measurements with or without continuous aeration, respectively, are the methods most commonly used. Additionally to several respirometry methods, different methods have also been developed to retrieve process parameters from respirometric data. Among them, methods based on model fitting and methods based on the injection of substrate pulse at increasing concentration are commonly used. An important question is then; what respirometry and data interpretation methods should be preferably used? So far, and despite a growing interest for respirometry, relatively little attention has been paid on the comparison between the different methods available. In this work, both static and dynamic respirometry methods and both interpretation methods; model fitting and pulses of increasing concentration, were compared to characterize an autotrophic nitrification process. A total of 60 respirometry experiments were done and exhaustively analysed, including sensitivity and error analyses. According to the results obtained, the substrate affinity constant (K _S ) was better determined by static respirometry with pulses of increasing concentration and the maximum oxygen uptake rate (OUR _ex.max ) was better determined by dynamic respirometry coupled to fitting procedure. The best method for combined K _S and OUR _ex.max determination was static respirometry with pulses of increasing concentration.     

Dynamics of microtubules visualized by darkfield microscopy: treadmilling and dynamic instability

Individual microtubules undergoing treadmilling in vitro were visualized by darkfield light microscopy, and the relationship between treadmilling and dynamic instability was studied as a function of microtubule-associated proteins (MAPs). In order to demonstrate treadmilling directly by real-time observation, we constructed three-block microtubules, the center-block of which was decorated with Tetrahymena dynein. The decorated block can easily be distinguished from undecorated blocks in the darkfield microscope because the decorated one appears much thicker. At steady-state conditions, the length of an undecorated block at one end increased and that at another end decreased, while the decorated center-block did not change in its length. The results from these direct observations show that calf brain 3X-microtubules exhibit a treadmilling flux of 0.9 micron/h. Using a similar microscopy technique, we previously demonstrated that phosphocellulose PC-microtubules existed in either the growing or the shortening phase and alternated quite frequently at steady-state conditions (dynamic instability). How does treadmilling relate to dynamic instability? An image recording of individual 3X-microtubules containing MAPs revealed that the microtubules undergo treadmilling and do not exhibit any dynamic instability. This evidence shows that MAPs suppress the dynamic instability of microtubules. That is, treadmilling can take place in the steady state only after microtubules have been stabilized by MAPs.     

Electron microscopy of decorated crystals for the determination of crystallographic rotation and translation parameters in large protein complexes.

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a core of 3 alpha subunits. The preparation of reconstituted hollow capsids consisting of 60 beta subunits and their crystallization in a hexagonal (space group P6(3)22) and in a monoclinic (space group C2) modification have been described. The rotational and translational parameters of the protein molecules in both crystal forms were studied by electron microscopy of freeze-etch replicas and by Patterson correlation techniques. Decoration with silver and image processing provided images with the positions of the 3-fold and 5-fold molecular axes being labelled by metal clusters. This allowed the unequivocal determination of the orientation and translational position of the protein molecules with respect to the crystallographic axes in the hexagonal modification. From inspection of the decoration images it was immediately obvious that the hexagonal crystal forms of alpha 3 beta 60 and of beta 60 are isomorphous. In the monoclinic crystals, a local icosahedral 2-fold coincides with the crystallographic 2-fold axis. The exact solution of the particle orientation was determined by interpretation of Patterson self-rotation functions for the icosahedral symmetry axes. Rotational and translational parameters for the monoclinic modification are given. A rational procedure for the efficient application of freeze-etching techniques in order to elucidate the packing in crystals of large proteins is described.     

Correlations between capillary microscopy and electrocardiographic results in the diabetic

Ninety-nine diabetics were simultaneously given a capillaroscopic examination of periungual walls and an electrocardiogram evaluated according to the Minnesota Code. From these tests the following data were recorded: a. the number of knobs revealed by capillaroscopy is inferior, in a statistically significant manner, in groups having "major abnormalities"; b. capillaroscopic abnormalities did not seem to be definitely correlated to age, but rather to the length of time diabetes mellitus was present; in particular, these abnormalities were quite evident in subjects diagnosed as diabetics for the first time. This procedure is therefore extremely useful for the early diagnosis of diabetic microangiopathy; c. electrocardiographic abnormalities are correlated to patient's age, whereas are not related to the period of the illness. Although the small sampling obliges us to maintain certain reservations, such abnormalities were more frequent in subjects taking antidiabetic drugs orally.