The rapid tracing of vasculature, and quantification of visual landmarks such as those corresponding to the branching and crossover points from images is extremely useful in a variety of fields including ophthalmology, radiology, and neurobiology. A novel algorithm that enables rapid, automatic, robust, adaptive, and accurate tracing of vasculature has been developed. Given an image (Fig 1), it automatically generates a labeled graph representation of the vasculature (Fig 2), and a table listing the locations of visual landmarks such as crossover and branching points (Fig 3). For each such point, it also lists the intersection angles, and the local thickness of the intersecting vasculature segments. This data is often useful by itself. They can also be used for image registration and montage building.

This algorithm greatly improves upon previous work in several ways. First, it is fully automatic, and free of parameters that need to be adjusted carefully (just needs optional sensitivity setting).

Certain disadvantages of traditional injection methods and difficulties arising during investigation of experimental and pathological material, limit the possibilities for extensive study of the microcirculatory bed (MCB). This has prompted the authors to develop new non-injection methods for revealing the MCB on histological sections by means of direct staining of the structural elements of the vessel wall. These methods are based on selective precipitation of the extra- or intracellular orthophosphates with ions of Zn, Cd, Co, Ca, Sr, and Pb in the vascular endothelium and smooth muscle cells. These methods (which we have called “histoangiological”) provide clear-cut contrast dyeing of the MCB in different organs and tissues as well as tumors in rat, cat,pig, dog, and in humans.

In these methods, the reaction end product is lead sulfide which provides a high degree of contrast. Vessels and capillaries are detected due to precipitation of the deposit in the vascular endothelium.

Fibrin clot formation is necessary for maintaining the integrity of the vasculature via physiological processes of hemostasis and wound healing and is also involved in pathological processes, such as thrombosis and atherosclerosis. A variety of structural and biophysical approaches has been used to examine intermediates in the formation of clots and to visualize in vitro clots and ex vivo thrombi.

Structures at all stages of polymerization have been examined to learn about molecular mechanisms of assembly. Fibrinogen is a polyfunctional, multi-domain protein that is essential for platelet aggregation and for the formation of the three-dimensional network of fibrin fibers which is the structural basis of the clot. Distinctive functions for several of fibrinogen's domains in the fibrin assembly process have been elucidated. Enzymatic removal of the fibrinopeptides exposes binding sites in the central region which then interact with complementary sites at the ends of a neighboring molecule to yield fibrin oligomers.

Atomic force microscopy (AFM) provides unique opportunities to study cell-surface and molecular scale interactions in three dimensions under aqueous conditions. In AFM, a small probe attached to cantilever (fig. 1) is used for interacting with a sample to obtain sensitive measurements of molecular structure, intermolecular forces (sub-nN) and interfacial properties. The data ouput can be presented in the form of a topographical image, force map or viscoelastic response (e.g., Figs. 2-4). Thus, AFM combines high resolution imaging with the ability to measure surface-dependent intermolecular forces and properties. AFM also is amenable for coupling with optical imaging methods including immunofluoresence and gold bead labeling.

This presentation will focus primarily on the use of AFM for molecular level imaging of plasma proteins, von Willebrand Factor (vWF) and fibrinogen. Both proteins play central roles in the regulation of hemostasis and thrombosis by participating in coagulation or by facilitating adhesion, spreading and aggregation of activated platelets.

In vivo the red blood cell reversibly and rapidly deforms as it passes through the small capillaries. Although the physiochemical basis of the red cell's deformability is unknown, it is regarded as being a property of the membrane protein spectrin. We have used cryoelectron microscopy to study the structure of spectrin in membrane skeletons to determine how its molecular structure confers elastic properties on the cell membrane. Cryomicroscopy has the virtue of minimally perturbing easily deformable structures such as spectrin.

Figure 1 is an electron micrograph of a portion of a frozen-hydrated red cell skeleton. The spectrin molecules are indicated by the arrow heads. They cross link the skeletal network by interacting with short rod like segments F-actin (indicated by the arrows) containing about 13 actin molecules. In such micrographs the spectrin appears to have a sinusoidal shape whereas in negatively stained preparations spectrin appears to be straight.

We have used the procedure of Margalef in order to determine the three dimensional trajectory of the spectrin molecule.

Nonlinear or multiphoton fluorescence microscopy utilizes the simultaneous absorption of two (or three) longer wavelength photons to excite fluorophores (Denk et.al., 1990). For example, UV absorbing fluorophores such as DAPI or lndo-1 are excited using 700 nm near infrared light rather than 350 nm UV excitation. This relatively new form of laser scanning fluorescence microscopy has excellent optical sectioning capabilities in thick, highly scattering tissues. The high degree of intrinsic optical sectioning arises from the spatial nature of the excitation dependence, rather than from a confocal aperture or deconvolution algorithm. The fluorescence arising from any point in the specimen depends on the second or third power of the intensity (i.e. two and three photon excitation, respectively). This squaring (or cubing) of the illumination point spread function effectively confines the excitation to a tenth of a femtoliter optical volume when using a high NA lens. 680 to 1100 nm mode-locked lasers producing pulses of 100 femtosecond duration at a repetition rate of 80 Mhz are used to efficiently produce fluorescence excitation at average powers that are usually in the 0.5 to 10 mW range at the sample.

This is an extension of our presentation from last year. We intend to continue the discussion and review several practical procedures developed in our laboratory for routine and immuno-electron microscopy.

TEM Vs IM-LM In the last decade, the use of diagnostic TEM in the medical field has experienced a drastic decline. The main reason is due to the advances of immuno-labeling techniques for light microscopy and also the decreased coverage of medical insurance for EM. Currently, immuno-histochemistry or in situ hybridization staining can be performed on large paraffin or frozen sections. These procedures can be performed quickly and much less expensively than TEM. However, TEM is still needed when PAP staining is not specific or minimum numbers of cells are stained. It is also possible to process immuno-labeled paraffin slides for TEM examination to confirm a diagnosis. In addition, when only a few microorganisms such as pneumocystic carinii or protothecosis are stained by Grocott’s methenamine silver in paraffin sections, a re-examination and confirmation by TEM can be performed without the use of uranium or lead staining.

The Tripod polishing technique is used to mechanically polish materials for further analysis by optical, SEM or TEM. The mechanical polishing is done using the Tripod Polisher (FIG. 1), which consists of three Delrin capped micrometers and an L-bracket. The micrometers provide accurate side-to-side and front-to-back adjustments of the polishing plane. The L-bracket is where the sample is attached to the polisher and it comes in a number of configurations, depending on the type of analysis needed. by attaching the sample at different locations on the L-bracket, either planar sections or cross sections can be made using the Tripod Polisher.

A sample to be cross sectioned is attached to the front of the L-bracket with wax . The micrometers are then adjusted so the bottom of the Delrin feet are aligned with the area of interest. The polisher is held on a rotating diamond wheel, with the Delrin feet and the sample in contact with the wheel.

Negative staining is the most frequently used procedure for preparing particulate specimens, e.g., cell organelles, macromolecules, and viruses, for electron microscopy (Figs. 1-4). The main advantage is that it is rapid, requiring only minutes of preparation time. Another is that it avoids some of the harsh chemicals, e.g., organic solvents, used in thin sectioning. Also, it does not require advanced technical skill. It is widely used in virology, both in classification of viruses as well as diagnosis of viral diseases. Notwithstanding the necessity for fairly high particle counts, virus identification by negative staining is advantageous in not requiring specific reagents such as antibodies, nucleic acid probes, or protein standards which necessitate prior knowledge of potential pathogens for selection of the proper reagent. Furthermore, it does not require viable virions as does growth in tissue culture. Another procedure that uses negative contrasting is ultrathin cryosectioning (Fig. 5).

In 1954 Farrant was the first to publish negatively stained material, ferritin particles.

Digital imaging is replacing conventional photography in many applications. As the quality of digital images improves, more applications for this technology will be found. This talk will examine the importance of gamma correction in digital imaging.

Although many researchers believe that digital imaging will soon replace photography, it is probably more correct to think of digital imaging as an enhancement to photography. The most critical problem with translating our knowledge of photography to digital imaging is that photography operates exclusively via logarithmic functions. The exposure versus density curves common to photography have an x axis that is logarithmic. The development curves for film and paper are also logarithmic. The slope of the log-linear portion of this curve is designated gamma. All operations normally performed in the darkroom whether processing film or prints manipulate the gamma functions to achieve the best recorded image. The first rule that should be obvious is that every image has a different optimal gamma and every different image medium (whether graded photographic paper or the density of print on a digital image printer) will change that optimal gamma.

Freeze-fracture techniques have contributed much to our understanding of membrane composition and macromolecular architecture. However, further substantive contributions from freeze fracture have seemed unlikely because: a) replicas typically fragmented, destroying histological orientation within samples; b) histochemical staining techniques were thought to be incompatible with bleach- or acid-cleaned freeze-fracture replicas; c) the limit of resolution was presumed to be 3-5 nm; and d) there were no methods for directing the fracture plane to specific components within a tissue. Recent developments in “grid-mapped freeze fracture” and freeze-fracture immunocytochemistry have addressed each of these limitations.

Using grid-mapped freeze fracture, samples are mapped in three dimensions using confocal microscopy before freeze-fracture (Fig. A), as well as after replication and stabilization in Lexan plastic on a gold “Finder” grid (Fig. B). After replica cleaning, areas of interest are correlated in confocal and TEM images —— for example, cilia of the ependymal layer of rat spinal cord (Fig. C).

It has been shown that a focused ion beam (FIB) instrument may be used to prepare site specific cross-sectioned specimens to within < 0.1 μm for both scanning and transmission electron microscopy (SEM and TEM, respectively). FIB specimen preparation has been used almost exclusively in the microelectronics industry. Recently, FIB specimen preparation has been utilized for other materials systems and applications.

A cross-sectioned SEM specimen is produced by sputtering away a trench of material from near the region of interest. Large amounts of material are sputtered using large ion beam diameters (e.g., l00’s nm) and high beam current (e.g., l000’s pA), while the final sputtering operations are achieved using smaller beam diameters (e.g., < 10 nm) and lower beam current (e.g., 10’s of pA). The SEM specimen may then be etched to reveal particular microstructural features of interest. A low magnification SEM image of a multi-layered device prepared for cross-section analysis by the FIB method is shown in FIG. 1.

Quantitative gold labeling studies allow the comparison of tissue antigens only if labeling conditions are the same. When labeling conditions deviate slightly, the labeling efficiency can also shift. Labeling efficiency can be determined on a standard containing known concentrations of the target antigen. When labeling this standard, the labeling density (LD) divided by the antigen concentration (C) gives the labeling efficiency (LE).(l) If the assumption is accepted that labeling efficiency is the same over the standard and tissue, then the antigen concentration in the tissue can be calculated from the labeling density. In this study, a labeling standard was constructed that contained collagen type IV in five concentrations and a blank agarose gel. Our design criteria specified that these six gels measure less than 2 mm across and the zones between protein concentrations be clearly delineated.

The labeling standard was constructed by diluting lyophilyzed collagen type IV in water to final concentrations of 1,2,3,4, and 5 mg/ml.

Various multichannel silicon electrodes have been developed for stimulating and recording in the central nervous system of experimental animals. These electrodes can be used in their planar form or can be assembled into 3-D structures for volume interaction with tissue. However, bio-compatibility issues arise concerning the introduction of any electrode into living tissue. From earlier observations, we noticed that the neuronal cells had a tendency to adhere to the chronically implanted silicon substrate. Routine microtechnique became an obstacle when attempting to section the implanted and embedded electrode. Most often, this was attempted by passing a stainless steel blade through paraffin, or plastic embedded tissue, resulting in disrupting the tissue material interface. We needed to develop a reproducible procedure which preserved the integrity of this interface for future LM and confocal microscopic studies.

In order to preserve the electrode/tissue interface, we used the Exakt microgrinding/micropolishing technique, where the electrode/brain block was embedded in Technovit 7200 resin and cut with a diamond impregnated band saw.

Models for the structure of the bovine casein micelle have been proposed in the past (1,2,3,4). These models are based on the chemical and physical properties of the micelles and fall into two general catagories, framework and submicelle models. Electron Microscopy techniques were one of the tools used in the development of these models. However, no definative model has been established. We have developed a new electron microscopy method and it is being applied to the re-examination of the protein structure of the casein micelle, with the goal of establishing a definative model for the structure of the casein micelle.

High resolution scannning electron microscopy has proven unsuccessful in revealing the protein structure in the casein micelle (Fig 1). To create high resolution scanning electron microscopic images, fine metal coatings are required in the 5 to l0nm range. Following the application of these coatings, images of casein micelles resemble a melting lumpy sphere. This is due to the metal layer obscuring the proteins from view.

Light, immuno, and electron microscopy are standard for evaluation of renal biopsies. For light microscopy (LM), special stains including PAS and Jones silver methenamine (JSM) are frequently used to enhance recognition of renal anomalies related to disease. We recently discovered that fluorescence microscopy (FM) extends the usefulness of frozen and paraffin sections stained with hematoxylin and eosin and may presage the findings of immuno and electron microscopy. With UV illumination and reflected light fluorescence excitation, standard histologie sections reveal pathologic glomerular and other renal changes not evident by conventional bright field microscopy and may obviate the need for additional special stains.

For frozen sections, tissues mounted on gum tragacanth were snap frozen in isopentane chilled with liquid nitrogen and cryosectioned at a thickness of 4-6 μm. For paraffin sections, tissues were fixed in neutral buffered 10% formalin or in Hollande’s fixative (3.6% formalin, 40% picric acid, 25% copper acetate, 1.5% acetic acid) and sectioned at 2-4 μm.

The demand for TEM analysis in semiconductor failure analysis is rising sharply due to the shrinking size of devices. A well-prepared sample is a necessity for getting meaningful results. In the past decades, a significant amount of effort has been invested in improving sample preparation techniques for TEM specimens, especially precision cross-sectioning techniques. The most common methods of preparation are mechanical dimpling & ion milling, focused ion beam milling (FIBXTEM), and wedge mechanical polishing. Each precision XTEM technique has important advantages and limitations that must be considered for each sample.

The concept for both dimpling & ion milling and wedge specimen preparation techniques is similar. Both techniques utilize mechanical polishing to remove the majority of the unwanted material, followed by ion milling to assist in final polishing or cleaning. Dimpling & ion milling produces the highest quality samples and is a relatively easy technique to master.

The usual method to prepare specimen for transmission electron microscopy (TEM) with an ultramicrotome (UM) is to embed a small piece of the sample in a resin which needs between hours and days to harden. Then the resin is trimmed and finally the thin sections are cut. We developed a method to produce cross sections with an UM without embedding the sample. This leads to first TEM pictures in less then 30 minutes after the end of the thin film production.

We produce neutron mirrors which consist of metallic monolayers (figl) or multilayers (fig.2). The materials we use are mostly cobalt-iron alloys and silicon. The quality of the mirrors critically depends on the structural and hence also on the magnetic properties which are strongly influenced by the settings of the sputter parameters (plasma current, pressure of the working gas, substrate temperature, sputtering high voltage). To find out the best parameters for each purpose a quick analysing method is needed for a fast feedback

Carbon-coated holey polymer film (HPF), or ‘holey carbon grid’ used to support fragile samples, is a crucial part of Cryo-TEM technique. A film with high porosity and uniform hole size as well as mechanical strength is needed. Efforts to develop a reliable preparation method date back to the 1950s. Today's favorite by far for routine application employs condensation, dip-coating, drainage and drying.it is not difficult, it affords control of hole size, and its yield is comparatively good. In this study, the mechanisms of hole generation and film formation were uncovered by means of in situ microscopy at medium working distance, super-VHS video recording and a high-speed motion analyzer, SEM and TEM.

Clean microscope slides were treated with Amine 105 (Kao-Atlas Co. Japan) to make the surface hydrophobic. A slide was then immersed in liquid nitrogen and exposed for 3 to 6 seconds to air with relative humidity of 30 to 60%.

The design and performance of a new instrument, based on improved Penning ion guns [1] for etching and coating samples for SEM and LM in a single vacuum chamber, are described. The instrument is based on an existing high resolution ion beam coating system, which is capable of producing high quality ultra-thin and amorphous conductive films, required for present high resolution electron microscopes. [2]. The fact that in this system both etching and coating processes are combined in one chamber, the specimen handling and specimen contamination are minimized. Furthermore, the system eliminates the traditional multiple mounting /dismounting of samples to various holders for mechanical polishing, etching, coating and microscopy purposes. The specimen can stay with the same holder throughout the entire process, increasing the sample through-put. Moreover, the system offers an alternative method to the traditional “wet chemical etching,” technique with its well known problems.