A simple tool for stereological assessment of digital images: the STEPanizer

STEPanizer is an easy-to-use computer-based software tool for the stereological assessment of digitally captured images from all kinds of microscopical (LM, TEM, LSM) and macroscopical (radiology, tomography) imaging modalities. The program design focuses on providing the user a defined workflow adapted to most basic stereological tasks. The software is compact, that is user friendly without being bulky. STEPanizer comprises the creation of test systems, the appropriate display of digital images with superimposed test systems, a scaling facility, a counting module and an export function for the transfer of results to spreadsheet programs. Here we describe the major workflow of the tool illustrating the application on two examples from transmission electron microscopy and light microscopy, respectively.     

High-resolution cryo-scanning electron microscopy study of the macromolecular structure of fibronectin fibrils

High-resolution cryo-scanning electron microscopy was used to examine fibronectin fibrils formed in culture by human skin fibroblasts and in a cell-free system by denaturing soluble plasma fibronectin with guanidine. These studies indicate that the conformation of fibrils formed in culture and in a cell-free system appeared to be similar and that fibronectin fibrils have at least two distinct structural conformations. Fibronectin fibrils can be very straight structures with smooth surfaces or highly nodular structures. The average diameter of the nodules in these fibrils is 12 nm. Both conformations can be seen within an individual fibril indicating that they are not different types of fibronectin fibrils but rather different conformational states. Immunolabeling studies with a monoclonal antibody, IST-2, to the heparin II binding domain of fibronectin revealed that the epitope was buried in highly smooth fibrils, but it was readily exposed in nodular fibrils. We propose, therefore, that fibronectin fibrils are highly flexible structures and, depending on the conformation of the fibril, certain epitopes on the surface may be buried or exposed.     

Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching

Fluorescence light microscopy (LM) has many advantages for the study of cell organization. Specimen preparation is easy and relatively inexpensive, and the use of appropriate tags gives scientists the ability to visualize specific proteins of interest. LM is, however, limited in resolution, so when one is interested in ultrastructure, one must turn to electron microscopy (EM), even though this method presents problems of its own. The biggest difficulty with cellular EM is its limited utility in localizing macromolecules of interest while retaining good structural preservation. We have built a cryo-light microscope stage that allows us to generate LM images of vitreous samples prepared for cryo-EM. Correlative LM and EM allows one to find areas of particular interest by using fluorescent proteins or vital dyes as markers within vitrified samples. Once located, the sample can be placed in the EM for further study at higher resolution. An additional benefit of the cryo-LM stage is that photobleaching is slower at cryogenic temperatures (−140°C) than at room temperature.     

Factors which influence light microscopic visualization of biological material in sections prepared for electron microscopy

Epoxy-embedded biological material, sectioned for conventional or high-voltage electron microscopy, can be visualized within the section with good contrast and detail by phase-contrast or dark-field light microscopy. The (phase) contrast of such material is not substantially influenced by the type of embedding resin or section support substrate. It is, however, influenced by the type of fixation, by heavy metal (uranyl and lead) staining and by the section thickness. After screening ultrathin and semithin sections for content with the light microscope, one need stain and examine only those grids containing sections of interest. This approach eliminates the need to screen sections with the electron microscope and, in some cases, the need to stain non-useful sections. This time-saving procedure is particularly useful for studies requiring ultrastructural examination of a selected area or structure which is large enough to be visualized with the light microscope but which comprises only a small volume of the embedded material.     

Morphometric study of the guinea pig small intestine during development

The mucosal surface area of the guinea pig duodenum, jejunum, and ileum was determined during development, in three age groups: 1-day-old and 2- and 12-week-old animals. The morphometric analysis was performed at three magnification levels. The nominal surface area was determined at the macroscopic level, from intestinal length and perimeter. Villus and microvillus amplification factors were estimated at light-microscopic and transmission electron-microscopic levels, respectively. We found: (1) an increase in the nominal surface area that is maximal in the ileum (7.7-fold); (2) an increase in the villus amplification factor in the duodenum (1.4-fold) and a decline in the jejunum and ileum (0.8-fold), although in the jejunum villus dimensions rise; and (3) a similar increase in the microvillus amplification factor in the three segments (1.1- to 1.4-fold). In conclusion, the total mucosal surface area increased from day 1 to week 12, by 3-fold in the duodenum and jejunum and by 8-fold in the ileum. Regionally, the jejunum shows the largest mucosal surface area, followed by the ileum, and, finally, the duodenum.     

Cytoplasmic cleavage in living zoosporangia of Allomyces macrogynus

We have used the vital fluorescent dye, FM4-64, as a marker of membrane development during zoospore formation in living zoosporangia of Allomyces macrogynus . Membrane development was visualized and documented using standard epifluorescence and laser scanning confocal microscopy. Video-enhanced light microscopy and transmission electron microscopy, using cryopreparation methods, were also employed in this study. In the first 10–12 min after the induction of zoospore formation, only the plasma membrane labelled with FM4-64. During this time, nuclei were strictly located in the cortical cytoplasm with their associated centrosomes positioned immediately adjacent to the plasma membrane ( Lowry & Roberson, 1997 ). Between 12 and 20 min post-induction, increased fluorescence appeared along regions of the plasma membrane adjacent to the nuclei. From these sites, membranes (i.e. cleavage elements) extended laterally within the cortex and then, in conjunction with nuclear migration, rapidly elongated into the sporangial cytoplasm. By 25–35 min post-induction, cleavage elements had ramified throughout the cytoplasm forming a complex, interconnected membranous network. Transmission electron microscopy revealed that cleavage elements were paired membrane sheets with a lumen consisting of an electron opaque, granular matrix. Cleavage elements developed into a highly ordered network by 35–40 min post-induction, which fully delimited zoospore initials into polyhedral-shaped cells. Zoospore discharge occurred between 40 and 50 min post-induction. Our results have shown that cleavage elements undergo four stages of development during zoospore formation in A. macrogynus : (i) development of membrane initials, (ii) cortical extension, (iii) cytoplasmic elongation and ramification and (iv) zoospore initial delimitation.     

Study of the tribologically transformed structure created during fretting tests

In fretting, without a lubricant, structures fail through wear involving debris. Various metallographic assessments show that this debris creation derives from a special structure, hereafter called the “tribologically transformed structure” (TTS). The latter is nucleated at the very beginning of a contact's life, and eventually masks the initial material's tribological behaviour. This TTS is very hard and brittle, and is quickly destroyed, which explains why TTS is rather difficult to observe. This study focuses on initiation mechanisms which, up to now, are still highly controversial. Large amounts of TTS are observed after fretting tests, which is the reason why they were chosen as the means of investigation. In this paper, the various possible creation mechanisms for TTS will be analysed. From this overview, two nucleation models are chosen: one is based on “mechanical alloying” considerations, and the other is linked to high-strain-recrystallisation phenomena. Transmission electron microscopy (TEM) investigations are used to check these assumptions.     

Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy

Using the tandem scanning microscope, in vivo confocal microscopic images of living eyes were compared to images obtained from ex vivo, freshly enucleated or fixed tissue in the rabbit. In the normal cornea, microscopic details of the superficial epithelium, basal lamina, stromal fibrocyte nuclei, nerves and endothelial cell borders were easily discernible. Removal of the eye from the intact animal resulted in loss of detail with distortion of the normal structural interrelationships within the corneal stroma whilst enhancing details of the corneal epithelium. Formalin fixation further enhanced details of the basal and suprabasal corneal epithelial cell nuclei and the stromal fibrocyte cell borders whilst inducing prominent brightly reflecting folds in the thickened stroma with concomitant enhancement of the edge contrast of the collagen lamellae. These changes appeared to be related, in part, to hydration of the cornea and artefactual pooling of water between structures that may enhance reflectivity by increasing the difference between the refractive index of the cellular and extracellular elements. We conclude that microscopic examination of ex vivo preparations of corneal tissue, although providing increased resolution similar to conventional light microscopic techniques, significantly altered the normal structural relationships and could lead to erroneous measurements of the physiological properties of the tissue as compared to in vivo microscopy of undisturbed, intact tissue.     

Direct imaging of paracrystalline phospholipid structure in the electron microscope

A long chain amphiphilic molecule—the phospholipid 1,2-dihexadecyl sn glycerophosphoethanolamine—has been crystallized epitaxially so that the interlamellar molecular periodicity is parallel to the substrate and hence normal to the electron beam in the electron microscope. This has permitted the direct resolution of the 55·6 Å lamellae in unstained crystals at room temperature. The lattice images have shown the presence of line dislocations and lenticular cracks in the crystals. Of significance to their biological properties is that the lattice is undulating with a periodicity of 0·1–0·5 μm. This would also account for the difficulties encountered by X-ray and electron diffraction techniques when examining these crystals.     

Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing

In this paper, the Rapid Transfer System (RTS), an attachment to the Leica EMPACT2 high‐pressure freezer, is described as a new tool for special applications within the cryofixation field. The RTS is an automated system that allows for fast processing of samples (<5 s) that make it possible for the first time to use high‐pressure freezing in combination with high time resolution correlative light and electron microscopy. In addition, with a working cycle of 30 s this rapid turn over time allows one to acquire more samples of biopsy material before it deteriorates than with other HPF machines with longer cycle times. With the use of the RTS it was possible to obtain three samples each of four different tissues in 6 min. Together with the finding that 90% of samples of cells grown on sapphire discs were well frozen, the RTS was both fast and reliable. Most important, together with other newly developed accessories, the RTS made it possible to capture specific events occurring live in the cell as observed by light microscopy, to cryofix that sample/event within 4 s, and then to analyze that event at high resolution in the electron microscope with excellent preservation of ultra‐structure. These developments should give us the tools to unravel intracellular processes that can be observed by live cell imaging but are too rare or fast to be picked up by routine EM methods or where the history of a structure is necessary to be able to discern its nature.     

Characterization of dentine structure in three dimensions using FIB-SEM

All biological tissues are three dimensional and contain structures that span a range of length scales from nanometres through to hundreds of millimetres. These are not ideally suited to current three-dimensional characterization techniques such as X-ray or transmission electron tomography. Such detailed morphological analysis is critical to understanding the structural features relevant to tissue function and designing therapeutic strategies intended to address structural deficiencies encountered in pathological states. We show that use of focused ion beam milling combined with scanning electron microscopy can provide three-dimensional information at nanometre resolution from biologically relevant volumes of material, in this case dentine.     

Effect of the metallic modifier is nature on the surface microstructure of the phenolic carboplastic-steel interface

Atomic force microscopy and scanning electron microscopy are used to explore the effect of the nature of fine-grained metallic modifiers (babbit B-83, nickel, copper) on the friction-surface morphology and triboengineering characteristics of phenolic carboplastics. It is demonstrated that introduction of a modifier reduces the wear rate of the composite by 2–10 times, while the mechanism of wear of carboplastics is determined by the modifier is nature.     

Determination of the size distribution of lecithin liposomes: a comparative study using freeze fracture, cryoelectron microscopy and dynamic light scattering

The size distribution of liposomes is often determined using freeze fracture, cryoelectron microscopy or dynamic light scattering. However, the resulting size distributions do not directly coincide owing to the different weighting of the techniques. We present several methods which correct for these effects and allow a comparison of liposome size distributions as obtained by freeze fracture, cryoelectron microscopy or dynamic light scattering. These methods are based on theoretical models for the weighting of the size distribution of liposomes, which result from the preparation procedure for freeze fracture electron microscopy and from the measurement by dynamic light scattering. The proposed transformation methods are then experimentally tested with a sample of lecithin liposomes, whose size distribution was determined by dynamic light scattering, freeze fracture and cryoelectron microscopy. Furthermore, the weaknesses of the experimental techniques and hence of the resulting size distributions are discussed.     

Reflection electron microscopy methods for the study of surface structure

The techniques of reflection electron microscopy (REM) using TEM instruments and scanning reflection electron microscopy (SREM) using STEM instruments have been explored as means for the observation of surface structure with high spatial resolution, better than 1 nm in each case. Under the ordinary environment of a commercial TEM instrument, we have studied the contrast in REM images of atomic steps and made comparison with the calculated results from the multi-slice dynamical diffraction theory. Comparison has also been made between the REM images of defects and the calculated images based on the column approximation. The influence of surface resonances on the contrast has been investigated. By SREM performed in a modified HB5 STEM with attached high vacuum preparation chamber, we have observed the formation of periodically distributed Pd particles on the surface of cleaved MgO.     

Scanning electron microscopy of the capillary loops in the dermal papillae of the hand in primates,including man

The microvasculature of the skin of the hand in primates, including man, was examined by means of scanning electron microscopy of corrosion casts. In this study, the microvascular patterns and structures in different areas of the hand, and the changes in vascular patterns that occur with age, have been described. The typical structure of the capillary loops in the hand can be observed in the ball of the finger of the young adult monkey. The capillary loops were formed out of not just one capillary vessel, but two or three vessels. Each capillary vessel arose and divided into several branches at the papillae, and these became descending limbs. After the loop passed a hairpin turn, the descending limbs were 1.5 times larger than the ascending limbs in the intrapapillary portion, and they became extrapapillary venules. The descending limbs connected with the postcapillary venules in the postpapillary portion and with the horizontal network. The postcapillary venules fused with each other to form the primary and secondary venous arcades. The secondary venous arcades anastomosed with each other and flowed into the subpapillary venules, which run along the dermal furrow in the fingerprint. Changes in vascular patterns with age could be observed. In the infant fingerprint, the vascular systems had not yet differentiated, especially the venous system in the dermis. In the old adult finger, the capillary loops presented complicated features deviating due to aging.     

A combined light and electron microscopic method for the visualization of the same in vitro neuron by radioautography and serial sections

We report here on a technical improvement which makes it possible to study, at the ultrastructural level, a dopaminergic neuron which has been previously identified by light microscopy. Primary cultures of virtually pure mesencephalic neurons from mouse embryos were obtained. These cultures were kept for 6 days, then incubated with tritiated dopamine. fixed and embedded in Epon. The dopaminergic neurons were firstly visualized by radioautography directly through Epon blocks in toto by light microscopy. In a second step, ultrathin sections of the identified dopaminergic cells were prepared and the neurons observed at the electron microscopy level. The dopaminergic nature of these neurons was regularly checked by radioautographic control on some selected ultrathin sections.     

Effect of streptozotocin on the ultrastructure of rat pancreatic islets

Our objective was to study the effects of three (30, 40, and 50 mg/kg) doses of Streptozotocin (STZ) on fasting plasma glucose level (FPG) and observe its effects at the cellular level in rat pancreas by electron microscopy. FPG was measured in rats before induction of diabetes and then on 3, 7, and 14 days after induction of diabetes with STZ. Keto diastix urine strips were used to check urine glucose and ketone bodies. Two weeks after the induction of diabetes, the rat pancreas was removed and fixed for light and electron microscopic studies. Three days after induction, the mean FPG level was 112 mg/dl in Group I (30 mg/kg STZ), 217 mg/dl in Group II (40 mg/kg STZ), and 376 mg/dl in Group III (50 mg/kg STZ). Histology was normal in Group I but revealed altered islet structure in Groups II and III. Ultrastructure revealed intact D cells in all three groups. The focal mitochondria and Golgi complex swelling found in A and B cells was occasional in Group I and frequent in Groups II and III. Swelling of other organelles and reduction in the size and number of granules was further observed in Group III. It is our conclusion that the 30-mg/kg body weight STZ produces mild changes while 50 mg/kg proves to be fatal. STZ at 40 mg/kg has a moderate effect on plasma glucose as well as on the islets of Langerhans at a cellular level.     

The transillumination possibility of imidazole-osmium postfixed muscle tissue and its consequences for the handling of muscle tissue samples

The osmium postfixation of tissue leads to good results for transmission electron microscopy, but also produces completely blackened tissue samples that do not allow the recognition of internal structures. With imidazole-osmium postfixation, one achieves comparable results in high electron microscopic resolution as with routine osmium postfixation. But the tissue samples are not blackened and can, therefore, be transilluminated with point light sources. The new postfixation technique makes it possible to recognize histological details such as vessels, nerve fibers, and the cross-banding pattern in an untrimmed block. This makes it possible to screen-embedded tissue samples for appropriate ultrastructural processing.     

Unravelling the structure of the lamellipodium

Summary Pushing at the cell front is the business of lamellipodia and understanding how lamellipodia function requires knowledge of their structural organization. Analysis of extracted, critical-point-dried cells by electron microscopy has led to a current dogma that the lamellipodium pushes as a branched array of actin filaments, with a branching angle of 70 degrees , defined by the Arp2/3 complex. Comparison of different preparative methods indicates that the critical-point-drying-replica technique introduces distortions into actin networks, such that crossing filaments may appear branched. After negative staining and from preliminary studies by cryo-electron tomography, no clear evidence could be found for actin filament branching in lamellipodia. From recent observations of a sub-class of actin speckles in lamellipodia that exhibit a dynamic behaviour similar to speckles in the lamella region behind, it has been proposed that the lamellipodium surfs on top of the lamella. Negative stain electron microscopy and cryo-electron microscopy of fixed cells, which reveal the entire complement of filaments in lamellipodia show, however, that there is no separate, second array of filaments beneath the lamellipodium network. From present data, we conclude that the lamellipodium is a distinct protrusive entity composed of a network of primarily unbranched actin filaments. Cryo-electron tomography of snap-frozen intact cells will be required to finally clarify the three-dimensional arrangement of actin filaments in lamellipodia in vivo.     

Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature

A survey of methods combining light microscopy and scanning electron microscopy is presented. A simple correlation is made when two preparations from adjacent parts of one specimen are investigated in two different microscopes. A more sophisticated method is the consecutive investigation of one specimen with two microscopes. A major problem in this method is the relocation of the area of interest. Several authors have presented solutions for this problem. It is preferable when one preparation is investigated in only one instrument, combining the two microscopical (LM and SEM) techniques, thus making relocation redundant.