Fine Structure of Botrytis fabae Sardina Conidia

The fine structure of Botrytis fabae conidia was studied usinga variety of electron-microscope techniques. The spore walllacks conspicuous ornamentation and consists of microfibrilsembedded in a granular matrix. The two distinct wall layersseen in chemically fixed sections cannot be detected in cross-fracturedreplicas; the two layers are probably structurally similar.The outer surface of the plasmalemma is covered with branchedinvaginations and two kinds of particles. Three distinct typesof particles are present on the inner surface of the plasmalemma.In freeze-etched replicas nuclei, vacuoles, and other organellesalways appear smoothly rounded. Small vesicles pass throughthe plasmalemma into the cell wall. Particles approximately10 nm in diameter occur in compact rows on the cristae of cross-fracturedmitochondria: dense spherical particles, probably of calciumphosphate, are present in chemically fixed mitochondria. Prevacuolesand vesicles with membranous inclusions can be seen in bothcross-fractured replicas and chemically fixed sections. In cross-fracturedreplicas vacuoles and lipid bodies are frequently joined bystrands of endoplasmic reticulum.     

Distribution of sialoglycoconjugates on the luminal surface of the endothelial cell in the fenestrated capillaries of the pancreas

Sialic acid-bearing molecules on the luminal surface of the vascular endothelium in mouse and rat pancreatic capillaries were detected electron microscopically by using a procedure with ferritin hydrazide (FH), after preferential oxidation of sialyl residues with sodium periodate. The distribution of FH on the endothelial surface demonstrated the existence of microdomains with various densities of sialoglycoconjugates oxidizable by sodium periodate and accessible to the tracer. On the plasmalemma proper, FH binding sites were heterogeneously distributed. Their concentration on various microdomains decreased as follows: plasmalemma proper greater than coated pits greater than stomal diaphragms of plasmalemmal vesicles and transendothelial channels, and fenestral diaphragms. The membrane of plasmalemmal vesicles and transendothelial channels was not labeled by FH. Nonspecific binding of FH to the nonoxidized endothelial surface or that oxidized after neuraminidase treatment was relatively low.     

Anionized and cationized hemeundecapeptides as probes for cell surface charge and permeability studies: differentiated labeling of endothelial plasmalemmal vesicles

To obtain small membrane markers easily accessible to the charged groups of the cell surface, we prepared, from hemeundecapeptide (HUP), three derivatives that maintain the peroxidatic activity: the anionized hemeundecapeptide, Mr 1,963, estimated diameter 1.68 nm, pl 3.5, for the detection of basic groups; and both a cationized hemeundecapeptide containing predominantly tertiary amino groups, Mr 2,215, estimated diameter 1.75 nm, pl 9.0, and a cationized hemeundecapeptide containing only primary amino groups, Mr 2,271, estimated diameter 1.75 nm, pl 10.6, for labeling acidic residues. The markers were perfused in situ in mice to label the luminal surface of fenestrated endothelium of pancreatic capillaries. Specimens were processed through the cytochemical reaction for peroxidatic activity and examined by electron microscopy. The anionized HUP and HUP (pl 4.85) marked the plasmalemma proper, the coated pits, and the membrane and diaphragms of plasmalemmal vesicles and transendothelial channels. The cationized HUP containing predominantly tertiary amino groups (pl 9.0) decorated all cell surface components with the exception of plasmalemmal vesicles and channels; the latter were, however, labeled by the cationized HUP containing only primary groups (pl 10.6), which suggests that these structures contain on their luminal surface very weak acidic residues of high pKa values. The fact that the membrane of plasmalemmal vesicles can discriminate against permeant cationic macromolecules only up to a pl of approximately 9.0 indicates that in the electrostatic restriction there is a charge limit. In the case of fenestrated capillary endothelium, the upper charge limit seems to be a pl of approximately 9.0. In these vessels, the charge discrimination is effective for molecules as small as 2 nm.     

Characean charasome-complex and plasmalemma vesicle development

Summary We report on an unusual phenomenon which occurs in some characean algae as a normal plasma membrane activity and also in association with charasome formation. The phenomenon of formation of coated invaginations of the plasma membrane was observed in twoChara and 6Nitella species. These invaginations are coated on their cytoplasmic surface, are 50–60 nm in diameter and rarely exceed 60 nm in length. They are abundant in the young cells ofChara andNitella and also occur in mature cells, but at a lower frequency.N. translucent is an exception in that coated invaginations were few in the young cells and absent in mature cells. Coated vesicles (50–60 nm diameter) were closely associated with these invaginations. Our observations suggest the vesicles may be derived from the invaginations by endocytosis.A close relationship was noted between the development of charasomes (plasmalemma modifications) and coated invaginations. Numerous coated invaginations are seen along the membranes of young charasomes; these invaginations appear to be associated with growth of the charasomes. Coated vesicles were not associated with the coated invaginations of the charasome membrane. The tubular network of cytoplasm and wall space seen in the mature charasome may be formed by fusion of coated invaginations of the developing charasomes, leaving cytoplasmic strands between the fused portions. Coated invaginations were not present along charasomes of the mature cells.     

Isolation and partial characterization of the luminal plasmalemma of microvascular endothelium from rat lungs.

This paper describes a procedure for isolating in high yield and at a high degree of purity the endothelial luminal plasmalemma from the microvasculature of the rat lung. The procedure relies on the modification of the density of the luminal plasmalemma obtained by coating it by perfusion in situ first, with cationized colloidal silica and then with Na polyacrylate. These steps generate a strongly adhering coat to the luminal plasmalemma that resists tissue homogenization to yield, upon repeated centrifugation through Nycodenz density gradients, a nearly homogeneous fraction of coated luminal plasmalemmal fragments still carrying their associated plasmalemmal vesicles. The fraction is enriched in the luminal plasmalemmal antigen, angiotensin converting enzyme, contains gp60, an antigen expected to occur on both plasmalemmal domains, is not enriched in either alkaline phosphatase or 5'-nucleotidase activity and is free of the mitochondrial and endoplasmic reticulum antigens so far tested. This procedure, that can be extended--in principle--to any vascular bed, obviates the use of cultured cells for studying the biochemistry of the endothelium, at least as far as the luminal endothelial plasmalemma is concerned.     

Maturation of myocardial capillaries in the fetal and neonatal rat: an ultrastructural study with a morphometric analysis of the vesicle populations

The ultrastructural morphology of the cellular and extracellular components of the developing myocardial capillary wall--from the 16-day-gestation fetus of the rat to the 21-day neonate--was examined. A morphometric analysis of plasmalemmal vesicles and of coated vesicles and pits of capillary endothelial cells was performed during the same developmental period. As the lateral extensions of the capillary endothelial cells change from irregular to regular in their thickness during development, there is an increase in the number of plasmalemmal vesicles and a progression from clusters of plasmalemmal vesicles to a uniform distribution in the endothelial cell. The ratio of vesicles which are open to the luminal front, which are "free" in the cytoplasm, or which are open to the abluminal front of the endothelial cell was consistent throughout development. The numerical density of plasmalemmal vesicles demonstrates a gradual and significant increase. In contrast, the numbers of coated vesicles and pits are variable within a very narrow range, and no pattern of increase or decrease is discernible during development. Similarly, there is no change in interendothelial cell junctions, which consist of occluding and primitive adhesive junctional types, during development. The lamina densa of the basal lamina gradually develops from discontinuous, patchy densities along the abluminal surface of the endothelial cells to a continuous and distinct layer by 21 days gestation. The presence of the proteoglycan species in the developing basal lamina was assessed with the cationic dye ruthenium red (RR), and the appearance of RR-marked proteoglycans was found to parallel the appearance of lamina densa material. found to parallel the appearance of lamina densa material. The RR sites appear discontinuously in patches; and later, the RR sites appear in a continuous and regular planar lattice in the lamina rara interna and externa at 21 days gestation. A complete array of RR-stainable anionic sites outside a continuous lamina densa near birth indicates that the basal laminae of developing capillaries in the heart are morphologically, and in part biochemically, mature by the end of the first neonatal week. Our results show that the endothelial cells and the subtending basal lamina of myocardial capillaries gradually mature morphologically during the final days of gestation and the first neonatal week.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron microscopic studies of coated membranes in two types of gill epithelial cells of lamprey

Summary Coated membranes in two types of gill epithelial cell of adult lamprey, Lampetra japonica, were studied by electron microscopy. The type 3 gill epithelial cells possess well-developed microvilli or microfolds, apical vesicles and abundant mitochondria. The cytoplasmic surface of the microvillous plasma membrane is covered by a coat of regularly spaced particles with a center-to-center distance of about 15 nm. Each particle consists of a bulbous free end, about 10 nm in diameter, and a connecting piece, about 5 nm long. Apical vesicles are covered by a surface coat which consists of fine filamentous material but lack any special coating on their cytoplasmic surface.The type 4 cells (chloride cells) are characterized by apical vesicles, abundant mitochondria and cytoplasmic tubules. These tubules possess a coat on their luminal surface which consists of spirally wound parallel rows of electron-dense materials. The rows are about 16 nm apart and wound at a pitch of about 45°. The cytoplasmic surface of these tubules does not display a special coat. These coated membranes are assumed to be the sites of active ion transport across the plasma membrane. In particular, particles in type 3 cells and linear coat materials in chloride cells may be either loci of transport enzymes or energy generating systems. Apical vesicles lack any coating on their cytoplasmic surface but a fine filamentous coat is present on their luminal surface. They contain intraluminal vesicles and are continuous with apical ends of cytoplasmic tubules.     

Depolarization redistributes synaptic membrane and creates a gradient of vesicles on the synaptic body at a ribbon synapse

We used electron tomography of frog saccular hair cells to reconstruct presynaptic ultrastructure at synapses specialized for sustained transmitter release. Synaptic vesicles at inhibited synapses were abundant in the cytoplasm and covered the synaptic body at high density. Continuous maximal stimulation depleted 73% of the vesicles within 800 nm of the synapse, with a concomitant increase in surface area of intracellular cisterns and plasmalemmal infoldings. Docked vesicles were depleted 60%-80% regardless of their distance from the active zone. Vesicles on the synaptic body were depleted primarily in the hemisphere facing the plasmalemma, creating a gradient of vesicles on its surface. We conclude that formation of new synaptic vesicles from cisterns is rate limiting in the vesicle cycle.     

Functional maturation of the capillary wall in the fetal and neonatal rat heart: permeability characteristics of developing myocardial capillaries

The development of the functional components of the myocardial capillary wall was characterized by time-course studies of transendothelial transport of intravascularly injected probes of graded size from 16 days of gestation in the fetal rat to seven days postpartum. Despite the morphological changes occurring in the developing endothelial cells, the interaction of the probes was similar throughout the developmental period studied. The carbon particles were retained within the capillary lumina without any association with interendothelial junctions or with plasmalemmal vesicles. Carbon also was associated with coated vesicles. In contrast to carbon, ferritin was localized sequentially, over 60 sec of circulation, in plasmalemmal vesicles on the lumenal surface, in the cytoplasm, and on the ablumenal surface of the endothelial cells as well as in the interstitial space. Ferritin was located also in coated pits and vesicles and, after 90 sec of circulation, in multivesicular bodies. Within 30 sec of circulation, reaction product of myoglobin was located in plasmalemmal vesicles, coated vesicles, and transendothelial cell channels. Also within 30 sec, myoglobin partially filled the interendothelial space from the capillary lumina to the level of the tight junction. At all developmental ages studied, the interendothelial cell junctions appeared structurally tight and were impermeable to all of the probes. Once ferritin or myoglobin had reached the ablumenal space, the basal lamina did not appear to restrain the passage of the probes. Plasmalemmal vesicles are the capillary structures which transendothelially transport ferritin and myoglobin in developing myocardial capillaries.     

Binding and transcytosis of glycoalbumin by the microvascular endothelium of the murine myocardium: evidence that glycoalbumin behaves as a bifunctional ligand

The binding and transport of glycoalbumin (gA) by the endothelium of murine myocardial microvessels were studied by perfusing in situ 125I-gA or gA-gold complexes (gA-Au) and examining the specimens by radioassays and EM, respectively. After a 3-min perfusion, the uptake of radioiodinated gA is 2.2-fold higher than that of native albumin; it is partially (approximately 55%) competed by either albumin or D-glucose, and almost completely abolished by the concomitant administration of both competitors or by gA. D-mannose and D-galactose are not effective competitors. Unlike albumin-gold complexes that bind restrictively to plasmalemmal vesicles, gA-Au labels the plasma-lemma proper, plasmalemmal vesicles open on the lumen, and most coated pits. Competing albumin prevents gA-Au binding to the membrane of plasmalemmal vesicles, while glucose significantly reduces the ligand binding to plasmalemma proper. Competition with albumin and glucose gives additive effects. Transcytosis of gA-Au, already detected at 3 min, becomes substantial by 30 min. No tracer exit via intercellular junctions was detected. gA-Au progressively accumulates in multivesicular bodies. The results of the binding and competition experiments indicate that the gA behaves as a bifunctional ligand which is recognized by two distinct binding sites: one, located on the plasma membrane, binds as a lectin the glucose residues of gA; whereas the other, confined to plasmalemmal vesicles, recognizes presumably specific domains of the albumin molecule.     

Vesicular transport in capillary endothelium: does it occur?

A revised picture of the organization of endothelial plasmalemmal vesicles is presented. Three-dimensional reconstructions of endothelial segments from frog mesenteric capillaries and rat heart capillaries based on ultrathin serial sectioning have shown that plasmalemmal vesicles are not true vesicles but parts of an elaborate system of invaginations of the surface membrane. The revised picture probably applies to capillary endothelia in general. The absence of free cytoplasmic vesicles implies that vesicular transport is unlikely to occur. A reinterpretation of previous studies of vesicular transport shows that they are equally compatible with the present view that plasmalemmal vesicles are static elements of invaginations of the endothelial surface membrane.     

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.     

Endocytic vesicles and surface invaginations in cultured vascular endothelium: a morphometric comparison

Ruthenium red staining of plasma membrane glycoproteins of confluent cultured arterial endothelial cells revealed that the limiting membrane of many apparently discrete cytoplasmic vesicles was continuous with the plasmalemma. Surface invaginations accessible to ruthenium red appeared as vesicles when sectioned out of the plane of attachment to the cell surface, Morphometric analysis of ruthenium red-positive (RR+) and ruthenium red-negative vesicles (RR-) indicated that 47.2% of the total apparent vesicle population was RR+ and that those infoldings accounted for 19.6 +/- 1.4% of the cell surface in transverse sections. Whereas 14.9% of the true vesicles (ruthenium red-negative) were coated vesicles, only 1.1% of RR+ "vesicles" were coated pits. These studies show that although many deep infoldings of the cell surface may be misinterpreted as vesicles, almost all are uncoated. The existence of discrete coated vesicles (independent of coated pits) in vascular endothelium in vitro is readily apparent.     

Microplicae: characteristic ridge-like folds of the plasmalemma

Scanning electron microscopy reveals that the free surfaces of stratified squamous epithelial cells lining the alimentary tract, cornea, and conjunctiva exhibit characteristic ridge-like folds of plasmalemma. These microplicae are approximately 0.1-0.2 micronm in width, of variable height (0.2-0.8 micronm) and length, may followstraight or winding paths, often branch, and exhibit a wide variety of patterns over the surfaces of cells. Transmission electron microscopy reveals that microplicae often have a fine (100-150 A) electron-dense zone subjacent to their plasmalemma and an intracellular matrix characterized by a disorderly arrary of fine filaments (40-60 A in diameter). Microplicae appear to arise from plasmalemmal fold which once provided for intercellular interdigitation and desmosome abhesion between adjacent cells. Ruthenium red staining demonstrates that microplicae and interplical grooves are covered with a polyanionic glycocalyx. Although free surface microplicae may merely represent the renmants of intercellular interdigitations or a modified expression of microvillous-like extensions, it is also possible that they serve another specific function. In this regard it is speculated that microplical and interplical grooves may function to hold a layer of lubricating and cushioning mucin designed to protect the underlying plasmalemma from abrasive abuse.     

Organelle relationships in cultured 3T3-L1 preadipocytes

In differentiating 3T3-L1 cells, lipid spheres, the endoplasmic reticulum (ER), microperoxisomes, and mitochondria form "constellations" that may reflect the interplay of lipid metabolizing enzymes in these organelles. ER cisternae are also situated very close to "rosettes,"plasmalemmal specializations found in mature adipocytes in vivo. As in hepatocytes and absorptive cells of the intestine, this spatial relationship of ER and plasmalemma suggests a role for rosettes in the uptake of exogenous lipid precursors. The morphological differentiation of 3T3-L1 preadipocytes includes the loss of "stress fibers" and the appearance of microfilament like structures that encase, in a complex manner, the cytosolic lipid spheres that appear during differentiation. Other features described for the first time in 3T3-L1 preadipocytes include: (a) the presence of an extensive acid phosphatase (AcPase) positive GERL from which coated vesicles apparently arise (these coated vesicles display AcPase activity and are much smaller and far more numerous than the coated vesicles that seem to arise from the plasmalemmal coated pits); (b) the abundance of AcPase-positive autophagic vacuoles; and (c) a high level of alpha- naphthyl-acetate-esterase activity which, by light microscopy cytochemistry, appears to be localized in the cytosol.     

Secretion of acid phosphatase in<Emphasis Type="Italic">Claviceps purpurea</Emphasis> — an ultracytochemical study

The lead phosphate precipitation method showed the reaction product of acid phosphatase (which reflects the presence of the enzyme glycoprotein) in peripheral cytoplasmic vesicles in the ascomycetous fungus Claviceps purpurea. The product appeared to diffuse from these vesicles (diameter 100-200 nm) towards the cell wall, usually to its sites covered by the capsular fibres exhibiting also acid phosphatase activity. This observation of diffusion of secretory glycoprotein in the cytoplasmic matrix and its orientation to the plasmalemma and capsular fibrils suggests an alternative to the well-described secretory mechanism of transport and exocytosis of glycoproteins via membrane-bound transport conveyors fusing with the cell membrane. It confirms and enlarges our previous finding of the reaction product of acid phosphatase performed by ultrastructural cytochemistry in vacuoles (lysosomes), in the growing cell septum, in cytoplasmic vesicles and in the fibres of the external capsule.     

REGULAR STRUCTURES IN MEMBRANES : I. Membranes in the Endocytic Complex of Ileal Epithelial Cells

An "apical endocytic complex" in the ileal lining cells of suckling rats is described. The complex consists of a continuous network of membrane-limited tubules which originate as invaginations of the apical plasma membrane at the base of the microvilli, some associated vesicles, and a giant vacuole. The lumenal surface of this tubular network of membranes and associated vesicles is covered with a regular repeating particulate structure. The repeating unit is an ~7.5-nm diameter particle which has a distinct subunit structure composed of possibly nine smaller particles each ~3 nm in diameter. The ~7.5-nm diameter particles are joined together with a center-to-center separation of ~15 nm to form long rows. These linear aggregates, when arranged laterally, give rise to several square and oblique two-dimensional lattice arrangements of the particles which cover the surface of the membrane. Whether a square or oblique lattice is generated depends on the center-to-center separation of the rows and on the relative displacement of the particles in adjacent rows. Four membrane faces are revealed by fracturing frozen membranes of the apical tubules and vesicles: two complementary inner membrane faces exposed by the fracturing process and the lumenal and cytoplasmic membrane surfaces revealed by etching. The outer membrane face reveals a distinct array of membrane particles. This array also sometimes can be seen on the outer (B) fracture face and is sometimes faintly visible on the inner (A) fracture face. Combined data from sectioned, negatively stained, and freeze-etched preparations indicate that this regular particulate structure is a specialization that is primarily localized in the outer half of the membrane mainly in the outer leaflet.     

Three classes of spiny-(Clathrin-) coated vesicles in rodent liver based on size distributions

Summary Clathrin-coated vesicles in rodent (rat and mouse) liver distribute into three distinct populations based on measurements of vesicle diameter. The first population consists of 60–80 nm vesicles found almost exclusively within the Golgi apparatus region. The second population is of 100–160 nm coated vesicles located within 100–500 nm of the cell surface. A third population of coated vesicles of intermediate diameter (ca. 90 nm) is present both at the Golgi apparatus and at the cell surface. We speculate that clathrin and clathrin-coated vesicles within the region of the Golgi apparatus and of the cell surface exist in two recycling populations. The third population of vesicles of intermediate diameter could represent a shuttle to link the two major compartments.     

The ultrafine structure of the body wall of sexually mature thorny-headed worms Echinorhynchus gadi (Acanthocephala)]

Studies of the fine structure of the adult acanthocephalan Echinorhynchus gadi have given a new information on the structure and organization of the body wall of these parasitic helminths. Their body surface is covered by glycocalyx of mucopolysaccharide nature. Just under it there is the surface membrane which has numerous invaginations forming a network of branching canals from which membrane vesicles are isolating. In their turn these canals pass through "the cytoplasmic canals" of the cortical matrix. Between the surface membrane and cortical matrix there is the base plate. These three structures form the striped layer underlain by the felt layer. It is formed by three layers of fibrous strands (one circular and two longitudinal), which are parallel to the body surface. These strands consist of loosely laid fibrils. The lowest layer is a radial one which occupies 2/3 of the body wall. It consists of the radial strands beginning from the cortical matrix and ending at the basement membrane. Numerous lipid droplets and glycogen granules are formed here. Two types of fibrils with 0.26 and 0.05 diameter have been detected for the first time. The radial layer in the cytoplasm was found to have crystalline structures and polymembrane bodies, numerous nuclei with light karyoplasm and distinct nucleoli. The location of the nuclei is of two types: either in the cytoplasm or in the "lacunae". We have shown that the "lacunae" are specialized sites of the cytoplasm whose boundaries are marked by the fibres of two types. Besides, this type of the acanthocephalan was found to have two "giant lacunae" extending along the body.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine Structures of Cell Envelopes of Chlamydia Organisms as Revealed by Freeze-Etching and Negative Staining Techniques

The cell walls of Chlamydia psittaci (meningopneumonitis strain) were examined by the freeze-etching and negative staining techniques. It was observed that the cleaved convex surface of the developmental, reticulate body was covered with numerous non-etchable particles 9 to 10 nm in diameter, these particles being rarely seen on the concave surface. Similarly, the convex surface of the mature, elementary body (EB) was covered with many particles but the concavity lacked these particles. After etching, the smooth concave surface of the EB appeared to have a hexagonally arrayed subunit structure, on which the button structure (B structure) was observed. Each B structure had a diameter of 27 nm and several B structures were grouped together in a hexagonal arrangement with a center-to-center spacing of 45 nm. In a limited area of the negatively stained EB cell wall, hexagonally arrayed rosette structures were present, with a center-to-center spacing similar to the B structures seen in the freeze-etched preparation. Each rosette, about 19 to 20 nm in diameter, appeared to be composed of a radial arrangement of nine subunits. The freeze-fractured cell wall-cytoplasmic membrane complexes indicated that the outer surface of the cytoplasmic membrane which appeared as the convex surface was covered with the fine particles, and thus it was likely that frozen EB was cleaved at the gap between the cell wall and ctyoplasmic membrane. On the cleaved inclusion, several groups of fine particles were observed. In each group, the particles were arranged hexagonally with the spacing ranging from 20 to 50 nm.