Characterization and implications of the cell surface reactivity of Calothrix sp. strain KC97

The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK(a)] between 5.0 and 6.2) and amine groups (mean pK(a) of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK(a) spectra similarly dominated by carboxyls (pK(a) of 4.6 to 6.1) and amines (pK(a) of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK(a) values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 x 10(-5) and 12.3 x 10(-5) mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.     

Characterization and Implications of the Cell Surface Reactivity of Calothrix sp. Strain KC97

The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK_a] between 5.0 and 6.2) and amine groups (mean pK_a of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK_a spectra similarly dominated by carboxyls (pK_a of 4.6 to 6.1) and amines (pK_a of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK_a values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 × 10⁻⁵ and 12.3 × 10⁻⁵ mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.     

Fine Structure of Listeria monocytogenes in Relation to Protoplast Formation

Among the eight strains of Listeria monocytogenes tested for lysozyme sensitivity, two were resistant to lysozyme but became sensitive after lipase pretreatment. Among the other six, one was very sensitive to lipase and another one was extremely susceptible to lysozyme. Stable protoplasts were formed from the lysozyme-resistant strain (42) by lipase and lysozyme treatment, which completely digested the cell wall. The cell wall (uranyl acetate-lead stained) was of a thick triple-layered profile, with the intermediate layer of low density. Lipase treatment for a short time (60 min) did not cause any alteration in structure, but prolonged treatment (180 min) caused extensive digestion of the plasma membrane and the cell wall, liberating cytoplasmic material. When the cells were treated with either lipase or lysozyme, a small number of protoplasts were extruded through the partly digested or weakened transverse cell wall, leaving an almost intact cell wall ghost. There were small vesicular structures in the interspace between cell wall and plasma membrane. Mesosomes of varied organization were prominent in electron micrographs, both in sections and in negatively stained preparations. These were largely everted during protoplasting in the form of tubules and as small peripheral buds; a few small vesicles also remained as intrusive structures, some of which were very unusual because they appeared to be enclosed by the inner layer of plasma membrane alone. Lysis of the protoplasts by dilution of the sucrose, while maintaining a constant ionic environment, liberated many small vesicular structures and fibrillar nuclear material.     

Cyst formation in a freshwater strain of the choanoflagellate Desmarella moniliformis Kent

Cyst formation in a freshwater strain of the colonial freshwater choanoflagellate Desmarella moniliformis Kent (Protozoa: Choanoflagellida) has been studied with light and electron microscopy for the first time. Batch cultures inoculated with motile vegetative cells start to produce cysts within 3 days during the exponential phase of growth. Cyst production proceeds until in late stationary phase there is a preponderance of cysts. Transfer of cysts to fresh medium results in limited excystment. Encystment involves the production of electron-dense fibrillar wall material, firstly around the neck of the cell and then around the posterior end. As the wall material is deposited the neck of the cell elongates and the dictyosome rotates from the horizontal to vertical plane. The number of mitochondrial profiles seen in individual sections of cells increases. Finally the neck of the cell is retracted, the flagellum and collar tentacles are withdrawn, and the bottom of the neck of the cyst wall is sealed with a diaphragm of wall material. Excystment, which has not been observed directly, appears to involve the disruption of the wall at the base of the neck, the remainder of the cyst wall remains intact. Comparisons are made between encystment in Desmarella and cyst development in other protists.     

Biaxial elastic material properties of porcine coronary media and adventitia

The importance of mechanical stresses and strains has become well recognized in vascular physiology and pathology. To compute the stress and strain on the various components of the vessel wall, we must know the constitutive equations for the different layers of the vessel wall. The objective of the present study is to determine the constitutive equation of the coronary artery treated as a two-layer composite: intima-media and adventitial layers. Twelve hearts were obtained from a local slaughterhouse, and the right coronary artery and left anterior descending artery were dissected free from the myocardium. The vessel wall was initially mechanically tested biaxially (inflation and axial extension) as a whole (intact wall) and subsequently as intima-media or adventitial layer. A Fung-type exponential strain energy function was used to curve fit the experimental data for the intact wall and individual layers for the right coronary artery and left anterior descending artery. Two methods were used for the determination of material constants, including the Marquardt-Levenberg nonlinear least squares method and the genetic algorithm method. Our results show that there were no statistically significant differences in the material constants obtained from the two methods and that either set of elastic constants results in good fit of the data. Furthermore, at an in vivo value of axial stretch ratio, we find that the stiffness is as follows: intima-media > intact > adventitia. These results underscore the composite nature of coronary arteries with different material properties in each layer. The present results are necessary for analysis of coronary artery mechanics and to provide a fundamental understanding of vessel physiology.     

Three-dimensional mechanical properties of porcine coronary arteries: a validated two-layer model

The normal coronary artery consists of two mechanically distinct layers: intima-media and adventitia. The objective of this study is to establish a two-layer three-dimensional (3-D) stress-strain relation of porcine coronary arteries. Experimental measurements were made by a series of biaxial tests (inflation and axial extension) of intact coronary arteries and, subsequently, their corresponding intima-media or adventitia layer. The Fung-type exponential strain energy function was used to describe the 3-D strain-stress relation for each layer and the intact wall. A genetic algorithm was used to determine the material constants in the Fung-type constitutive equation by curve fitting the experimental data. Because one layer must be sacrificed before the other layer can be tested, the material property of the missing layer was computed from the material constants of the intact vessel and the tested layer. A total of 20 porcine hearts were used: one group of 10 hearts for the left anterior descending artery and another group of 10 hearts for the right coronary artery. Each group was further divided into two subgroups of five specimens tested for the intact wall and the intima-media layer and for the intact wall and the adventitia layer. Our results show statistically significant differences in the material properties of the two layers. The mathematical model was validated by experimental stress-strain data for individual layers. The validated 3-D constitutive model will serve as a foundation for formulation of layer-specific boundary value problems in coronary physiology and cardiology.     

Role of elastin anisotropy in structural strain energy functions of arterial tissue

The vascular wall exhibits nonlinear anisotropic mechanical properties. The identification of a strain energy function (SEF) is the preferred method to describe its complex nonlinear elastic properties. Earlier constituent-based SEF models, where elastin is modeled as an isotropic material, failed in describing accurately the tissue response to inflation–extension loading. We hypothesized that these shortcomings are partly due to unaccounted anisotropic properties of elastin. We performed inflation–extension tests on common carotid of rabbits before and after enzymatic degradation of elastin and applied constituent-based SEFs, with both an isotropic and an anisotropic elastin part, on the experimental data. We used transmission electron microscopy (TEM) and serial block-face scanning electron microscopy (SBFSEM) to provide direct structural evidence of the assumed anisotropy. In intact arteries, the SEF including anisotropic elastin with one family of fibers in the circumferential direction fitted better the inflation–extension data than the isotropic SEF. This was supported by TEM and SBFSEM imaging, which showed interlamellar elastin fibers in the circumferential direction. In elastin-degraded arteries, both SEFs succeeded equally well in predicting anisotropic wall behavior. In elastase-treated arteries fitted with the anisotropic SEF for elastin, collagen engaged later than in intact arteries. We conclude that constituent-based models with an anisotropic elastin part characterize more accurately the mechanical properties of the arterial wall when compared to models with simply an isotropic elastin. Microstructural imaging based on electron microscopy techniques provided evidence for elastin anisotropy. Finally, the model suggests a later and less abrupt collagen engagement after elastase treatment.     

Characterization of the wound-induced material in Citrus paradisi fruit peel by carbon-13 CP-MAS solid state NMR spectroscopy

Grapefruit, Citrus paradisi, were injured, inoculated with Penicillium digitatum and incubated under conditions favourable for the accumulation of defence related material. Histochemical examination revealed that tissues adjacent to inoculated injuries contained phloroglucinol-HCl (PG-HCl) reactive material. Solvent washed cell wall preparations of intact and injured-inoculated peel were further purified using a mixture of cell wall degrading enzymes. Samples from injured inoculated tissue contained PG-HCl reactive globular material in addition to the fragments of xylem and cuticle found in controls. The principal chemical moieties of the material that accumulates in grapefruit injuries during wound-healing were studied by solid state 13C cross-polarization magic angle spinning NMR. A complete assignment of the NMR signals was made. From the analysis evidence was found that cellulose and hemicellulose are the biopolymers present in the intact peel samples, in addition, relevant quantities of cutin were found in the residues of enzyme digest. The NMR difference spectrum intact- wounded peels showed resonances which were attributed to all major functional groups of the aromatic-aliphatic suberin polyester of new material produced by the wounds. Information on the latter polyester was obtained by analyzing the T(1)rho (1H) relaxation.     

Mechanical Consequences of Cell-Wall Turnover in the Elongation of a Gram-Positive Bacterium

A common feature of walled organisms is their exposure to osmotic forces that challenge the mechanical integrity of cells while driving elongation. Most bacteria rely on their cell wall to bear osmotic stress and determine cell shape. Wall thickness can vary greatly among species, with Gram-positive bacteria having a thicker wall than Gram-negative bacteria. How wall dimensions and mechanical properties are regulated and how they affect growth have not yet been elucidated. To investigate the regulation of wall thickness in the rod-shaped Gram-positive bacterium Bacillus subtilis, we analyzed exponentially growing cells in different media. Using transmission electron and epifluorescence microscopy, we found that wall thickness and strain were maintained even between media that yielded a threefold change in growth rate. To probe mechanisms of elongation, we developed a biophysical model of the Gram-positive wall that balances the mechanical effects of synthesis of new material and removal of old material through hydrolysis. Our results suggest that cells can vary their growth rate without changing wall thickness or strain by maintaining a constant ratio of synthesis and hydrolysis rates. Our model also indicates that steady growth requires wall turnover on the same timescale as elongation, which can be driven primarily by hydrolysis rather than insertion. This perspective of turnover-driven elongation provides mechanistic insight into previous experiments involving mutants whose growth rate was accelerated by the addition of lysozyme or autolysin. Our approach provides a general framework for deconstructing shape maintenance in cells with thick walls by integrating wall mechanics with the kinetics and regulation of synthesis and turnover.     

Ultrastructure of soybean nodules. I: release of rhizobia from the infection thread

Root nodules on soybeans (var. Clark 63) were examined by transmission electron microscopy 10-12 days after seed inoculation and planting. The cell infection process appeared identical in both effective nodules, induced by Rhizobium japonicum strain 138 (USDA) and in ineffective nodules, induced by strain 8-0 (Iowa). Electron micrographs are presented which suggest that rhizobia are freed from the infection thread by disintegration of the thread wall and compartmentalization of the distintegrated wall material in membrane-bound vesicles derived from the membrane surrounding the thread. As the thread wall is removed in this manner, the bacteria are released into the host cytoplasm by a process which encloses each in an envelope also dervide from the thread membrane. Any thread wall material remaining around a bacterium after it has dissociated from the thread is removed from the envelope space by vesiculation of the membrane envelope. thus, it appears that endocytosis of both the bacteria and the material composing the infection thread wall occurs during release of rhizobia into the host cell.     

The legume Rhizosphere

Summary Examination of the root surfaces of Medicago tribuloides Desr. with phase contrast microscopy or electron microscopy using thin sections revealed the presence of a layer of material outside the root surface. In thin sections of KMnO₄ fixed roots this layer was composed of a thin electron dense layer, an electron dense granular matrix of varying width and an enclosing electron dense membrane. After inoculation with an effective Rhizobium strain, rhizobia were found aggregated in a definite zone adjacent to the root surface when either living roots were examined by phase microscopy or thin sections by electron microscopy. This layer was also found in inoculated and uninoculated roots of Trifolium fragiferum and T. pratense. The bacteria were packed with inclusion granules and lay enclosed by a membrane layer adjacent to the granular matrix seen in uninoculated roots. The ultrastructural organisation of root hairs is essentially similar to that of other differentiated root cells. The replicated surface of the uninoculated root hair wall is largely amorphous with a few sculptured portions resembling a cuticle layer. The inoculated root hair wall often shows areas of exposed, open microfibrillar meshwork with rhizobia sitting on the microfibrils. The rhizobia resemble a flagellated, coccoid swarmer form of Rhizobium which is found in the barrel medic rhizosphere.     

Diurnal difference in the amount of immunogold-labeled glucomannans detected with field emission scanning electron microscopy at the innermost surface of developing secondary walls of differentiating conifer tracheids

The differences between cell wall formation at night, when the tangential strain used as an index of the volumetric changes in differentiating cells is high, and in the day, when the tangential strain is low, were investigated in Cryptomeria japonica D. Don. Samples containing differentiating xylem were collected at 0500 hours and 1400 hours. The innermost surface of developing secondary walls in differentiating tracheids was observed by field emission scanning electron microscopy. In the specimens collected at 0500 hours, an amorphous material was observed covering the cellulose microfibrils. The cell wall surface was immunogold-labeled with an anti-glucomannan antiserum. After chlorite treatment, the amorphous material disappeared, and immunogold labeling was rarely observed. In the specimens collected at 1400 hours, cellulose microfibrils were clearly evident, and amorphous material and immunogold labeling were rarely observed. We thus confirmed that much amorphous material containing glucomannans is observed at night, when differentiating tracheids are turgid due to the increase in their volume, while the amorphous material was rarely observed during the day when cellulose microfibrils are clearly observed.     

Epicardial suction: a new approach to mechanical testing of the passive ventricular wall

The lack of an appropriate three-dimensional constitutive relation for stress in passive ventricular myocardium currently limits the utility of existing mathematical models for experimental and clinical applications. Previous experiments used to estimate parameters in three-dimensional constitutive relations, such as biaxial testing of excised myocardial sheets or passive inflation of the isolated arrested heart, have not included significant transverse shear deformation or in-plane compression. Therefore, a new approach has been developed in which suction is applied locally to the ventricular epicardium to introduce a complex deformation in the region of interest, with transmural variations in the magnitude and sign of nearly all six strain components. The resulting deformation is measured throughout the region of interest using magnetic resonance tagging. A nonlinear, three-dimensional, finite element model is used to predict these measurements at several suction pressures. Parameters defining the material properties of this model are optimized by comparing the measured and predicted myocardial deformations. We used this technique to estimate material parameters of the intact passive canine left ventricular free wall using an exponential, transversely isotropic constitutive relation. We tested two possible models of the heart wall: first, that it was homogeneous myocardium, and second, that the myocardium was covered with a thin epicardium with different material properties. For both models, in agreement with previous studies, we found that myocardium was nonlinear and anisotropic with greater stiffness in the fiber direction. We obtained closer agreement to previously published strain data from passive filling when the ventricular wall was modeled as having a separate, isotropic epicardium. These results suggest that epicardium may play a significant role in passive ventricular mechanics.     

Investigation of cell wall components in actinomycin D resistant Staphylococcus aureus]

Cell walls in 2 strains of Staphylococcus aureus 209P, i.e. actinomycin D susceptible and resistant ones were comparatively investigated. The resistant cells contained much more wall material per a unit of the biomass weight vs the susceptible strain cells, that conformed to thickening of the resistant cell walls detected by electron microscopy and a sharp increase of their electron density. Investigation of peptidoglycans and teichoic acids did not reveal any significant alterations in the structure of the wall components in the actinomycin D resistant cells. Only some increase of glucosamine in the peptidoglycan fraction of the resistant cells vs the susceptible ones was observed. It was shown that preparations of the resistant cell walls and peptidoglycan isolated from the resistant cells were able to bind somewhat lower quantities of actinomycin D vs the analogous preparations of the susceptible cells. The significant decrease of the antibiotic binding by live cells of the resistant strain probably slightly depended on the structure characteristics of the main wall components. The barrier properties of the walls in resistant staphylococci are most likely defined by the wall thickening and consolidation while adapting to actinomycin D.     

Ultrastructural features of phagocytosis and intracellular killing of Candida albicans by mouse polymorphonuclear phagocyte monolayers

Phagocyte monolayers provided a simple method of following ultrastructural events associated with phagocytosis and intracellular killing of Candida albicans. Preformed monolayers of mouse polymorphonuclear (PMN) phagocytes attached to glass coverslips were incubated with blastospore phase C. albicans and then examined by scanning and transmission electron microscopy. Scanning electron microscopy revealed phagocytosis of C. albicans by mouse phagocytes. Ingestion of the organism was facilitated by the production of lamellipodia by the phagocytes. Transmission electron microscopy revealed complete phagocytosis of C. albicans and the fusion of lysosomal granules with loose and tight phagosomes. Ingested C. albicans remained structurally intact after 2 hr incubation in blastospore-free medium. However, cytoplasmic alterations were clearly evident, with a patchy loss of electron density. Alterations of the blastospore cell wall were also observed, with complete disruption of the plasma membrane but the wall remaining morphologically intact.     

Ultrastructure of the cell wall of a Synechocystis strain

The ultrastructure of the cell wall of a Synechocystis strain, isolated from the Gulf of Finland, was studied using several electron microscopic techniques. This cyanobacterium has numerous projections which were observed to penetrate the cell wall complex. An additional layer (AL) was associated with the outer membrane. An additional external wall layer (EL) was connected to the outer membrane complex by thin fibers as revealed by ruthenium red staining. A hexagonal arrangement of the subunits in the additional external wall layer with a lattice constant of 15.5 nm was found.     

Comparative ultrastructure of a mucoid strain of Pseudomonas aeruginosa isolated from cystic fibrosis patient and its spontaneous non-mucoid mutant

The ultrastructure of a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin and its spontaneous non-mucoid variant was compared by transmission electron microscopy. Negatively-stained preparations of the mucoid strain obtained from plate cultures demonstrated dense, fibrous material projecting from the cell. No such material was observed in thin-sections or in negatively-strained preparations from liquid cultures. Thin-sections of ethanol-precipitated extracellular material from liquid cultures of the mucoid-strain revealed a cottony mesh of thin electron dense fibres. The non-mucoid strain did not produce such material. When prefixed with glutaraldehyde/malachite green mixture, cells of both strains demonstrated electron dense intracellular and extracellular malachite green-stainable structures. The internal complexes were frequently associated with the nucleoid or cell membrane and were replaced by electron transparent areas in cells prefixed with glutaraldehyde alone. Aeruginocins of the R-type were observed in mitomycin C induced cultures of both strains. Bacteriophages with 'claw-shaped' tail-tips were observed in the mucoid strain. Crystalline material was produced by the mucoid strain but only when plated on certain media.     

Maceration of Clover and Grass Leaves by Lachnospira multiparus

A strain of Lachnospira multiparus, a pectin-hydrolyzing bacterium from the rumen, was incubated in nutrient media in the presence of surface-disinfected clover leaflets. When the culture flasks containing the leaflets together with Lachnospira were shaken after overnight incubation, extensive maceration of the leaflets was seen, although uninoculated control leaflets remained intact during a similar treatment. Examination of inoculated leaflets by transmission electron microscopy showed extensive invasion of intercellular areas of the mesophyll tissue but only minor invasion of vascular tissue. Cutting the leaves before incubation greatly increased the ability of L. multiparus to colonize and macerate the leaflets. Similar experiments with grass leaves are also described, and the possible role of maceration in the digestion of plant material in the rumen is discussed. Although Lachnospira stains gram variable and often gram negative, the ultrastructure of the cell wall was that of a gram-positive bacterium.     

CELL WALL STRUCTURE AND IRON DISTRIBUTION IN THE GREEN ALGA STAURASTRUM LUETKEMUELLERI (DESMIDIACEAE)1

The cell wall of Staurastrum luetkemuelleri Donnat & Ruttner was examined with scanning electron microscope (SEM) using whole cells, in thin sections with transmission electron microscope (TEM), and in air dried whole cells and unstained thin sections with X-ray microanalysis in the scanning-transmission electron microscope (STEM). The cell wall was ornamented with spines and wartlike structures. Spines were solid structures, consisting of deposits of cell wall material between two main cell wall layers. The wart-like structures were pore organs extending through the cell wall and the mucilaginous layer outside the cell wall. The pore cylinder was surrounded by deposits of cell wall material similar to the ones in the spines. X-ray microanalysis of selected areas on whole cells from a natural population showed iron accumulation in discrete locations on the cell extensions of S. luetkemuelleri. In the unstained thin sections iron was found only in the cell wall deposits in the spines. Cells grown in laboratory cultures failed to show iron accumulation regardless of readdition of iron-EDTA (Fe-EDTA) to the culture medium.     

Opening angle and residual strain in a three-layered model of pig oesophagus

Studies of various biological tissues have shown that residual strains are important for tissue function. Since a force balance exists in whole wall thickness specimens cut radially, it is evident that layer separation is an important procedure in the understanding of the meaning of residual stresses and strains. The present study investigated the zero-stress state and residual strain distribution in a three-layer model of the pig oesophagus. The middle part of the oesophagus was obtained from six slaughterhouse pigs. Four 3-mm-wide rings were serially cut from each oesophagus. Two of them were used for separating the wall into mucosa-submucosa, inner and outer muscle layers. The remaining two rings were kept as intact rings. The inner and outer circumferences and wall thickness of different layers in intact and separated rings were measured from the digital images in the no-load state and zero-stress state. The opening angle was measured and the residual strain at the inner and outer surface of different layers and the intact wall were computed. Compared with intact sectors (62.8+/-9.8 degrees ), the opening angles were smaller in the inner muscle sectors (37.2+/-11.4 degrees , P<0.01), whereas the opening angles of mucosa-submucosa (63.9+/-6.8 degrees ) and outer muscle sectors (63.9+/-6.8 degrees ) did not differ (P>0.1). Referenced to the zero-stress state of the intact sectors, the inner and outer residual strains of the intact rings was -0.128+/-0.043 and outer residual strain was 0.308+/-0.032. Referenced to the "true" zero-stress state of separated three-layered sectors, the inner residual strain of intact rings were -0.223+/-0.021 (P<0.01) and 0.071+/-0.022 (P<0.01). Referenced to the "true" zero-stress state, the residual strain distribution of different layers in intact rings was shown that the inner surface residual strain was negative at mucosa-submucosa and inner muscle layers and was positive at outer muscle layer, whereas the outer surface residual strain was negative at the mucosa-submucosa layer and positive at the inner and outer muscle layers. For the separated different layered rings, the inner residual strain was negative and outer residual strain was positive; however, the absolute values did not differ (P>0.1). In conclusion, it is possible to microsurgically separate the oesophagus into three layers, i.e., mucosa-submucosa, inner muscle and outer muscle layers, the residual strain differ between the layers, and the residual strain distribution was more uniform after the layers were separated.