The structure of the developing chick retinal pigment epithelium revealed by high resolution scanning electron microscopy

The retinal pigment epithelium (RPE) in the developing eye of chick embryos has been studied during the early stages of development by high resolution scanning electron microscopy (HRSEM). Specimen preparation techniques which involve removal of the cytoplasmic matrix permitted visualization of organelles and other subcellular structures within RPE cells in detail and in three dimensional (3-D) stereo HRSEM. Using this technique, we were able to examine changes in melanosome structures during development and demonstrate that pigmentation in the RPE was present by day 4 of development. RPE plasma cell membranes showed extensive folding of the apical portion of the membrane closest to the developing neural retina by day 9. Examination of RPE photoreceptor junction revealed photoreceptor inner segments by day 6 and an outer segment by day 9. Mitochondria in the RPE were found to contain tubular cristae only. The ultrastructure in 3-D of the Golgi apparatus, smooth and rough endoplasmic reticulum, lysosomes and nuclear chromatin of the RPE, and Bruch's layer was revealed by the HRSEM method.     

Tridimensional ultrastructure of perfusion fixed gastrointestinal epithelial cells by high resolution scanning electron microscopy

Improvements in the design of modern scanning electron microscopes (SEM) and new methods of specimen preparation incorporating chemical removal of the cytosol and cytoskeleton, now make it possible to view cells and their organelles in three dimensions (3D) at high magnification. In this experiment, high resolution SEM (HRSEM) utilizing new methods of tissue preparation was used to study the intracellular structures of the mouse ileum. In addition, in vivo intestinal perfusion was used to further enhance cellular preservation. Using these modifications it was possible to visualize, in 3D, the fine structure of intestinal epithelial cells and intracellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi complex, as well as microvilli and cell membrane. Whole mitochondria appeared as irregularly shaped organelles which contained tubular cristae. Plate-like cristae were not observed. The brush border was found to be a closely packed array of cylindrical projections. The extensive folding and structural intricacy of lateral cell membranes between absorptive cells could only be appreciated by viewing this tissue with 3D HRSEM. The use of HRSEM to study 3D ultrastructure of cells and their organelles will improve our understanding of the structure-function relationships in both the healthy and diseased gastrointestinal tract.     

Brefeldin A effects in plant and fungal cells: something new about vesicle trafficking?

Whilst the function and organization of the secretory machinery in eukaryotic cells exhibit basic similarities, the compartmentation of the endomembrane system can show significant differences between the fungal, plant and animal kingdoms. The use of the antibiotic brefeldin A (BFA) as an inhibitor of secretion in both animal and yeast cells has resulted in a remarkable advance in our understanding of the modes of action of vesicle shuttles between the endoplasmic reticulum and Golgi apparatus and within the Golgi apparatus itself. It is now apparent that application of the drug to filamentous fungi and plants will also help unravel the workings of the secretory system in these organisms. In this paper we review recent progress in our laboratories on elucidating the effects of BFA on the morphology of the Golgi apparatus and compare these with recently published data on fungal and plant cells. Variation in the response to BFA are reported, which may not all be attributed to differences in drug concentration and time of treatment. These may reflect differences in cellular sensitivity or multiple sites of action of the drug, and the existence of a specific molecular target for BFA is questioned.     

ER-Tracker dye and BODIPY-brefeldin A differentiate the endoplasmic reticulum and Golgi bodies from the tubular-vacuole system in living hyphae of Pisolithus tinctorius

Two fluorochromes, ER-Tracker^TM Blue-White DPX dye and the fluorescent brefeldin A (BFA) derivative, BODIPY-BFA, label the endoplasmic reticulum (ER) in hyphal tips of Pisolithus tinctorius and allow its differentiation from the tubular-vacuole system at the light microscope level in living cells. The ER-Tracker dye labels a reticulate network similar in distribution to ER as seen in electron micrographs of freeze-substituted hyphae. BODIPY-BFA stains a thicker axially aligned structure with an expanded region at the apex, which is similar to that seen when hyphae are stained with ER-Tracker dye in the presence of unconjugated BFA. This structure is considered to be ER modified by BFA, a view supported by ultrastructural observations of the effect of BFA on the fungal ER. Both fluorescent probes also stain punctate structures, which are most likely to be Golgi bodies. Neither probe labels the tubular-vacuole system.     

Ultrastructure of submandibular salivary glands of mouse: TEM and HRSEM observations

The fine structure of submandibular glands of mouse were analyzed using light microscopy (LM), high resolution scanning electron microscopy (HRSEM), and transmission electron microscopy (TEM) methods. For LM, the specimens were embedded in Spurr resin, stained by toluidin blue solutions. For TEM, the tissues of submandibular salivary glands were fixed with modified Karnovsky solution and postfixed with osmium tetroxide. For HRSEM, the tissues were fixed with 2% osmium tetroxide solution in 1/15M sodium phosphate buffer (pH 7.4). The samples were immersed successively in dymethylsulphoxide and freeze cracked. The maceration was made in diluted osmium tetroxide for 24-48 h. The samples were examined by high resolution scanning electron microscopy. The intracellular components of acinar and ductal cells revealed clearly the Golgi apparatus, rough endoplasmic reticulum, secretory granules, and mitochondria. The end bulbs of Golgi lamellae and flattened cisterns of rough endoplasmic reticulum showed the luminal surface. A few mitochondria were identified intermingling between the rough endoplasmic reticulum and the mitochondriales cristae in three-dimensional HRSEM images. Secretory granules were numerous and presented different sizes. Small granules of ribosomes were attached on cistern surface, measuring 20-25 nm in diameter. Numerous arranged microvilli were found on the luminal surface of secretory canaliculus. The contact surfaces of acinar cells revealed complicated interdigitations by cytoplasmic processes. The mitochondria of duct cells were disposed vertically and surrounded by basal infoldings of plasma membranes. Basement membrane showed a spongy-like structure having an irregular surface with various strands and meshes of fine collagen fibrils.     

Chemical extraction of the cytosol using osmium tetroxide for high resolution scanning electron microscopy.

Detailed examination of subcellular structures in three dimensions (3D) by high resolution scanning electron microscopy (HRSEM) is now possible due to improvements in the design of the scanning electron microscope and the introduction of methods of specimen preparation using chemical removal of the cytosol and cytoskeleton by dilute osmium tetroxide. Cells which have been fixed, frozen, cleaved, thawed, and subjected to cytosol extraction display intact intracellular structures in 3D including nuclear chromatin, endoplasmic reticulum, mitochondria, and the Golgi complex at a resolution close to that of conventional biological transmission electron microscopy (TEM). Small changes in the 3D structure of subcellular components can be conveniently examined in this way in development, in a variety of physiological processes and in disease. Broad areas of the specimen can be quickly surveyed by HRSEM since sectioning is not required and specimens of comparatively large size (up to 5 mm3) can be placed in the microscope. Extraction of the cytosol with dilute osmium tetroxide (OsO4) exposes subcellular structures in relief, permitting their examination in 3D from several aspects. However, the OsO4 extraction technique is limited, since significant intracellular structures, such as the cytoskeleton, vesicles, and antibody binding sites can be removed or inactivated during the cytosol removal steps.     

Assessment of chloride secretion in human nasal epithelial cells by X-ray microanalysis

The genetic disease cystic fibrosis (CF) is due to defective epithelial chloride transport. Different treatments have been proposed that could restore chloride transport in CF patients. A new method is proposed for measuring the chloride secretion in easily accessible epithelial cells.
Fresh nasal epithelial cells were obtained by nasal brushing and made to attach to titanium grids for electron microscopy. Chloride efflux through the cystic fibrosis transmembrane regulator channel was stimulated by 20 µ m forskolin and 100 µ m isobutyl-methylxanthine (IBMX), in standard Ringer's solution (SR). Chloride efflux through the calcium-regulated channel was stimulated by 200 µ m adenosine triphosphate (ATP) in SR. The cells were rinsed after the exposure, in order to remove the experimental medium, frozen and freeze-dried. The elemental composition of the cells was determined by X-ray microanalysis.
Rinsing with distilled water or ammonium acetate appeared to cause damage to the cells, whereas rinsing with isotonic mannitol preserved the ionic composition. Stimulation of cells from healthy controls with forskolin and IBMX in a chloride-containing medium caused a significant (28 ± 6%) decrease in chloride concentration, which is indicative of net chloride efflux. In similar conditions, stimulation with ATP induced a 29 ± 5% decrease in the chloride concentration.
Stimulation of cells from CF patients with forskolin and IBMX in a chloride-containing medium caused no significant change in the intracellular chloride concentration, whereas ATP stimulation induced a response similar to that obtained in cells from healthy controls.
It is concluded that X-ray microanalysis of nasal epithelial cells may be used to determine chloride secretion in CF patients in an easily accessible cell type.     

Imaging of ferroelectric thin films by X-ray photoemission electron microscopy (XPEEM)

We performed X-ray photoemission electron microscopy (XPEEM) measurements at the Nanospectroscopy Beamline of the synchrotron light source ELETTRA, Trieste, Italy, to demonstrate the principal possibility of imaging ferroelectric thin films by low-energy photoelectrons. Due to the insulating properties of ferroelectric films, severe surface charging was the major experimental challenge to overcome. This was achieved by grounding an array of gold inter-digital electrodes (with 5 microm blank intervals between them) deposited on top of the films. The images taken with BaTiO(3) films revealed 50-100 nm-sized holes (material discontinuities) on the surface, an observation confirmed by high-resolution scanning electron microscopy (HRSEM). Finer details, e.g. a granular structure, which has been resolved with HRSEM, could not be observed in the XPEEM images. Our measurements indicate that despite some residual charging, a 50 nm lateral resolution can be achieved in XPEEM measurements with ferroelectric films.     

A tilting device for three-dimensional microscopy: Application to in situ imaging of interphase cell nuclei

The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the focal plane (x-y plane). This is true of conventional as well as confocal microscopes. For quantitative microscopy, for instance studies of the three-dimensional (3-D) organization of chromosomes in human interphase cell nuclei, the 3-D image must be reconstructed by a point spread function or an optical transfer function with careful consideration of the properties of the imaging system. To alleviate the reconstruction problem, a tilting device was developed so that several data sets of the same cell nucleus under different views could be registered. The 3-D information was obtained from a series of optical sections with a Zeiss transmission light microscope Axiomat using a stage with a computer-controlled stepping motor for movement in the z-axis. The tilting device on the Axiomat stage could turn a cell nucleus through any desired angle and also provide movement in the x-y direction. The technique was applied to 3-D imaging of human lymphocyte cell nuclei, which were labelled by in situ hybridization with the DNA probe pUC 1.77 (mainly specific for chromosome 1). For each nucleus, 3-D data sets were registered at viewing angles of 0°, 90° and 180°; the volumes and positions of the labelled regions (spots) were calculated. The results also confirm that, in principle, any angle of a 2p geometry can be fixed for data acquisition with a high reproducibility. This indicates the feasibility of axiotomographical microscopy of cell nuclei.     

Fine Structure of Bacterial Adhesion to the Epithelial Cell Membranes of the Filiform Papillae of Tongue and Palatine Mucosa of Rodents: A Morphometric,TEM, and HRSEM Study

The palatine mucosa and filiform papillae of the dorsal tongue mucosae of rodents were examined using transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HRSEM). In the HRSEM method, the samples were fixed in 2% osmium tetroxide, dehydrated in alcohol, critical point‐dried, and coated with gold‐palladium. In addition, the HRSEM technique was used for morphometric analysis (length, width, and length/width ratio of cocci and bacilli). For the TEM method, the tissues were fixed in modified Karnovsky solution (2.5% glutaraldehyde, 2% formalin in 0.1M sodium phosphate buffer, pH 7.4) and embedded in Spurr resin. The results demonstrated that there are thick polygonal keratinized epithelial cells where groups of bacteria are revealed in three‐dimensional images on the surface of filiform papillae in these animals. The bacterial membranes are randomly attached to the microplicae surface of epithelial cells. Morphometrics showed higher values of length and width of cocci in newborn (0 day) as compared to newborn (7 days) and adults animals, the bacilli showed no differences in these measurements. At high magnification, the TEM images revealed the presence of glycocalyx microfilaments that constitute a fine adhesion area between bacterial membranes and the membranes of epithelial microplicae cells. In conclusion, the present data revealed the fine fibrillar structures of bacteria that facilitate adhesion to the epithelial cell membranes of the oral cavity and morphometric changes in newborn (0 day) rats as compared with other periods. Microsc. Res. Tech. 76:1226–1233, 2013. © 2013 Wiley Periodicals, Inc.     

Changes in the biomechanical properties of a single cell induced by nonthermal atmospheric pressure micro‐dielectric barrier discharge plasma

Mechanical properties of a single cell are closely related to the fate and functions of the cell. Changes in mechanical properties may cause diseases or cell apoptosis. Selective cytotoxic effects of nonthermal atmospheric pressure micro‐dielectric barrier discharge (DBD) plasma have been demonstrated on cancer cells. In this work, changes in the mechanical properties of a single cell induced by nonthermal atmospheric pressure micro‐DBD plasma were investigated using atomic force microscopy (AFM). Two cervical cancer cell lines (HeLa and SiHa) and normal human fibroblast cells (HFBs) were exposed to micro‐DBD plasma for various exposure times. The elasticity of a single cell was determined by force–distance curve measurement using AFM. Young's modulus was decreased by plasma treatment for all cells. The Young's modulus of plasma‐treated HeLa cells was decreased by 75% compared to nontreated HeLa cells. In SiHa cells and HFBs, elasticity was decreased slightly. Chemical changes induced by the plasma treatment, which were observed by Raman spectroscopy, were also significant in HeLa cells compared to SiHa cells and HFBs. These results suggested that the molecular changes induced by micro‐DBD plasma were related to cell mechanical changes.     

Angiotensin II stimulates expression of transcription factors c-Jun and c-Fos in cyclosporine induced human gingival fibroblasts

The present study demonstrates that the expression of c-Jun and c-Fos are elevated in gingival fibroblast cells treated with angiotensin II and cyclosporine. The healthy human gingival tissues were collected and gingival fibroblasts were isolated and cultured. We used RT-PCR and Western blot analysis to identify the expression of c-Jun and c-Fos in cyclosporine and angiotensin II treated human gingival fibroblast cells. We found that angiotensin II in combination with cyclosporine induces c-Jun and c-Fos expressions significantly; however, the angiotensin II antagonist losartan inhibits the expression of c-Jun and c-Fos (p < 0.01). The data suggest that angiotensin II in combination with cyclosporine modulates the expression of c-Jun and c-Fos in human gingival fibroblast cells.     

Dynamic three-dimensional visualization of collagen matrix remodeling and cytoskeletal organization in living corneal fibroblasts

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing, as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the dynamic process of 3-D matrix remodeling at the cellular and subcellular level. We recently developed an experimental model for investigating cell-matrix mechanical interactions by plating green fluorescen protein (GFP)-zyxin transfected cells inside fibrillar collagen matrices and performing high-magnification time-lapse differential interference microscopy (DIC) and wide-field fluorescent imaging. In this study, we extend this experimental model by performing four-dimensional (4-D) reflected light and fluorescent confocal imaging (using either visible light or multiphoton excitation) of living corneal fibroblasts transfected to express GFP-zyxin or GFP-alpha-actinin, 18 h after plating inside 3-D collagen matrices. Reflected light confocal imaging allowed detailed visualization of the cells and the fibrillar collagen surrounding them. By overlaying maximum intensity projections of reflected light and GFP-zyxin or GFP-alpha-actinin images and generating stereo pair reconstructions, 3-D interactions between focal adhesions and collagen fibrils in living cells could be visualized directly. Focal adhesions were generally oriented parallel to the direction of collagen fibril alignment in front of the cell. Killing the cells induced relaxation of transient cell-induced tension on the matrix; however, significant permanent remodeling always remained. Time-lapse 3-D imaging demonstrated an active response to the Rho-kinase inhibitor Y-27632, as indicated by cell elongation, extracellular matrix relaxation, and extension of pseudopodial processes. It is interesting that, at higher cell densities, groups of collagen fibrils were compacted and aligned into straps between neighboring cells. Overall, the continued development and application of this new approach should provide important insights into the basic underlying biochemical and biomechanical regulatory mechanisms controlling matrix remodeling by corneal fibroblasts.     

Three-dimensional localisation of fluorescence resonance energy transfer in living cells under two-photon excitation

Three-dimensional (3-D) imaging of fluorescence resonance energy transfer (FRET) in human cells under two-photon excitation was demonstrated in this study. A sample was prepared by expressing a donor and an acceptor in living cells and using an antibody to secure the proximity of contact between the donor and the acceptor. The quenching of fluorescence emission of a donor in the double-labelled cells indicates the presence of FRET that occurred in these living cells. Because of the quadratic relation of the excitation power, 3-D localisation of FRET becomes possible.     

Histatin-induced alterations in Candida albicans: a microscopic and submicroscopic comparison

Despite the numerous studies performed in an attempt to clarify the issue, the mechanism of action of salivary histatins remains unclear. The aim of the present study was to correlate histatin-induced morphological changes in Candida albicans by fluorescence microscopy (FM), transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM). Each of the fluorescent dyes used by FM (i.e., tetramethylrhodamine methyl ester perchlorate for mitochondrial potential, Lysotracker for lysosome acidic compartment, and 4',6-diamino-2-phenylindole dihydrochloride for DNA) exhibited a specific staining in control cells. Following histatin treatment, we observed a recurring staining pattern, corresponding to fluorescence concentration along the cell periphery, suggesting a loss of dye specificity. To assess histatin-induced cytoplasmic modifications, ultrastructural analysis was then carried out. After treatments with histatins, TEM revealed characteristic intracellular modifications including: vacuole overgrowth, nuclear disappearance, loss of organelle identity, as well as the appearance of electron-dense membranes, likely of mitochondrial origin. Additionally, structures resembling autophagosomes were occasionally observed. By HRSEM, mitochondrial swelling was invariably the first sign of a histatin-induced effect. Other modifications included intracellular membrane disarrangement, organelles in disarray, and a large central cavity with deformed bodies displaced to the cell periphery, similar to what was detected by TEM. In summary, our study illustrates the occurrence of ultrastructural modifications following administration of histatins. Observations made with FM, TEM, and HRSEM provided different views of the same signs, demonstrating a definite action of histatins on C. albicans morphology. The possible functional meanings of these morphological results is discussed in light of the most recent biochemical data on histatin fungicidal activity.     

Comparisons of golgi structure and dynamics in plant and animal cells

The Golgi apparatus of both higher plant and animal cells sorts and packages macromolecules which are in transit to and from the cell surface and to the lysosome (vacuole). It is also the site of oligosaccharide and polysaccharide synthesis and modification. The underlying similarity of function of plant and animal Golgi is reflected in similar morphological features, such as cisternal stacking. There are, however, several fundamental differences between the Golgi of plant and animal cells, reflecting, in large part, the fact that the extracellular matrices and lysosomal systems differ between these kingdoms. These include (1) the form and replication of the Golgi during cell division; (2) the disposition of the Golgi in the interphase cell; (3) the nature of “anchoring” the Golgi in the cytoplasm; (4) the genesis, extent, and nature of membranes at the trans side of the stack; (5) targeting signals to the lysosome (vacuole); and (6) physiological regulation of secretion events (constitutive vs. regulated secretion). The degree of participation of the Golgi in endocytosis and membrane recycling is becoming clear for animal cells, but has yet to be explored in detail for plant cells.     

Three-dimensional distribution of damaged cells in cryopreserved pancreatic islets as determined by laser scanning confocal microscopy

The technique of serial optical sectioning by confocal microscopy, in conjunction with off-line digital image analysis, was used to quantify the radial distribution of damaged cells in rat pancreatic islets following cryopreservation. The process consists of imaging frozen-thawed islets of Langerhans using laser scanning confocal microscopy (LSCM). The three-dimensional (3-D) distribution and analysis of the two populations of viable and damaged cells was visualized via acridine orange/propidium iodide (AO/PI) fluorescent staining. In preparation for cryopreservation, isolated and cultured rat pancreatic islets were brought to a 2 m concentration of dimethyl sulphoxide (DMSO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at ?10°C, and individual specimens were frozen to ?70°C at cooling rates of 1, 3, 10 and 30°C/min in a programmable bulk freezer and subsequently stored in liquid nitrogen. After rapid thawing and serial dilution to remove DMSO, individual islets were prepared with AO/PI stains for imaging on the LSCM. Serial sections of the islets, 2–7 μm in thickness, were obtained and processed to obtain high-contrast images. Analysis algorithms consisted of template masking, grey-level thresholding, median filtering and 3-D blob colouring. The radial distribution of damaged cells in the islets was determined by isolating the cell and computing its distance from the centroid of the 3-D islet volume. An increase in the number of blobs corresponding to single and/or aggregates of damaged cells was observed progressively with distance from the centre towards the periphery of the islet. This pattern of freeze-induced killing of cells within the islet was found to occur consistently in the numerous individual specimens processed.     

切削速度对精密干式车削淬硬工具钢切削力的影响

通过使用PCBN刀具精密干式车削淬硬Cr12MoV工具钢(62±1 HRC)的试验,分析了切削速度对三向切削力的影响,得出了最优切削速度。试验表明:随切削速度提高,三向切削力先急剧增大,后急剧减小,再又缓慢增大。若从最小车削合力与提高加工效率两个角度来优化切削速度,则226 n/min是最优切削速度。试验结果也对精密干式切削淬硬工具钢具有实际指导意义与参考价值。     

Study of Golgi-impregnated material using the confocal tandem scanning reflected light microscope

The tandem scanning reflected-light microscope (TSM) is a real-time, direct-view confocal microscope. Only those points in the specimen situated in the focal plane contribute information to the image. A Tracor Northern TMS with piezo-electric control of the objective lens was used to generate 3-D images from Golgi-impregnated hamster cerebral cortex. Stereoscopic pairs of images were recorded as 35-mm colour film transparencies by photographing while automatically through-focusing along inclined axes. Transferring the image via a TV camera to the computer, stereo-pairs were obtained by oblique through-focusing and summing, displaying maximum intensity data in each line of sight. Pseudocolour topographic displays were generated by assigning the pixel value in a z map image as the focal depth at which the back-scattered light signal was maximal. The TSM was also modified so that a conventional transmitted-light image with a large depth of field could be obtained simultaneously as the very shallow depth of field confocal back-scattered-light image seen at any focus level. The conventional image is a silhouette of the impregnated neurons: the top surface of the cell is not visible and the relationships of processes that cross over cell bodies cannot be discerned. TSM gives a high-contrast image. The Golgi precipitate over the neuronal surface is resolved as globular or ovoid, coloured particles. The smaller particles also cover the dendritic spines. All the confocal range (extended focus) image display methods satisfactorily demonstrated the 3-D arrangement of cell bodies and processes in the chosen volume.     

Morphometrical study of plant vacuolar dynamics in single cells using three-dimensional reconstruction from optical sections

In higher plants, vacuoles increase their volumes in accordance with cell enlargement and occupy most of the cell volume. However, quantitative analyses of vacuolar contributions during changes in cell morphology have been hampered by the inadequacies and frequent artifacts associated with current three-dimensional (3-D) reconstruction methods of images derived from light microscopy. To overcome the limitations of quantifying 3-D structures, we have introduced 3-D morphometrics into light microscopy, adopting a contour-based approach for which we have developed an interpolation method. Using this software, named REANT, the morphological and morphometrical changes in protoplasts and vacuoles during plasmolysis could be investigated. We employed the tobacco (Nicotiana tabacum) BY-2 cell line No.7, expressing a GFP-AtVam3p fusion protein, BY-GV7, using GFP as a marker of vacuolar membranes (VMs). By vital staining of the plasma membrane (PM) of cells, we simultaneously obtained optical sections of both the PM and VM. We, therefore, reconstructed the 3-D structures of protoplasts and vacuoles before and after plasmolysis. We were able to identify the appearance of elliptical structures of VMs in the vacuolar lumen, and to determine that they were derived from cytoplasmic strands. From the 3-D structures, the volumes and surface areas were measured at the single cell level. The shrinkage of vacuoles accounted for most of the decrease in protoplast volume, while the surface area of the vacuoles remained mostly unchanged. These morphometrical analyses suggest that the elliptical structures are reservoirs for excess VMs that result from the response to rapid decreases in vacuolar and protoplast volumes.