An ultrastructural study of cryofractured myocardial cells with special attention to the relationship between mitochondria and sarcoplasmic reticulum

A comparative study of various cryofracturing techniques has been conducted on the mammallian myocardial cell. Quench freezing of fresh or fixed tissue in melting Freon 22 resulted in severe cellular damage due to ice crystallization. Fixation with Karnovsky's fixative prior to quenching had no modifying effect on the size and distribution of the ice crystals. The crystals were orientated primarily in the direction of the long axis of the myofibrils, manifested as empty tube-like structures in the scanning electron microscope (SEM). Regular cross-bridging often seen at the Z-band levels indicated that ice crystals, at least in some portions of the cells, were confined within the sarcomere. Within the same cell the size of the ice crystals could vary considerably. Treatment of the tissue with polyvinylpyrrolidone (PVP) prior to rapid freezing had no noticeable cryoprotective effect. The surface of the thin layer of PVP surrounding the freeze dried tissue appeared amorphous in the SEM. However, the first evidence of ice crystallization was found a few micrometres under the surface. The freezing artefacts were completely circumvented if the cryofracturing was carried out on ethanol-impregnated or on critical point dried material. While the first method resulted in a smooth fracture plane passing through the cell structures, the intracellular fracture plane of the critical point dried material followed the surface of the cell organelles. Separation of the cell organelles caused by freezing or by critical point drying revealed thread-like structures extending from the mitochondrial surface. Re-examination of SEM-processed material in the transmission electron microscope (TEM) revealed that these structures were part of the sarcoplasmic reticulum (SR), and that a close contact between the SR and the outer mitochondrial membrane existed. TEM of conventional prepared material revealed that strands of electron-dense material, here named ‘mito-reticular junctional fibres’, bridged the narrow gap between the mitochondrial surface and the SR. It is suggested that these fibres have a specific anchoring function.     

Detection of wood cell wall porosity using small carbohydrate molecules and confocal fluorescence microscopy

A novel approach to nanoscale detection of cell wall porosity using confocal fluorescence microscopy is described. Infiltration of cell walls with a range of nitrophenyl‐substituted carbohydrates of different molecular weights was assessed by measuring changes in the intensity of lignin fluorescence, in response to the quenching effect of the 4‐nitrophenyl group. The following carbohydrates were used in order of increasing molecular weight; 4‐nitrophenyl β‐D‐glucopyrano‐side (monosaccharide), 4‐nitrophenyl β‐D‐lactopyranoside (disaccharide), 2‐chloro‐4‐nitrophenyl β‐D‐maltotrioside (trisaccharide), and 4‐nitrophenyl α‐D‐maltopentaoside (pentasaccharide). This technique was used to compare cell wall porosity in wood which had been dewatered to 40% moisture content using supercritical CO_2, where cell walls remain fully hydrated, with kiln dried wood equilibrated to 12% moisture content. Infiltration of cell walls as measured by fluorescence quenching, was found to decrease with increasing molecular weight, with the pentasaccharide being significantly excluded compared to the monosaccharide. Porosity experiments were performed on blocks and sections to assess differences in cell wall accessibility. Dewatered and kiln dried wood infiltrated as blocks showed similar results, but greater infiltration was achieved by using sections, indicating that not all pores were easily accessible by infiltration from the lumen surface. In wood blocks infiltrated with 4‐nitrophenyl α‐D‐maltopentaoside, quenching of the secondary wall was quite variable, especially in kiln dried wood, indicating limited connectivity of pores accessible from the lumen surface.     

Surface and internal structure correlation: high-voltage and scanning electron microscopies of wholemount alveolar walls of human lung

We found that the high-voltage electron microscope (HVEM) operating at 1–5 MeV was able to transilluminate and form a focused transmission image of whole-mounts of alveolar walls from human lung, a tissue sufficiently thin to require no embedment and sectioning. Resultant micrographs resembled a composite of scanning and transmission electron microscope images: surface and internal structure of the alveolar wall were visualized in a single micrograph. Although the scanning electron microscope extracts some subsurface information in the secondary electron mode, the HVEM produced better images of both surface and subsurface features. Lungs were fixed, dehydrated, critical point dried, and metal coated as for conventional scanning electron microscopy, then individual alveolar walls were excised by hand and mounted on transmission electron microscope grids. Regions of the alveolar wall up to 10 μm thick were delineated with the high-voltage electron microscope. Cell surface characteristics were correlated with cell type as identified by underlying cell internal structure. Whole white blood cells within capillaries of the alveolar wall were identified by the configuration of their nuclei. Features of the nucleus and surface of alveolar type II cells were recorded simultaneously. Whole red blood cells were imaged within intact capillaries that branched and wove from one alveolar surface to the other. HVEM analysis of excised alveolar septa allows definitive correlation of surface and underlying structures in single micrographs of broad portions of the alveolar wall and is an alternative to embedment, microtomy and serial section reconstruction for this uniquely thin tissue.     

Preparation of cultured cells for SEM: air drying from organic solvents.

Air evaporation from organic solvents of differing polarities and surface free energies was used in the preparation of cultured murine peritoneal macrophages for scanning electron microscopy (SEM). The surface structural features of these cells were compared to the surfaces of similar cells prepared by the critical-point procedure. In general, all organic solvents produced a marked collapse of cell structure resulting in an increase in surface irregularity. Fracture surfaces and sharply defined contours including numerous flaps and ridges were characteristic of the non-polar solvent dehydrated samples. Polar solvent dehydrated samples, including those dried from solvents of low surface free energy, exhibited a secondary flowing and settling of the cell membrane. Primary collapse was apparent but cell contours were smoothed and rounded. Overall cell shape and cell-to-cell relationships were retained regardless of solvent type. It is suggested that solvent evaporation may prove useful in certain cases, though investigators are advised to use caution when interpreting the results obtained by such procedures.     

Preparation of cell membranes for high resolution imaging by AFM

Studies of cell membrane structure by atomic force microscopy (AFM) have been limited because of the softness of cell membranes. Here, we utilize a new technique of sample preparation to lay red blood cell membranes on the top of a mica surface to obtain high resolution images by in-situ AFM on both sides of cell membranes. Our results indicate that the location of oligosaccharides and proteins in red blood cell membranes might be different from the current membrane model. The inner membrane leaflet is covered by dense proteins with fewer free lipids than expected. In contrast, the outer membrane leaflet is quite smooth; oligosaccharides and peptides supposed to protrude out of the outer membrane leaflet surface might be actually hidden in the middle of hydrophilic lipid heads; transmembrane proteins might form domains in the membranes revealed by PNGase F and trypsin digestion. Our result could be significant to interpret some functions about red blood cell membranes and guide to heal the blood diseases related to cell membranes.     

Comparison of different preparation methods of biological samples for FIB milling and SEM investigation

When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.     

Photoelectron microscopy of erythrocyte ghosts; imaging of lectin binding sites with colloidal gold markers

Images of human red cell ghosts have been obtained by photoelectron microscopy (photoemission electron microscopy or PEM) without any staining, metal coating or shadowing. Membrane folding, when it occurs in these collapsed structures, shows up clearly even though the membrane itself is only 5–10 nm in thickness. Using red cell ghosts as a model system, it is shown that colloidal gold can act as a photoemissive marker. Specific lectin binding sites on the cell surface were labeled with a colloidal gold-wheat germ agglutinin complex. The gold particles were readily detectable against the weaker emission from the cell surface.     

Photoelectron microscopy of erythrocyte ghosts: imaging of lectin binding sites with colloidal gold markers

Images of human red cell ghosts have been obtained by photoelectron microscopy (photoemission electron microscopy or PEM) without any staining, metal coating or shadowing. Membrane folding, when it occurs in these collapsed structures, shows up clearly even though the membrane itself is only 5-10 nm in thickness. Using red cell ghosts as a model system, it is shown that colloidal gold can act as a photoemissive marker. Specific lectin binding sites on the cell surface were labeled with a colloidal gold-wheat germ agglutinin complex. The gold particles were readily detectable against the weaker emission from the cell surface.     

超高效液相色谱-四极杆-静电场轨道阱质谱法分析不同炮制红参的化学成分差异

采用超高效液相色谱-四极杆-静电场轨道阱质谱法（UPLC-Q-Orbitrap MS）对蒸制红参、醋制红参和烘干红参中的人参皂苷类成分进行分析研究。基于三醇型皂苷Re、Rg1、Rg2、Rh1标准品和二醇型皂苷Rb1、Rb2、Rc、Rd、Rg3、Rh2标准品的加热降解规律和质谱特征,鉴定出不同人参炮制品中的32个人参皂苷。其中,人参皂苷mRb1、Rh1、F1、20(R)-Rh1、Rg5和Rs5仅在醋制红参中检出,notoR1仅在蒸制红参中检出。该方法明确了3种人参炮制品的化学成分差异,可为指导人参不同炮制品的分类应用提供技术支撑。     

Mapping functional sites on biological macromolecules

Electron microscopy of macromolecules dried from glycerol and rotary shadowed from a low angle can reveal the structure of individual molecules, or groups of molecules, with remarkable clarity. We used this technique to examine the interaction of the red blood cell cytoskeletal proteins spectrin, a 500,000 dalton protein which is long (750 A) and flexible;actin, a 43,000 dalton protein capable of polymerizing into double helical filaments; and band 4.1, an 82,000 dalton globular protein. By examining binary and ternary complexes of these molecules, the binding sites for actin, band 4.1 and a fourth protein ankyrin, which links the cytoskeleton to the membrane, have been mapped along the length of the spectrin molecule. These findings, which have enabled us to construct a model of the red cell cytoskeleton, show that low angle shadowing is a powerful but simple method for investigating associations among macromolecules.     

Quantification of red blood cells using atomic force microscopy

For humans the sizes and shapes of their red blood cells are important indicators of well being. In this study, the feasibility of using the atomic force microscope (AFM) to provide the sizes and shapes of red blood cells has been investigated. An immobilisation procedure has been developed that enabled red blood cells to be reliably imaged by contact AFM in air. The shapes of the red blood cells were readily apparent in the AFM images. Various cell quantification parameters were investigated, including thickness, width, surface area and volume. Excellent correlation was found between the AFM-derived immobilised mean cell volume (IMCV) parameter and the mean cell volume (MCV) parameter used in current haematological practice. The correlation between MCV and IMCV values has validated the immobilisation procedure by demonstrating that the significant cell shrinkage that occurs during immobilisation and drying does not introduce quantification artifacts. Reliable IMCV values were obtained by quantifying 100 red blood cells and this typically required 3-5 AFM images of 100 microm x 100 microm area. This work has demonstrated that the AFM can provide in a single test the red blood cell size and shape data needed in the assessment of human health.     

红色固态荧光碳量子点的制备及在指纹检测中的应用

荧光碳量子点是一种新型的光致发光纳米材料,由于其具有稳定的发光性能、丰富的表面官能团、安全无毒、生物相容性好、成本低廉等优势,在潜指纹检测和识别领域有着极大的应用前景。潜指纹是指人的手指分泌物留在固体接触面上靠肉眼难以分辨的痕迹,需要借助物理或化学方法以有效地显现和提取。到目前为止,关于碳量子点显影潜指纹并与计算机技术结合精确识别指纹的研究鲜有报道。以邻苯二胺为前驱体,以草酸锌为修饰剂,采用一步溶剂热法成功合成了红色荧光碳量子点,将红色荧光碳量子点与聚乙烯吡咯烷酮混合,干燥研磨后制备出均匀分散、量子产率高达27%的红色固态荧光碳量子点,并成功应用于多种基底上潜指纹的增强检测。为了精确评价显影潜指纹与目标对照指纹之间的相似度,通过结构相似度算法进行相似度分析,锡纸上潜指纹的匹配度高达90.5%,表明红色固态荧光碳量子点结合数字处理程序能很好地显影和精确识别潜指纹,在刑事侦查领域具有极大的应用前景。     

Surface topography and intracellular organization of human cells in suspension as revealed by scanning electron microscopy

This report presents a new procedure to study the ultrastructure of human cells in suspension by means of scanning electron microscopy. Living cells were maintained in suspension within cell culture flasks located on a rotating tilting table within an incubator. These cells were injected into warm glutaraldehyde/formaldehyde fixative. After washing in buffer, fixed cells were attached to propylamine-derived glass carriers. However, saturating free aldehyde groups of fixed cells and blocking amine groups of the derived glass carriers prevented cells from attachment to these carriers. Thus, we postulated that bonds between the fixed cell-free aldehyde groups and the carrier amine groups were responsible for cell-to-carrier attachment. Fixed cells attached to the carriers were subsequently dehydrated, dried, and coated for surface topography studies. For studies of internal cell organization, these attached cells were immersed in agar or gelatin as extracellular embedments and infused with sucrose or polyvinyl-pyrrolidone as cryoprotectants. Cells then were frozen and fractured. Fractured cells were either thawed, dehydrated, critical point dried, and ion beam sputter-coated, or freeze-substituted, dried, and planar magnetron sputter-coated. Finally, cell preparations were observed in the scanning electron microscope. Due to high cell attachment yield, both approaches samples observed in the electron microscope were representative of the entire cell population.     

Morphological polarity in oral epithelial cells revealed by boric acid partial dissociation

Layers of epithelium were stripped with adhesive tape from the surface of rat palatal mucosa after that tissue had been fixed in OsO₄, treated in 0.2 M boric acid and critical point or freeze dried. Cell layers stripped off evenly with very few intracellular fractures. The oral surface of cells in the stratum corneum was characterized by small pits and raised cell boundaries. By contrast the deep aspect of these cells displayed microvilli and linear grooves which exactly matched the pits and raised boundaries on the opposing cell beneath. Partial dissociation with boric acid after OsO₄ fixation greatly assists the demonstration of complementary cell surfaces deep in the epithelium.     

Air-drying of human leucocytes for scanning electron microscopy using the GTGO procedure

The utilization of tannic acid and guanidine hydrochloride as mordants for better osmium binding has been shown to serve as an excellent alternative to metal coating of organ tissue specimens for scanning electron microscopy (SEM). The present report describes the GTGO procedure, a modification of the TAO technique introduced by Murakami et al. (1977, 1978), which we have found successful for the preparation of air dried peripheral blood leucocytes for SEM studies. Air dried, GTGO-treated leucocytes show excellent preservation of surface features with minimal cell shrinkage. When critical point dried, GTGO-treated cells are examined, they also show less shrinkage than cells prepared with standard glutaraldehyde fixation and critical point drying. The potential application of this air drying procedure (GTGO-AD) to other soft biological specimens is currently under investigation. This technique is recommended as a new and effective air drying procedure for the successful preparation of cells for SEM.     

Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro

Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.
AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (−15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).     

High-resolution scanning electron microscopy of immunogold-labelled cells by the use of thin plasma coating of osmium

The feasibility of plasma coating of a thin osmium layer for high‐resolution immuno‐scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro‐muscular preparations were fixed with formaldehyde and labelled with a neurone‐specific antibody and 10 or 5 nm colloidal gold‐conjugated secondary antibodies. The specimens were post‐fixed with osmium tetroxide and freeze‐dried. Then they were coated with a 1–2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three‐dimensionally complex surface structures of cellular processes with a resolution of several nanometres.     

Imaging via widefield surface plasmon resonance microscope for studying bone cell interactions with micropatterned ECM proteins

The widefield surface plasmon resonance microscope has recently been used to monitor label free antibody/antigen binding events and focal contacts in HaCaT cells at high special resolutions. Thus the aim of this study was to examine MG63 bone cell attachment and alignment to microcontact printed extracellular matrix proteins. Collagen, fibronectin and laminin were stamp patterned onto glass slides using templates consisting of 5-, 10-, 25-, 50- and 100-μm-wide repeat grating. MG63 bone cells were seeded at 50,000 cells per 25 cm(2) and cell alignment was determined from micrographs taken at time-points 2, 5 and 18 h after cell seeding. Cells on the fibronectin pattern attached and elongated at early stages after seeding. In the case of collagen and laminin, cells did not adhere readily and appeared more rounded until 18 h after seeding. This indicated MG63 cells attach mostly via fibronectin specific integrins. The cells aligned well on the fibronectin-patterned cover slips especially to the 50- and 100-μm-wide patterns, although in this case cells did not position themselves in the middle of each fibronectin-coated region, but instead aligned to the small features associated with the edges of the fibronectin-coated regions. Patterned and un-patterned cells also had quite different morphologies. The un-patterned cells had a more rounded morphology and lengths of 25 to 35 μm, whereas patterned cells elongated in the direction of the pattern and had lengths of 50-70 μm. The widefield surface plasmon resonance imaging indicated that cells on un-patterned surfaces had a rounded morphology in which the focal contacts were evenly distributed around the periphery of the cell. However, MG63 bone cells on fibronectin-patterned substrates organized most of their focal contacts along the periphery of the cell distal to the edge of the fibronectin patterns. This suggests that the interaction between the cell and the edge of the pattern induces a reorganization of focal contacts such that the region of the cell guided by the edge of the fibronectin pattern is relatively loosely coupled to the cell culture substrate, but the region of the cell positioned away from that edge is quite tightly coupled to the fibronectin-coated region of the culture substrate. This in turn suggests that guidance is not necessarily associated with enhanced cell substrate coupling along the guidance cue, but may be more associated with a decreased coupling at the guidance cue. Such an arrangement may influence cytoplasmic streaming and as such modulate cell extension. Verification of this finding is required; as such a response to a guidance cue is quite unexpected because it is believed that cells cluster their focal contacts along a guidance cue.     

Preparation of a new red dye from Janus black and its use in the staining of DNA-aldehyde molecules

This communication presents a method for the preparation of a new red dye from an aqueous solution of Janus black by adding NHC1 and sodium thiosulphate to it. This new red dye when used on acid-hydrolysed tissue sections reveals the presence of red nuclei when sections after staining are dried between folds of filter paper, differentiated in n-butanol, cleared in xylene and mounted. Similarly stained sections when treated with SO2 water show partial leaching of the dye from the nuclei. Tissue sections when treated with cold concentrated phosphoric acid for 20 min and then stained with an aqueous solution of Janus black reveal the presence of orange-red nuclei. The new red dye obtained from Janus black does not respond to treatment under UV rays. The in vitro absorption data of the red dye indicate peaks at 210, 270 and 545 nm. The in situ absorption spectra of nuclei stained with the new red dye following Feulgen procedure reveal the peak of maximum absorption at 560 nm and those of nuclei treated with cold concentrated phosphoric acid and then stained with this red dye reveal peak at 530--540 nm. Some relevant points raised out of this investigation have been discussed.