Scanning and Transmission Electron Micrographs of the Same Frozen Fractured Surface

The freeze-etch technique was applied to scanning and transmission electron-microscopy by freezing samples of tissue from 3-wk-old laboratory white mice in Freon 12 cooled with liquid nitrogen. The samples were fractured and placed into a rotary stage cold-block device and rotary shadowed with platinum and then carbon at a 45° angle. They were dried overnight in a vacuum at approximately 5 × 10^-6 torr and then exposed to OsO₄ vapors for 48 hr. After being viewed in a scanning electron microscope, the carbon-platinum replica was removed from the sample by dissolving away the tissue with Clorox. The replica was mounted on a grid and viewed in a transmission electron microscope.     

Two Convenient Washing Devices for Tissues and Slides

Two improved mechanisms are described: (1) For washing fixed tissues, a device which utilizes a downward movement of water with a consequent thoro washing is most convenient, because the washing tubes may be removed or replaced instantly. (2) A pan for washing microscope slides, fitted with a valve stem from an automobile inner tube, produces unusually effective washing and accommodates as many as 80 slides at once.     

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.     

Ultrastructure of the capsule of Klebsiella pneumoniae and slime of Enterobacter aerogenes revealed by freeze etching

Summary The capsule of Klebsiella pneumoniae type I and slime of Enterobacter aerogenes strain A3 (SL) was examined by electron microscopy using the freeze etch technique. The capsules of K. pneumoniae were found to be composed of several layers of polysaccharide 10 nm thick; while the polysaccharide slime of E. aerogenes strain A3 (SL) was found to be composed of a diffuse network of fibrils. This work represents the first effort to visualize the replica of the unfixed, partially hydrated bacterial capsule or slime in the electron microscope. The slime of E. aerogenes strain A3 (SL) which was purified, and then freeze etched, resembled the layered structure of the capsule of K. pneumoniae. It is suggested that the charge or dielectric constant of the slime polysaccharide polymers was altered during purification, thereby permitting the layering to occur.Paper presented at the Annual Meeting of the American Society for Microbiology, Philadelphia, Pa. (U.S.A.), 1972.     

Electron Microscopy of Free-Floating Cells: Thin-Layering Technique, and Selection of Individual Cells for Ultramicrotomy

A rapid and accurate procedure for electron microscopy of individual cells from suspensions (blood, peritoneal exudate, etc.) is described. After fixation of the sample with standard techniques, the particulate constituents are suspended in buffered 5% bovine serum albumin, thin-layered by gravity on clear supports (cover glasses or polyester slips) in which an orientation grid had been scored, and then immobilized by exposure of the preparations to acrolein vapors. The specimens are examined for cells of interest under a light microscope using interference or phase contrast; individual cells to be sectioned are documented in three photomicrographs taken at different magnifications. After this the specimens are embedded like ordinary cover slip preparations. When examining the face of the polymerized block under a light microscope, the position of the selected cell beneath the orientation grid relief can readily he relocated by the aid of the pre-embedding reference micrographs.     

Method for Collecting Air-Water Interface Microbes Suitable for Subsequent Microscopy and Molecular Analysis in both Research and Teaching Laboratories

A method has been developed for collecting air-water interface (AWI) microbes and biofilms that enables analysis of the same sample with various combinations of bright-field and fluorescence light microscopy optics, scanning and transmission electron microscopy (TEM), and atomic force microscopy. The identical sample is then subjected to molecular analysis. The sampling tool consists of a microscope slide supporting appropriate substrates, TEM grids, for example, that are removable for the desired protocols. The slide with its substrates is then coated with a collodion polymer membrane to which in situ AWI organisms adhere upon contact. This sampling device effectively separates the captured AWI bacterial community from the bulk water community immediately subtending. Preliminary data indicate that the AWI community differs significantly from the water column community from the same sample site when both are evaluated with microscopy and with 16S ribosomal DNA sequence-based culture-independent comparisons. This microbe collection method can be used at many levels in research and teaching.     

Method for collecting air-water interface microbes suitable for subsequent microscopy and molecular analysis in both research and teaching laboratories

A method has been developed for collecting air-water interface (AWI) microbes and biofilms that enables analysis of the same sample with various combinations of bright-field and fluorescence light microscopy optics, scanning and transmission electron microscopy (TEM), and atomic force microscopy. The identical sample is then subjected to molecular analysis. The sampling tool consists of a microscope slide supporting appropriate substrates, TEM grids, for example, that are removable for the desired protocols. The slide with its substrates is then coated with a collodion polymer membrane to which in situ AWI organisms adhere upon contact. This sampling device effectively separates the captured AWI bacterial community from the bulk water community immediately subtending. Preliminary data indicate that the AWI community differs significantly from the water column community from the same sample site when both are evaluated with microscopy and with 16S ribosomal DNA sequence-based culture-independent comparisons. This microbe collection method can be used at many levels in research and teaching.     

Simultaneous visualization of LDL receptor distribution and clathrin lattices on membranes torn from the upper surface of cultured cells

We have developed a method for simultaneous visualization by electron microscopy of both the distribution of cell surface receptors and architectural features of the inner membrane surface, such as clathrin-coated pits. Electron microscope grids were covered with formvar and coated with poly-L-lysine. These grids were then placed on a piece of buffer-impregnated cellulose acetate membrane filter maintained at 4 degrees C on an ice bath. Cells of interest were grown on glass coverslips and incubated with either a ligand-gold or an antibody-gold conjugate specific for the membrane determinant of interest. The coverslip with gold-labeled cells was then overlaid on the grids and pressure was applied. When the grid was removed, large areas of the upper cell surface, which had labeled determinants, remained adherent to the formvar support. With the proper staining, both the gold particles and internal membrane features could be seen at the same time in the electron microscope. This method is rapid, does not require extensive experience with electron microscopic technique, and permits viewing of membrane samples that are large enough to perform quantitative analysis of gold distribution in relation to membrane specializations.     

Cytological studies of the nematocysts of Hydra. I. Desmonemes,isorhizas, cnidocils,and supporting structures

Entire hydras or tentacles were fixed in OsO(4) or in KMnO(4) and thereafter washed, dehydrated, and embedded in a methacrylate mixture. Ultrathin sections were cut on an experimental model, thermal expansion type ultramicrotome or on a Porter-Blume microtome. The sections were examined in an RCA electron microscope. Type EMU-2 D. "Squash preparations" for light microscopy, were made from the hydra mouth region and the attached tentacles. These were observed with an AO Baker interference microscope. In the mature organism, three of the four types of nematocysts normally found in hydra could be positively identified with the electron microscope. The desmonemes, the smallest type, have a dense matrix and a thin capsule. The two different types of mature isorhizas could not be distinguished with certainty. They are intermediate in size between the desmonemes and stenoteles and have a capsule with a dense matrix. The cnidocil, or triggering hair, which is composed of a dense core and a fibrillar sheath has nine supporting elements arranged in a semi-circle near its base. Twenty "supporting structures" are arranged around the nematocyst capsule and interconnections between the supporting elements and these latter structures have been observed. Development of the nematocysts involves an increase in density of the matrix. Spines can be seen in the interior of tubular structures within the capsules of the holotrichous isorhizas.     

Multiple infection in bovines from the tropics: observation of blood parasites by scanning and transmission electron microscope

Blood samples from a splenectomized bovine, experimentally inoculated with blood from a field cow living in southwestern Venezuela, were processed for transmission and scanning electron microscopy. The blood sample showed multiple infection with hemoparasites of the genera Anaplasma marginale, Eperythrozoon wenyonii and Trypanosoma vivax. Scanning electron microscope showed that the blood from bovines with multiple infection had profound deformation in knob-like protruding structures with reduced cellular volume similar to echinocyte red blood cells. E. wenyonii parasites appear associated with the membrane, grouped in shallow to severe invaginations at the surface of the erythrocytes. The morphology of the parasites is predominantly rod-like; they also appear as coccoid-shaped and bifurcate or triskelion-shaped organisms. The organisms are present in pairs or clusters. T. vivax appeared with double flagella, which indicates active cellular division and infection processes. Transmission electron microscope study showed erythrocytes infected with intracytoplasmic bodies of A. marginale and with E. wenyonii embedded in the external membrane cell, with mature, juvenile and dividing forms present.     

Cytological Studies of the Nematocysts of Hydra : I. Desmonemes, Isorhizas, Cnidocils, and Supporting Structures

Entire hydras or tentacles were fixed in OsO₄ or in KMnO₄ and thereafter washed, dehydrated, and embedded in a methacrylate mixture. Ultrathin sections were cut on an experimental model, thermal expansion type ultramicrotome or on a Porter-Blume microtome. The sections were examined in an RCA electron microscope. Type EMU-2 D. "Squash preparations" for light microscopy, were made from the hydra mouth region and the attached tentacles. These were observed with an AO Baker interference microscope. In the mature organism, three of the four types of nematocysts normally found in hydra could be positively identified with the electron microscope. The desmonemes, the smallest type, have a dense matrix and a thin capsule. The two different types of mature isorhizas could not be distinguished with certainty. They are intermediate in size between the desmonemes and stenoteles and have a capsule with a dense matrix. The cnidocil, or triggering hair, which is composed of a dense core and a fibrillar sheath has nine supporting elements arranged in a semi-circle near its base. Twenty "supporting structures" are arranged around the nematocyst capsule and interconnections between the supporting elements and these latter structures have been observed. Development of the nematocysts involves an increase in density of the matrix. Spines can be seen in the interior of tubular structures within the capsules of the holotrichous isorhizas.     

Serial Sections of Smears for Electron Microscopy

Ultrathin sections for electron microscopy may be prepared from smears or squashes embedded in methacrylate. The cover slip or glass slide with the attached fixed cellular material is passed through alcohols to methacrylate monomer and finally to monomer containing a catalyst. The portion of the smear to be sectioned is covered with a gelatin capsule containing partially polymerized methacrylate. When polymerization is completed at 47°C, the hardened block is separated from the cover slip and trimmed under the compound microscope so as to encompass the desired area. Photographs are made of the intact smear to afford a basis for identification of cellular materials in electron micrographs of the individual ultrathin sections.     

The ultrastructure of the capsule surrounding Pomphorhynchus laevis (Acanthocephala) in its intermediate host Echinogammarus stammeri (Amphipoda).

The capsule or the membranous layer surrounding larvae of Pomphorhynchus laevis Müller, 1776 in its intermediate host, Echinogammarus stammeri S. Karaman, 1931, was studied by electron microscopy. Some observations were also made with a light microscope. Capsule components mainly show a tubular profile; capsule thickness can reach up to 8 microns. In some instances, within the hemocoel of E. stammeri, the parasite was encapsulated by amphipod hemocytes.     

A PLastic MIcrostrainer for HAndling MAmmalian BLastocysts

Friend (1963) has described a microstrainer for handling microscopic marine ova which permits direct transfer of batches of ova through various histological solutions without centrifugation or decanting. This strainer consists of a length of glass tubing with an electron microscope grid cemented over one end. In our laboratory, several modifications of this device have been made for use in handling mammalian ova and blastocysts. The most satisfactory model, which can be made easily, was contructed from BEEM plastic capsules (Better Equipment for Electron Microscopy, Inc., P.O. Box 132 Jerome Avenue Station, Bronx, New York 10468) and circular electron microscope grids of suitable mesh size.     

Spatially and temporally synchronized atomic force and total internal reflection fluorescence microscopy for imaging and manipulating cells and biomolecules

The atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution.     

Persistence of low hypothalamic dopaminergic activity after removal of chronic estrogen treatment

The purpose of this study was to determine whether inhibition of tuberoinfundibular dopaminergic (TIDA) neuron function which occurs during chronic estrogen administration persists after removal of the estrogen. Ovariectomized (OVX) Fischer 344 (F344) rats were implanted for 4 weeks with a Silastic capsule containing estradiol-17 beta (E2) and controls with an empty capsule for 4 weeks. Other rats which received E2 for 4 weeks had the capsule removed and experiments performed 4 weeks later. At the end of 4 weeks of E2 treatment, anterior pituitary (AP) weight was increased sixfold, serum prolactin (PRL) 65-fold, and AP DNA content fivefold over OVX control rats. Four weeks after removal of E2, AP weight, serum PRL, and AP DNA content declined, but remained significantly above OVX control values. At the end of 4 weeks of E2 treatment and after E2 withdrawal, release of [3H]dopamine (DA) from median eminence (ME) tissue superfused in vitro was lower than from ME of OVX control rats although [3H]DA accumulation was not significantly different among the treatment groups. Administration of apomorphine (APO), a dopamine agonist, significantly reduced plasma prolactin levels in OVX control rats, in rats at the end of 4 weeks E2 treatment, and in rats after 4 weeks of E2 withdrawal. Injection of haloperidol (HALO) produced similar increases in plasma PRL/estimated PRL-cell DNA in OVX controls, at the end of E2 treatment or after E2 withdrawal. However, injection of morphine (MOR), a drug which increases the release of PRL by inhibiting hypothalamic dopaminergic activity, resulted in a rise in plasma PRL/estimated PRL-cell DNA in OVX control rats that was significantly greater compared to rats at the end of E2 treatment or after E2 withdrawal. Since rats treated with E2 released less [3H]DA from ME tissue in vitro, and were less responsive to MOR, it can be that animals treated for 4 weeks with E2 show a decreased ability to release DA from TIDA neurons which persists even after termination of E2 treatment. These results suggest that chronic high circulating E2 levels result in a depression of TIDA neuronal activity which is sustained after E2 is removed.     

MALDI-MS-imaging of whole human lens capsule

The ocular lens capsule is a smooth, transparent basement membrane that encapsulates the lens and is composed of a rigid network of interacting structural proteins and glycosaminoglycans. During cataract surgery, the anterior lens capsule is routinely removed in the form of a circular disk. We considered that the excised capsule could be easily prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) analysis. MALDI-MSI is a powerful tool to elucidate the spatial distribution of small molecules, peptides, and proteins within tissues. Here, we apply this molecular imaging technique to analyze the freshly excised human lens capsule en face. We demonstrate that novel information about the distribution of proteins by MALDI-MSI can be obtained from this highly compact connective tissue, having no evident histo-morphological characteristics. Trypsin digestion carried out on-tissue is shown to improve MALDI-MSI analysis of human lens capsules and affords high repeatability. Most importantly, MALDI-MSI analysis reveals a concentric distribution pattern of proteins such as apolipoprotein E (ApoE) and collagen IV alpha-1 on the anterior surface of surgically removed lens capsule, which may indicate direct or indirect effects of environmental and mechanical stresses on the human ocular lens.     

Cover Picture

Imaging biological tissue in 3D with a serial block‐face electron microscope. In an automated acquisition cycle, a thin layer of sample surface is removed with a diamond knife and imaged at high resolution with the microscope's electron beam.     

A Precision Hand Trimmer for Electron Microscope Tissue Blocks

The construction and operation of a precision device for hand-trimmiig electron microscope tissue blocks to the pyramidal shape essential to successful thin sectioning are described. The hand trimmer permits very delicate control of the pyramid shaping operation which can be carried out with a speed rivaling or exceeding free-band trimming. It can be utilized with any stereomicroscope provided with epi-illumination.     

Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry.