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 be relocated by the aid of the pre-embedding reference micrographs.     

The Processing of Mammalian Haemopoietic Cells in Thin Layer Agar Cultures for Electron Microscopy

Human and mouse haemopoietic cells cultured by the thin layer agar technique have been studied with the electron microscope. To process colonies of haemopoietic cells or individual cells which appeared in these colonies, a special technique had to be developed. The technique presented covers methods of selection, isolation, and sectioning that were devised for this purpose.

Haemopoietic cells are cultured in small plastic Petri dishes containing a culture system with 0.25% agar. Cell colonies and individual cells intended for light as well as for electron microscopic study are examined and selected microscopically with the aid of a numbered grid which is placed under the closed Petri dish.

Cells in the agar gel are fixed with glutaraldehyde which is pipetted directly onto the cultures. In order to facilitate their removal from the medium, the consistency of the agar solution is increased by evaporating liquid with controlled mild warming.

Pieces of agar containing colonies or single cells are cut out with a fine trephine and postfixed in osmium tetroxide. Agar pieces are embedded cell side up in a thin layer of Epon. After polymerization, the Epon-embedded pieces of agar are appropriately oriented at the head of flat embedding molds filled with fresh Epon. After another polymerization procedure, the top of the Epon blocks containing the cells are trimmed to a smooth surface with a glass knife.

The exact distance between the smooth surface of the blocks and the cells is measured by use of the vertical micrometer of a standard light microscope. The Epon layer around the specimen is trimmed away to expose selected cells for subsequent semi-thick and ultrathin sectioning. Sections are stained and examined microscopically.

With minor modifications the technique described also enables the processing of extremely small quantities of biological materials derived from other experiments for both light and electron microscopic observation.     

Photoengraving of coverslips and slides to facilitate monitoring of micromanipulated cells or chromosome spreads

A simple photolithographic technique has been developed which can be used to produce microscopic grid patterns on glass coverslips. The grid pattern is first photo-reduced onto film, and the resulting photographic negative is then used as a mask. A glass slide or coverslip, coated with a layer of photoresist, is then exposed to tungsten light through the mask. After developing and etching, the grid pattern is transferred permanently onto glass. This simple and rapid procedure allows one to mass-produce very small, high resolution grids which are useful for monitoring individual microinjected cells or chromosomal spreads under the microscope.     

Epoxy-Slide Embedment of Cytochemically Stained Tissues and Cultured Cells for Light and Electron Microscopy

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cost-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.     

An improved method for preparing microorganism laden alginate bead specimens for accurate Scanning Electron Microscope examination

Summary The distribution of biomass encapsulated within alginate beads can be examined using a Scanning Electron Microscope (SEM). Existing methods for the preparation of suitable specimens are extremely time consuming, involving fixing of samples with glutaraldehyde, dehydrating with successive acetone washings and final drying using a critical point technique. These preparations are necessary to avoid significant specimen shrinkage, however, the resultant specimens are sometimes difficult to analyse using an SEM or light microscope. A reliable quick method has been developed where alginate specimens are directly mounted using a water based adhesive, then dried under controlled conditions. These specimens were found to be robust enough for SEM processing and gave true measurements of the original alginate beads including microorganism orientation.     

A Simple Permanent Aceto-Orcein Stain for Free Cells, Cultures and Homogenates

A simplified technic for preparation of the aceto-orcein stain permits the storage of cells in the stain or squash preparations at room temperature for long periods without in* jury to or distortion of the cells and mitotic plates. Fresh cells from tumor ascites, tissue culture cells growing in free suspension or over cover slips, and homogenates of whole tissues are stained directly in a test tube in either (1) regular aceto-orcein and subsequently mounted in glycerol, or (2) aceto-orcein-glycerol mixture. These preparations are squashed for chromosome counts, and the permanent slides are kept from drying out by ringing the cover slip with Damar or Permount.     

Measurement of live bacteria by Nomarski interference microscopy and stereologic methods as tested with macroscopic rod-shaped models.

A new method is proposed to measure bacterial cells under growth conditions. Bacterial cells, suspended in their growth medium, were attached to a cover slip with poly-L-lysine. The cover slip was inverted and placed on a glass microscope slide. To prevent dehydration of the medium, the edges of the cover slip were sealed to the microscope slide with clear fingernail polish. The bacteria on the slide were then quickly photographed with a Leitz light microscope, using Nomarski optics. The photographic negatives were then projected at a standard distance through a lens system, and the projected images of the whole cells were outlined by hand onto graph paper. The profile images so transcribed onto the graph paper were in effect transverse sections of each of the cells. Using stereologic grid and point counting techniques, the area of the cell transverse section as well as the perimeter or circumference of the transverse section were estimated. Formulae were developed so that both the volume and surface area of the whole cell could be ascertained from these area and circumference measurements. Since the efficacy of any measurements of surface area and volume of microscopic rod-shaped bacterial cells could be questioned, macroscopic rod-shaped models were used to test the theory and formulae and to compare this method with other commonly used cell-sizing techniques. This technique could be used in any study of bacterial cell size or changes in cell size (e.g., osmotic shifts).     

Measurement of live bacteria by Nomarski interference microscopy and stereologic methods as tested with macroscopic rod-shaped models

A new method is proposed to measure bacterial cells under growth conditions. Bacterial cells, suspended in their growth medium, were attached to a cover slip with poly-L-lysine. The cover slip was inverted and placed on a glass microscope slide. To prevent dehydration of the medium, the edges of the cover slip were sealed to the microscope slide with clear fingernail polish. The bacteria on the slide were then quickly photographed with a Leitz light microscope, using Nomarski optics. The photographic negatives were then projected at a standard distance through a lens system, and the projected images of the whole cells were outlined by hand onto graph paper. The profile images so transcribed onto the graph paper were in effect transverse sections of each of the cells. Using stereologic grid and point counting techniques, the area of the cell transverse section as well as the perimeter or circumference of the transverse section were estimated. Formulae were developed so that both the volume and surface area of the whole cell could be ascertained from these area and circumference measurements. Since the efficacy of any measurements of surface area and volume of microscopic rod-shaped bacterial cells could be questioned, macroscopic rod-shaped models were used to test the theory and formulae and to compare this method with other commonly used cell-sizing techniques. This technique could be used in any study of bacterial cell size or changes in cell size (e.g., osmotic shifts).     

Morphological types of bipolar cells identified by regular grids of their axonal expansions in the inner plexiform layer of the herring retina

The morphological structure of the inner plexiform layer in the region of sharp vision was investigated under the light microscope in the retina of five species of herring. This layer is a three-dimensional regular grid formed by the club-shaped expansions (synaptic complexes) of the axons of the bipolar cells. These expansions, located at different levels of the inner plexiform layer, form mutually conforming periodic grids differing in their orientation and periods. Analysis of this structure shows that at least three types of bipolar cells participate in its organization.     

Orientation of Red Blood Cells and Rouleaux Disaggregation in Interference Laser Fields

The effect of interference laser fields on red blood cells (RBCs) was investigated both theoretically and experimentally. The optical trapping and orientation of individual RBC in interference fringes were observed. It was found that RBC rouleaux undergo disaggregation under the action of interference laser fields. To describe the effect of RBC orientation in interference fringes, we used the equation for torque exerted on a discoid dielectric particle in a gradient light field. The experimental results are in agreement with the predictions of the developed theoretical model.     

Epoxy-slide embedment of cytochemically stained tissues and cultured cells for light and electron microscopy

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cast-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.     

Size measurements on isolated rat heart cells using coulter analysis and light scatter flow cytometry

Isolated ventricular muscle cells from the adult rat heart have been examined by both Coulter analysis and light scatter flow cytometry. The dispersed cell preparations contain two main cell types: viable, rod-shaped cells and damaged, round cells. Coulter analytical techniques provided statistical data on cell volume for both cell types. The contribution of each population to the Coulter pulse height distributions were separated by a subtraction method using data obtained from digitonin-treated preparations that contain only round cells. A shape factor for cells aligned with the flow direction was computed from light microscope measurements and the effects of cell orientation within the Coulter aperture were approximately assessed. The estimated volumes for intact myocytes compare favourably with those reported in the literature. No significant size difference was observed between fresh and fixed cells.Narrow angle, forward light scatter measurements were made on individual cells flowing across a focused laser beam. Both scatter pulse height and pulse width (pulse duration) distributions were collected. Values for myocyte length calculated from pulse width information agree well with published data and confirm that the hydrodynamic forces in the flow system produced alignment of the cells with the flow direction. Scatter pulse width distributions reveal two distinct peaks assignable to either rod or round cells. Preliminary electronic gating experiments, using pulse height signals, suggest that signals derived from round cells could be eliminated entirely using a gating regime based on pulse width. This would enable flow cytometric measurements to be made on only the intact myocytes present in heterogeneous preparations.     

A simple method for counting Rickettsia cells]

A simple modification of the method for counting Rickettsiae is described. The Escherichia coli cells (ECC) which served as reference particles were stained in suspension with methylene blue mixed with Rickettsia prowazekii (RP) and quickly sprayed over the glass slide. After fixation the samples were stained according to the technique of Gimenez and examined in the light microscope under oil immersion. Through a grid in the eye-piece it was not so difficult to count red-coloured RP and dark-blue ECC against a background formed by impurities. To calculate RP concentration, the reference particles' concentration was multiplied by the dilution factor of RP suspension by the ratio of RP to ECC enumerated. The statistical approach has shown that the wash of the slides during staining procedure does not change this ratio. Differential staining of Rickettsiae with fuchsin is the main clue of this new method to count them even in the crude preparations of infected yolk sacs.     

压片法检查蓝藻液泡

鱼腥藻7120（Anabaena sp.PCC7120）在含0.1mol/L NaCl的条件下培养4d,90％以上细胞液泡化。若在正常条件下培养,1个多月之后部分细胞开始液泡化,随着培养时间延长,液泡化细胞比例逐渐提高。液泡化藻丝材料在玻片上经手指轻轻施压,细胞破袭,液泡从细胞破裂处释出。所释放液泡完全透明,大小不等,可在相关显微镜下显示,个别液泡可弹至细胞外远外。无机盐诱导的液泡化和细胞衰老引起的液泡化之间具有明显的平行性。     

Albumen Embedding and Individual Mounting of One or Many Mammalian Ova on Slides for Fluid Processing

For studies of ova with the light or electron microscope, as well as for autoradiographic and histochemical studies, these cells need to be sectioned. The handling of individual, often hard-to-obtain, cells through fluid processing by micropipettes is time-consuming and can easily cause damage or loss of valuable specimens. A number of interesting methods have been described for handling ova or free-floating cells. In these methods cells are commonly handled in containers with fine-mesh, wire cloth bottoms, when a number of cells are involved. Unfortunately they all require special equipment not readily or easily available (Buchanan 1965; Rinaldi et al. 1966; Izquierdo 1967; Shands 1968). In our method, egg white provides a supporting matrix for mouse ova and allows one or several specimens to be mounted on a slide.     

Squash Method for Meiosis in Ovules

The ordinary Feulgen or acetic-lacmoid squash tech-nic following fixation in freshly made Carnoy's fluid (alcohol, 6: chloroform, 3: glacial acetic acid, 1), provides an easy and reliable method of studying meiosis in ovules. After fixation for 1 day, the material was hardened in 95% ethyl alcohol for 1-2 days and taken to water by gradual hydration. For staining by the Feulgen method, the material was hydrolyzed 8-10 minutes in 1 N HO at 58-60°C., followed by staining in decolorized leuco basic fuchsin for 2 hours. The staining was intensified by transferring the material to water. After 15-20 minutes the water was replaced by 45% acetic acid. For staining by acetic-lacmoid, the ovules after fixation, hardening and hydration were transferred to standard acetic-lacmoid stain to which was added 1 drop of 1 N HCl to every 10 drops of stain. Gentle heat was applied till the stain started to give fumes. After allowing 20 minutes at room temperature the material was transferred to fresh acetic-lacmoid. Some 6-12 ovules were mounted either in a drop of 45% acetic acid or acetic-lacmoid, depending upon the Feulgen or acetic-lacmoid staining respectively. Gentle and repeated tapping over the cover glass by a blunt needle loosened the cells of integument and nucellus and finally left the megaspore mother cells undergoing meiosis, fully exposed to view. The process was carried out under constant observation using the low power of the microscope. The desired amount of flattening was brought about by light pressure over the cover glass and gentle heating. The preparations were made permanent by dehydrating in ethyl alcohol and mounting in Euparal.     

Scanning electron microscope analysis of structural changes and aberrations in human chromosomes associated with the inhibition and reversal of inhibition of ultraviolet light induced DNA repair

Metaphase chromosomes appear decondensed in preparations from mitotic cells that have been irradiated with ultraviolet light (UV) and incubated with inhibitors of DNA synthesis; under these conditions DNA repair is inhibited and both single and double strand DNA breaks accumulate. After reversal of the inhibition chromosomes are condensed, but are often damaged. In this paper we show by scanning electron microscopy (SEM) that decondensed HeLa chromosomes are composed of fibre clusters similar to those previously described for the large chromosomes of the Indian muntjac. This suggests that the clusters may be a universal higher order packing unit in mammalian metaphase chromosomes. We also examine by SEM the nature of the aberrations that appear after the reversal of periods of inhibited repair; these include gaps, breaks, deletions and telomeric packing abnormalities. SEM analysis allows an extension and reconsideration of conclusions about chromatid continuity based on the study of conventional light microscope (LM) preparations.     

The Membrane Method of Electron Microscope Autoradiography

Thin sections of methacrylate and Araldite embedded tissues labelled with radioactive isotopes were transferred with a wire loop or brush from the knife edge onto thin formvar membranes which covered 7 mm holes in 76 × 25 × 1.5 mm or 76 × 38 × 1.5 mm plastic slides. To facilitate the mounting of sections, a platform supported the plastic slides close to the ultramicrotome knife. Photographic emulsion diluted 1:5 or 1:10 with water was applied with a pipette to the upper surface of each formvar membrane to cover the mounted sections. Excess emulsion was drained off and the remaining thin film was dried on a warm plate at 45 C to produce a uniform layer over the sections. After storing in the dark for several weeks, preparations were processed in photographic solutions and washed, and sometimes stained, before applying electron microscope grids to the underside of each formvar membrane. To detach each grid with its adherent formvar, section and emulsion, the membrane was pierced around the perimeter of the grid. Grain counts made over nuclei of cells labelled with tritiated thymidine indicate that emulsion is uniformly distributed over each section and that quantitative comparison is possible between labelled areas.     

Imaging the live plant cell

Observing a biological event as it unfolds in the living cell provides unique insight into the nature of the phenomenon under study. Capturing live cell data differs from imaging fixed preparations because living plants respond to the intense light used in the imaging process. In addition, live plant cells are inherently thick specimens containing colored and fluorescent molecules often removed when the plant is fixed and sectioned. For fixed cells, the straightforward goal is to maximize contrast and resolution. For live cell imaging, maximizing contrast and resolution will probably damage the specimen or rapidly bleach the probe. Therefore, the goals are different. Live cell imaging seeks a balance between image quality and the information content that comes with increasing contrast and resolution. That "lousy" live cell image may contain all the information needed to answer the question being posed--provided the investigator properly framed the question and imaged the cells appropriately. Successful data collection from live cells requires developing a specimen-mounting protocol, careful selection and alignment of microscope components, and a clear understanding of how the microscope system generates contrast and resolution. This paper discusses general aspects of modern live cell imaging and the special considerations for imaging live plant specimens.     

Open-Face, Epoxy Embedding of Single Cells for Ultrathin Sections

Intact stamens of Tradescantia were fixed, dehydrated, and infiltrated with an epoxy resin. Each stamen was then put into a drop of resin on a microscope slide, which was transferred to the stage of a dissecting microscope so that individual hairs could be detached from the filament with fine tungsten needles. The detached hairs were transferred to drops of resin ca. 2 mm in diameter (6 or 7 in each of two rows) lying on a slide heavily coated with evaporated carbon. Polymerization was carried out in an oven until the resin attained a degree of viscosity that permitted orientation of the isolated hairs (by using a compound microscope) without their subsequent dislocation. When the small drops of resin had hardened after further polymerization, the positions of the hairs were marked by circumscribing the cells with India ink. The block was pried from the slide after rapid cooling with solid CO₂, and was then trimmed and sectioned. Cells suspended in culture medium were embedded in much the same way; they were centrifuged to obtain a pellet, which was fixed, dehydrated, and infiltrated. A small fragment of the pellet with a little resin was placed on a microscope slide, where the cells were dissociated under a dissecting microscope at ca. 100 × magnification. Individual cells were then picked up with tungsten needles and transferred to droplets of resin on a carbon-coated slide. The subsequent steps were similar to those described for the staminate hairs. Pieces of tissue in the 50-500 μ range were also handled by the foregoing technique. However, after infiltration they were put into large drops of resin on a slide coated with silicone mold-release rather than on a surface coated with carbon.