Scanning Electron and Phase-Contrast Microscopy of Bacterial Spores

The three-dimensional immages of free and intrasporangial spores produced by scanning electron microscopy show surface structures not visible by phase-contrast microscopy. Although fine surface detail is not elucidated by scanning electron microscopy, this technique does afford a definitive picture of the general shape of spores. Spores of Bacillus popilliae, B. lentimorbus, B. thuringiensis, B. alvei, B. cereus, and Sarcina ureae have varying patterns of surface ridge formation, whereas spores of B. larvae, B. subtilis, and B. licheniformis have relatively smooth surfaces.     

Luxol Fast Scarlet C as a Stain for Phase-Contrast Microscopy

Neutral red stains both normal and cancer mitotic cells, but uptake by living mitotic cancer cells is distinctly higher than in normal cells. This new approach to cancer cell identification is demonstrated in 4 established tumorigenic cancer cell lines: human skin epidermoid carcinoma A431, mouse Cloudman malignant melanoma, human oral epidermoid carcinoma and rat hepatoma. Human Chang liver cells served as normal controls. With epidermal growth factor (EGF) prepulse, neutral red uptake is dramatically enhanced. The possibility of a causal relationship with M-phase specific phosphorylation is discussed.     

Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

Germination of Bacillus spores with a high pressure (HP) of ∼150 MPa is via activation of spores'' germinant receptors (GRs). The HP germination of multiple individual Bacillus subtilis spores in a diamond anvil cell (DAC) was monitored with phase-contrast microscopy. Major conclusions were that (i) >95% of wild-type spores germinated in 40 min in a DAC at ∼150 MPa and 37°C but individual spores'' germination kinetics were heterogeneous; (ii) individual spores'' HP germination kinetic parameters were similar to those of nutrient-triggered germination with a variable lag time (T_lag) prior to a period of the rapid release (ΔT_release) of the spores'' dipicolinic acid in a 1:1 chelate with Ca²⁺ (CaDPA); (iii) spore germination at 50 MPa had longer average T_lag values than that at ∼150 MPa, but the ΔT_release values at the two pressures were identical and HPs of <10 MPa did not induce germination; (iv) B. subtilis spores that lacked the cortex-lytic enzyme CwlJ and that were germinated with an HP of 150 MPa exhibited average ΔT_release values ∼15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average T_lag values; and (v) the germination of wild-type spores given a ≥30-s 140-MPa HP pulse followed by a constant pressure of 1 MPa was the same as that of spores exposed to a constant pressure of 140 MPa that was continued for ≥35 min; (vi) however, after short 150-MPa HP pulses and incubation at 0.1 MPa (ambient pressure), spore germination stopped 5 to 10 min after the HP was released. These results suggest that an HP of ∼150 MPa for ≤30 s is sufficient to fully activate spores'' GRs, which remain activated at 1 MPa but can deactivate at ambient pressure.     

Electron Microscopy of Mummified Material

It has proved possible to cut ultrathin sections of mummified material obtained from an American Indian burial (approximate age unknown). Small pieces of tissue were placed for 48 hr in a softening fluid consisting of 96% ethyl alcohol, 30 vol.; 1% aqueous formalin, 50 vol.; 5% aqueous Na₂CO₃, 20 vol. During this period the fluid was changed twice. The tissue was then cut with a razor blade into cubes of 1 mm per side or less, dehydrated in graded ethanols, infiltrated and embedded in methacrylate and the plastic polymerised by placing in the oven at 58°C overnight. The blocks were trimmed to a truncated cone leaving a surface area of 0.5 mm² or less, and cut on a Porter Blum ultramicrotome using a glass or a diamond knife.     

Rapid,High-Throughput Tracking of Bacterial Motility in 3D via Phase-Contrast Holographic Video Microscopy

Tracking fast-swimming bacteria in three dimensions can be extremely challenging with current optical techniques and a microscopic approach that can rapidly acquire volumetric information is required. Here, we introduce phase-contrast holographic video microscopy as a solution for the simultaneous tracking of multiple fast moving cells in three dimensions. This technique uses interference patterns formed between the scattered and the incident field to infer the three-dimensional (3D) position and size of bacteria. Using this optical approach, motility dynamics of multiple bacteria in three dimensions, such as speed and turn angles, can be obtained within minutes. We demonstrated the feasibility of this method by effectively tracking multiple bacteria species, including Escherichia coli, Agrobacterium tumefaciens, and Pseudomonas aeruginosa. In addition, we combined our fast 3D imaging technique with a microfluidic device to present an example of a drug/chemical assay to study effects on bacterial motility.     

Germination and Outgrowth of Single Spores of Saccharomyces cerevisiae Viewed by Scanning Electron and Phase-Contrast Microscopy

Single spores of Saccharomyces cerevisiae were examined during germination and outgrowth by scanning electron and phase-contrast microscopy. Also determined were changes in cell weight and light absorbance, trehalose utilization, and synthesis of protein and KOH-soluble carbohydrates. These studies reveal that development of the vegetative cell from a spore follows a definite sequence of events involving dramatic physical and chemical modifications. These changes are: initial rapid loss in cellular absorbance followed later by an abrupt gain in absorbance; reduction in cell weight and a subsequent progressive increase; modification of the spore surface with concomitant diminution in refractility; elongation of the cell and augmentation of surface irregularities; rapid decline in trehalose content of the cell accompanied by extensive formation of KOH-soluble carbohydrates; and bud formation.     

Application of Polarizing Microscopy to Medical Mycology

The hyphae of 15 species of fungi were cultivated by conventional slide-culture technique and then examined by polarizing microscopy. Extinction angles exhibited by the anisotropic chitin in the cell walls of hyphae were, as a rule, measurable within a minimal growth range of 2 to 3 days after inoculation to slide culture. Of the 15 species examined, 8 saprophytic fungi were found to possess a range of common extinction angles (10° ± 2°). The remaining seven organisms implicated as pathogens were found to possess a range of common extinction angles (20° ± 2°). There appears to be a correlation existing between the pathogenic nature of the examined fungi and their extinction angles, as demonstrated by the polarizing microscope.     

利用散射光声微分成像技术实现弱吸收物质显微成像

光声显微成像技术依赖于样品的内源性光吸收,对强散射弱吸收样品成像效果差,甚至无法进行成像。为了实现强散射弱吸收高透明生物样品的光声显微成像,以及获得图像的边缘增强效果,使光声显微成像技术在实际的生物医学研究中更有应用价值,本文首次将散射光声技术引入到光声微分显微技术中,研制了新型的散射光声微分成像技术。该技术不仅可以获得强散射弱吸收高透明生物样品的散射光声显微图像,还可以获得对应的边缘清晰的散射光声微分图像,对在生物医学研究领域有重要的应用意义。     

Application of Electron Diffraction to Biological Electron Microscopy

Three methods by which electron diffraction may be applied to problems in electron microscopy are discussed from a fundamental point of view, and experimental applications with biological specimens are demonstrated for each case. It is shown that wide-angle electron diffraction provides valuable information for evaluating specimen damage that can occur either during specimen preparation or while in the electron beam. Dark-field electron microscopy can be used both to enhance the image contrast and to provide highly restricted and therefore highly specific information about the object. Low-angle electron diffraction provides quantitative information about the object structure in the range from 20 A to ~ 1000 A. Lowangle electron diffraction also demonstrates the important role of Fourier contrast with biological specimens, which are usually characterized by structural features with dimensions of 20 A or larger.     

Application of Fluorescence Microscopy and Photomicrography to Woody Tissues

A procedure is described for making preparations of woody tissues for visual observation or photography by incident-light fluorescence microscopy. The chief advantages of the technic are the following:

(1) Reliable recognition of anatomical characteristics in wood without ordinary time-consuming histological technics.

(2) Examination of relatively larger surface areas of wood blocks than by usual methods.

(3) Visual observation and, if desired, photography of tissues and cell structure in dry or in nearly natural or fresh condition.

(4) Marked color contrast without the use of stains in many tissues, including specific types of cells comprising them.

(5) Improved color contrast by use of Congo red with aspects not usually obtained by other methods.     

Toward Clinically Compatible Phase-Contrast Mammography

Phase-contrast mammography using laboratory X-ray sources is a promising approach to overcome the relatively low sensitivity and specificity of clinical, absorption-based screening. Current research is mostly centered on identifying potential diagnostic benefits arising from phase-contrast and dark-field mammography and benchmarking the latter with conventional state-of-the-art imaging methods. So far, little effort has been made to adjust this novel imaging technique to clinical needs. In this article, we address the key points for a successful implementation to a clinical routine in the near future and present the very first dose-compatible and rapid scan-time phase-contrast mammograms of both a freshly dissected, cancer-bearing mastectomy specimen and a mammographic accreditation phantom.     

A Method for Improved Resolution for Fluorescence Microscopy Using Plastic-Embedded Material Subjected to Resin Extraction

A protocol is given that uses NaOH, benzene, acetone and methanol to extract epoxy resins from semithin sections. Such sections appear superior to paraffin or unsectioned materials for fluorescence microscopic observations. Use of ultrarapid films (e.g., Kodak T-Max P3200) at ISO 3200 minimizes fading without use of antifading agents and without introducing unacceptable photographic grain size.     

X-Ray Phase-Contrast Tomography of Renal Ischemia-Reperfusion Damage

Purpose

The aim of the study was to investigate microstructural changes occurring in unilateral renal ischemia-reperfusion injury in a murine animal model using synchrotron radiation.

Material and Methods

The effects of renal ischemia-reperfusion were investigated in a murine animal model of unilateral ischemia. Kidney samples were harvested on day 18. Grating-Based Phase-Contrast Imaging (GB-PCI) of the paraffin-embedded kidney samples was performed at a Synchrotron Radiation Facility (beam energy of 19 keV). To obtain phase information, a two-grating Talbot interferometer was used applying the phase stepping technique. The imaging system provided an effective pixel size of 7.5 µm. The resulting attenuation and differential phase projections were tomographically reconstructed using filtered back-projection. Semi-automated segmentation and volumetry and correlation to histopathology were performed.

Results

GB-PCI provided good discrimination of the cortex, outer and inner medulla in non-ischemic control kidneys. Post-ischemic kidneys showed a reduced compartmental differentiation, particularly of the outer stripe of the outer medulla, which could not be differentiated from the inner stripe. Compared to the contralateral kidney, after ischemia a volume loss was detected, while the inner medulla mainly retained its volume (ratio 0.94). Post-ischemic kidneys exhibited severe tissue damage as evidenced by tubular atrophy and dilatation, moderate inflammatory infiltration, loss of brush borders and tubular protein cylinders.

Conclusion

In conclusion GB-PCI with synchrotron radiation allows for non-destructive microstructural assessment of parenchymal kidney disease and vessel architecture. If translation to lab-based approaches generates sufficient density resolution, and with a time-optimized image analysis protocol, GB-PCI may ultimately serve as a non-invasive, non-enhanced alternative for imaging of pathological changes of the kidney.     

A Procedure for Embedding Plant Material in Araldite for Electron Microscopy

Plant material is embedded in Araldite made up according toLuft's compositional formula but with casting resin M replacedby type N. After fixation specimens are dehydrated rapidly inacetone of increasing 10 per cent concentrations. At the absolute-acetonestage specimens are left for I h with three changes of absoluteacetone during that time. Infiltration is initiated by replacinghalf the last change of absolute acetone by an equal volumeof Araldite mixture. Specimens are left to be infiltrated forat least I h on a rotary agitator. Half of the first infiltrationmixture is then replaced by the same volume of fresh Aralditeand the specimens left to agitate overnight. Specimens are thenembedded.     

Epoxy Embedding, Sectioning and Staining of Plant Material for Light Microscopy

By using a formula which gives a relatively soft epoxy embedding medium, it is possible to cut sections of plant material with a sliding microtome equipped with a regular steel knife. Blocks having a cutting face of 10 × 10 mm, giving sections of 4-10 μm, can be used. Tissues are fixed in Karnovsky's fluid, postfixed in 1 or 2% OsO₄, embedded in Spurr's soft epoxy resin, Araldite, or Epon mixtures. 5% KMnO₄, followed by 5% oxalic acid, then neutralized in 1% LiCO₃, are used to mordant the sections. Some of the stains used are Mallory's phosphotungstic acid-hemotoxylin, acid fuchsin and toluidine blue, or toluidine blue. Mounting is done with whichever soft epoxy resin was used in casting the blocks.     

The Application of Freeze-Dry Methods to Plant Material

The freeze-dry method of preparing tissue for histological observation has been applied to a large variety of plant material with good results. This method involves freezing the tissue rapidly and dehydrating at a temperature below -30°C. under vacuum with a desiccant. The tissue, when dry, is infiltrated with paraffin under vacuum. The apparatus uses a liquid nitrogen-cooled condenser as the desiccant and the tissue is infiltrated with paraffin while in the original vacuum. Using root, stem, leaf, and reproductive tissue of common experimental species such as Vicia faba, Zea mays, Allium cepa, Lillium longiflorium, Pisum sativum and Phaseolus vulgaris, the effect of thickness on drying time was studied. It was found that there was greater difference between types of tissue than between species so that regardless of species, leaf tissue was easiest and root tips the most difficult to freeze and dry. Leaf segments required 1-2 days to dry while root material required 6 days or more. In all tissue except leaf, 1-2 mm. segments were optimal for freezing and drying although 4 mm. segments could occasionally be used.     

Wire-Loop Application of Liquid Emulsion to Slides for Autoradiography in Light Microscopy

This is an adaptation of procedures used to prepare autoradiographs for electron microscopy to light microscopy. A rectangular wire loop slightly larger than the slide is used. The liquid emulsion is prepared by mixing 1 volume of a 0.1% solution of Dreft or Ivory Snow with 2 volumes of Kodak NTB3 nuclear track emulsion. The slide to be coated is placed on a cork stopper supported by a glass plate 61 cm from the safelight. The loop was withdrawn from the liquid emulsion, with the plane of the loop parallel to the surface of the liquid, placed directly over the slide and slowly lowered so that the film (in the sol state) was broken by the slide. If desired, the film can be allowed to gel before application to the slide by waiting approximately 40 sec after the loop is removed from the liquid emulsion. That the developed emulsion is consistently uniform was indicated by a thickness of 0.4 ± 0.02 μm. Diluted emulsion can be stored after use for 1-2 wk at 2-8 C and used a second time before discarding. The identification of human D group chromosomes labeled with tritiated thymidine as well as prescreening patients for the Lesch-Nyhan syndrome, which utilizes tritiated hypoxanthine labeling, have been successfully carried out by applying the emulsion in the sol state.