A new method of preparing fish chromosomes for scanning electron microscopy

A preparative ion-etching technique has been developed which enhances the images of fish chromosomes obtained by scanning electron microscopy.     

A simplified method for preparing rotifer trophi for scanning electron microscopy

A simple, quick technique for the preparation of rotifer trophi for scanning electron microscopy is described. The method permits visual monitoring of the extraction process and does not require critical point drying of the specimens. Micrographs showing fine, structural detail of the hard parts of the mastax of representatives of the following genera are presented:Asplanchna, Conochilus, Filinia, Hexarthra, Keratella, Proalides, Synchaeta, andTrichocerca.     

A rapid technique for preparing microorganisms for transmission electron microscopy.

A rapid and efficient method of preparing microorganisms for transmission electron microscopy is reported. In developing the method Salmonella, streptococcal, and protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

Atomic-force microscopy imaging of plasma membranes purified from spinach leaves

Summary Plasma membranes purified from spinach leaves by aqueous two-phase partitioning were examined by atomic-force microscopy (AFM) in phosphate buffer, and details on their structure were reported at nanometric scale. Examination of the fresh membrane preparation deposited on mica revealed a complex organization of the surface. It appeared composed of a first layer of material, about 8 nm in thickness, that practically covered all the mica surface and on which stand structures highly heterogeneous in shape and size. High-resolution imaging showed that the surface of the first layer appeared relatively smooth in some regions, whereas different characteristic features were observed in other regions. They consisted of globular-to-elliptical protruding particles of various sizes, from 4–5 nm x-y size for the smallest to 40–70 nm for the largest, and of channel-like structures 25–30 nm in diameter with a central hole. Macromolecular assemblies of protruding particles of various shapes were imaged. Addition of the proteolytic enzyme pronase led to a net roughness decrease in regions covered with particles, indicating their proteinaceous nature. The results open fascinating perspectives in the investigation of membrane surfaces in plant cells with the possibility to get structural information at the nanometric range.Abbreviations AFM atomic-force microscopy - EM electron microscopy - TMAFM tapping-mode atomic-force microscopy     

A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy

Stimulated emission depletion (STED) microscopy is a prominent approach of super‐resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time‐gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub‐diffraction spatial resolution. If the time‐gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW‐STED microscope. However, time‐gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti‐Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti‐Stokes background. The method hinges on the uncorrelated nature of the anti‐Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two‐photon‐excitation with gated CW‐STED microscopy is demonstrated. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A high-yield technique for preparing cells fixed in suspension for scanning electron microscopy

Human leukocytes fixed in suspension were allowed to settle onto poly-L-lysine-coated glass coverslips and prepared for observation with the scanning electron microscope (SEM). The coverslips were dehydrated in ethanol, critical point dried with CO2, and coated with gold-palladium. With the aid of a locator grid, several fields were photographed with light microscopy after the cells had settled onto the poly-L-lysine-coated coverslips and again after completion of the processing before SEM observation. Quantitative comparison of the number of cells present after settling with the number retained for final viewing with the SEM revealed a cell yield approaching 100%. This simple, reproducible, high-yield technique for processing cells fixed in suspension for SEM prevents changes in surface architecture induced by collecting live cells onto various substrates before fixation and also avoids potentially selective cell losses. Such a technique should allow quantitative correlations between SEM and other morphological and functional parameters.     

A new method of preparing insects for scanning electron microscopy.

Acidified 2,2-dimethoxypropane (DMP), used as an alternative to regular fixation and dehydration methods for insects, was found to be the only successful means of preparing the sugarbeet root maggot larva, Tetanops myopaeformis (R?der) (Diptera:Otitidae), for the scanning electron microscope. No morphological changes were evident when DMP treated sugarbeet root maggot adults were compared to fresh (unfixed) adults and glutaraldehyde-osmium tetroxide fixed adults. The method has been used with success on several gropus of insects. Acidified CMP is quickly hydrolyzed by water in tissue to acetone and methanol. DMP is advantageous in that it penetrates water impermeable cuticles rapidly and saves several steps and time in the fixation and dehydration process.     

A replication technique for scanning electron microscopy: Applications for anthropologists

Scanning electron microscopy has become an increasingly useful tool for anthropologists, particularly because of the development of improved methods of replicating specimens. One of the best replication techniques involves silicone-based dental impression materials to make negative impressions, in conjunction with epoxy resins, which are used to make positives or casts. The technique outlined here is particularly useful for anthropologists. Using this technique allows the examination of bone surfaces, teeth, and fossils for taphonomic, microwear, and experimental studies. Reproduction of detail is faithful at magnifications of × 1,500 to × 2,000, routinely giving resolutions of .1 to .25 μm.     

A new technique for visualizing proanthocyanidins by light microscopy

We describe a new technique for visualizing proanthocyanidin-containing elements in plant tissues. Our innovation is the fixation of condensed tannins with an exogenous protein prior to alcohol dehydration. In this way, tannins do not undergo partial solubilization during the dehydration sequence and appear as sharply contoured globules of various diameters.     

Energy compensated synthetic aperture focusing technique for photoacoustic microscopy

We report an adaptive energy-compensated synthetic aperture focusing technique (eC-SAFT) for improving the imaging performance of photoacoustic microscopy (PAM) in terms of depth of field (DOF), spatial resolution (both axial and lateral), and SNR. In addition to coherency and time-delay (in conventional SAFT), our beamforming-based reconstruction algorithm takes into account acoustic energy loss—a primary physical parameter in acoustic wave propagation—following Beer-Lambert's law. Experimental validation studies were performed in tissue-mimicking (Agar) phantoms, complex leaf veins, and chicken breast tissues. Results demonstrate that our proposed eC-SAFT+CF outperforms conventional SAFT+CF to improve axial resolution (up to $\sim 5\%$), lateral resolution (up to $\sim 5\%$), SNR (up to $\sim 6\%$) and CR (up to $\sim 8\%$).     

观察球孢白僵菌侵染烟粉虱若虫过程的新方法——荧光显微法

【目的】介绍一种观察白僵菌Beauveria bassiana侵染烟粉虱Bemisia tabaci(Gennadius)若虫的荧光显微方法。【方法】将被白僵菌侵染的烟粉虱若虫用荧光素二乙酸酯(FDA)染色,并于波长为450~490 nm的蓝光下显微观察。【结果】在接种白僵菌12 h和24 h的烟粉虱若虫上分别观察到昆虫表皮上分生孢子的萌发和芽管穿透表皮。结合裸眼和荧光显微观察结果证实了被真菌侵染的烟粉虱若虫体色变红和菌体在虫体内增殖是同时发生的。【结论】应用荧光显微方法能观察到白僵菌在烟粉虱若虫体表和体内的侵染。