Specimen Preparation For Microscopy Research Articles

Self-pressurized rapid freezing at arbitrary cryoprotectant concentrations.

Self-pressurized rapid freezing (SPRF) has been proposed as a simple alternative to traditional high pressure freezing (HPF) protocols for vitrification of biological samples in electron microscopy and cryopreservation applications. Both methods exploit the circumstance that the melting point of ice reaches a minimum when subjected to pressure of around 210 [MPa], however, in SPRF its precise quantity depends on sample properties and hence, is generally unknown. In particular, cryoprotective agents (CPAs) are expected to be a factor; though eschewed by many SPRF experiments, vitrification of larger samples notably cannot be envisaged without them. Thus, in this study, we address the question of how CPA concentration affects pressure inside sealed capillaries, and how to design SPRF experiments accordingly. By embedding a fiber-optic probe in samples and performing Raman spectroscopy after freezing, we first present a direct assessment of pressure buildup during SPRF, enabled by the large pressure sensitivity of the Raman shift of hexagonal ice. Choosing dimethyl sulfoxide (DMSO) as a model CPA, this approach allows us to demonstrate that average pressure drops to zero when DMSO concentrations of 15 wt % are exceeded. Since a trade-off between pressure and DMSO concentration represents an impasse with regards to vitrification of larger samples, we introduce a sample architecture with two chambers, separated by a partition that allows for equilibration of pressure but not DMSO concentrations. We show that pressure and concentration in the fiber-facing chamber can be tuned independently, and present differential scanning calorimetry (DSC) data supporting the improved vitrification performance of two-chamber designs. Lay version of abstract for "Self-pressurized rapid freezing at arbitrary cryoprotectant concentrations" Anyone is familiar with pipes bursting in winter because the volume of ice is greater than that of liquid water. Less well known is the fact that inside a thick-walled container, sealed and devoid of air bubbles, this pressure buildup will allow a fraction of water to remain unfrozen if the sample is also cooled sufficiently rapidly far below the freezing point. This phenomenon has already been harnessed for specimen preparation in microscopy, where low temperatures are useful to immobilize the sample, but harmful if ice-formation occurs. However, specimen preparation cannot always rely on this pressure-based effect alone, but sometimes requires addition of chemicals to inhibit ice-formation. Not enough is known directly about how these chemicals affect pressure buildup: Indeed, rapid cooling below the freezing point is only possible for small sample volumes, typically placed inside sealed capillaries, so that space is generally insufficient to accommodate a pressure sensor. By means of a compact sensor, based on an optical fiber, laser and spectrometer, we present the first direct assessment of pressure inside sealed capillaries. We show that addition of chemicals reduces pressure build-up and present a two-chambered capillary to circumvent the resulting trade-off. Also, we present evidence showing that the two-chambered capillary design can avoid ice-formation more readily than a single-chambered one. This article is protected by copyright. All rights reserved.

Procedures for preparing metallic specimens for submission to optical microscopy

Microscopy analyzes are very important for metallography, they serve to reveal the microstructural constituents, the distribution of present phases and other important properties to characterize the materials. It is important that the specimens follow a rigorous preparation process to ensure that they are free from defects that could compromise the results of the characterization. The work presents the stages of preparation of specimens for optical microscopy. Works related to the research proposal were analyzed in literature texts and thus it was possible to identify the most common and effective practices in the preparation phase of metallic samples.

Spermatozoon head size - the main differentiating feature between spermatozoa of blue and white Arctic fox (Vulpes lagopus).

Morphology and sperm morphometry, this is an important determinant of male reproductive capacity. Morphometric data may provide relevant information in studies focused on evolutionary biology, sperm quality assessment, including prediction of the potential fertility, semen cryopreservation, or the effect of reprotoxicants. The paper presents the morphometric analysis of spermatozoa from two colour morphs of Arctic fox (Vulpes lagopus), and attempts to determine the relationship between selected quality indicators and dimensions and shape of spermatozoa. The research material consisted of ejaculates collected once by manual stimulation from 20 one-year-old Arctic foxes (10 individuals of the blue morph and 10 of the white morph). Ejaculates were analysed for standard parameters (volume, sperm concentration, total number of spermatozoa in the ejaculate) and used for the preparation of microscopic specimens. It was found that, the dimensions of spermatozoa from Arctic foxes depend on the male colour morphs. Spermatozoa from white Arctic foxes were significantly longer (by 1.82 µm) and had larger heads (0.32 µm longer and 0.15 µm wider) compared to spermatozoa from blue Arctic foxes (P<0.05). The interactions between particular sperm dimensions indicated the occurrence of gametes differing in shape. The all correlation coefficients between the morphometric traits of spermatozoa were statistically significant. Our research proved that in the blue Arctic foxes, sperm dimensions (tail length and total sperm length) can be related to the percentage of spermatozoa with primary changes (respectively: r = -0.68 and r = -0.75; at P <0.05). However, in the case of white Arctic foxes, these characteristics depend on the ejaculate volume (respectively: r = 0.65 and r = 0.68; at P <0.05).

Application of focused ion-beam sampling for sidewall-roughness measurement of free-standing sub-μm objects by atomic force microscopy.

In the present study, a free-standing object-sampling technique for microelectromechanical systems (MEMS) is developed to measure their sidewall surface roughnesses by atomic force microscopy (AFM). For this purpose, a conventional focused ion beam (FIB) sampling technique widely used for cross-sectional transmission electron microscope specimen preparation was applied. The sub-nm-order roughness parameters were quantitatively measured for sidewalls of Si-bridge test samples. The roughness parameters were compared before and after H2 annealing treatment, which induced smoothing of the surface by migration of the Si atoms. The reduction in the surface roughness by a factor of approximately one-third with 60-s H2 annealing was quantitatively evaluated by AFM. The present study confirms that the developed FIB-AFM technique is one potential approach for quantitatively evaluating the surface-roughness parameters on the oblique faces of free-standing objects in MEMS devices.

Direct measurement of TEM lamella thickness in FIB-SEM.

Transmission electron microscope (TEM) specimen preparation by focused ion beam (FIB) milling requires delicate polishing of a thin window of material during the final stages of the procedure. Over or underpolishing is common and requires extra microscope resources to correct. Despite some methods for lamella thickness measurement being available, the majority of users judge the final polishing step subjectively from scanning electron microscope (SEM) images acquired between milling steps. Here we demonstrate successful thickness determination of thin silicon lamellae using calibrated secondary electron detectors in a FIB-SEM dual-beam chamber. Unlike previous thickness measurement methods it does not require long acquisition times, the use of in-chamber scanning transmission electron microscope (STEM) or energy dispersive x-ray spectroscopy detectors. The calibration aligns a SEM image to an electron energy loss spectroscopy (EELS) map of lamella thickness acquired in a TEM. This calibration reveals the greyscale-thickness dependence of two secondary electron SEM detectors: the through-lens detector (TLD) and the in-chamber electron detector (ICE). It was found that lamella thickness estimation for TLD images is accurate for areas thinner than 0.4 t/λ, whilst ICE images are most accurate for areas thicker than 0.5 t/λ up to 1.1 t/λ. The procedure presented here allows objective lamella thickness determination during the final stages of FIB specimen preparation using conventional imaging modes for common secondary electron detectors. LAY DESCRIPTION: Successful analysis of a material in a transmission electron microscope requires very thin windows of the material to be fabricated. Despite the quality of this analysis relying heavily on the thickness of the window, measuring thickness during window fabrication is not common practice. The authors show that it is possible to measure the thickness of the window directly in a focused-ion-beam chamber with a scanning electron microscope without altering the fabrication procedure, and using electron detectors common to most microscopes.

Liftout of High-Quality Thin Sections of a Perovskite Oxide Thin Film Using a Xenon Plasma Focused Ion Beam Microscope.

It is shown that a xenon plasma focused ion beam (FIB) microscope is an excellent tool for high-quality preparation of functional oxide thin films for atomic resolution electron microscopy. Samples may be prepared rapidly, at least as fast as those prepared using conventional gallium FIB. Moreover, the surface quality after 2 kV final polishing with the Xe beam is exceptional with only about 3 nm of amorphized surface present. The sample quality was of a suitably high quality to allow atomic resolution high-angle annular dark field imaging and integrated differential phase contrast without any further preparation, and the resulting images were good enough for quantitative evaluation of atomic positions to reveal the oxygen octahedral tilt pattern. This suggests that such xenon plasma FIB instruments may find widespread application in transmission electron microscope and scanning transmission electron microscope specimen preparation.

Transmission Electron Microscope Specimen Preparation of Si-Based Anode Materials for Li-Ion Battery by Using Focused Ion Beam and Ultramicrotome

Transmission Electron Microscope Specimen Preparation of Si-Based Anode Materials for Li-Ion Battery by Using Focused Ion Beam and Ultramicrotome

Density profile and morphology of viscoelastic thermal compressed wood

The viscoelastic thermal compression (VTC) of low-density hybrid poplar (Populus deltoides × Populus trichocarpa) from fast growing trees was performed in order to produce specimens with three different degrees of densification (63, 98, and 132%). The morphology and density profile of the VTC specimens were studied. Three different methods for the preparation of specimens for microscopy were used in order to find a technique that makes it possible to examine the VTC wood microscopically in the completely deformed state. It was found that the abrasive surface preparation of oil-embedded blocks was the most promising technique. Microscopic observation revealed that the deformations in the VTC wood were mostly the result of the viscous buckling of cell walls without fracture. The volume of the void areas in the specimens decreased with the degree of densification. The results showed that the density profile of the VTC wood varied with the degree of densification as a consequence of different temperature and moisture gradients formed before and during wood compression. The density profile is also visible on the cross-section of the VTC specimens.

Osler's lost thesis

William Osler's thesis was undertaken in 1871 to fulfil the graduation requirements of the McGill Faculty of Medicine. The thesis was based upon 20 autopsies and the preparation of gross and microscopic specimens. To the despair of historians, only a portion, the Introduction, was thought to have survived and part of which appeared in Cushing's biography. In the Osler Library is the final version of the Introduction together with parts of newly found earlier drafts and several incomplete autopsy reports and commentaries from the body of the thesis. Osler's early literary and classical erudition is clearly seen, and his philosophy of pathology as the foundation of clinical medicine strongly expressed. In addition, from a fragment of commentary on one of the postmortems, a venture into parasitology is seen representing what appears to be the first identification of the beef tapeworm in Canada.

Transmission electron microscope specimen preparation for exploring the buried interfaces in plan view

A relatively easy and convenient process for the preparation of transmission electron microscope specimens of buried interfaces is described. The method is based on the alignment and realignment of the specimen rotation centre during ion milling. The ion-milling time interval in which good samples are obtained is substantially extended in this way.

Rapid Cross-Section TEM Specimen Preparation of III-V Materials

Abstract Cross-section transmission electron microscope (TEM) specimen preparation of Ill-V materials using conventional methods can be a painful and time-consuming activity, with a day or more from receipt of a sample to examination in the TEM being the norm. This article describes the cross-section TEM specimen preparation technique used at Bookham Caswell. The usual time from start to finish is &amp;lt;1 hour. Up to 10 samples can be prepared at once, depending upon sample type. Most of the tools used are widely available and inexpensive, making the technique ideal for use in institutions with limited resources.

The effect of the gold sputter-coated films in minimising damage in FIB-produced TEM specimens

A sputter-coated gold film, of variable thickness, was used to protect the near surface region structure of an AlAs–InAs superlattice from damage during transmission electron microscope (TEM) specimen preparation using a focused ion beam (FIB) miller. This coating was applied before the deposition of any platinum protective film in the FIB. The extent of any damage in this specimen was investigated by TEM. Where the gold film was either locally absent or of low thickness (<15 nm), ion beam damage of this near surface region was evident. It was concluded that the deposition of platinum in the FIB may lead to surface modification of TEM specimens and that the application of a gold layer at least 60 nm in thickness should be made prior to any FIB processing.

Tem Characterisation of Electrolytic Separated Specimens of Strontium Bismuth Tantalate Ferroelectric Thin Films

Preparation of specimens for Transmission Electron Microscopy usually involves elaborate and aggressive procedures that can produce artefacts in the observations. In this work we use an original preparation method for thin films that involves the electrolytic separation of the film from the substrate. Developed initially to study the characteristics of films isolated from the substrate, this method proves to be easier, less time consuming and in principle less intrusive than the traditional ones. The materials analysed are Strontium Bismuth Tantalate (SBT) ferroelectric films, processed by multiple deposition onto Pt/TiO 2 /SiO 2 substrates of sol-gel derived solutions and crystallisation by Rapid Thermal Processing (RTP) at 650°C. Final thickness range between 70 and 500 nm, which test the versatility of the method to obtain TEM specimens from films with varying thickness, and, at the same time, allows us to study the differences that the deposition of several layers may induce. Several issues concerning the optimisation of the SBT thin films processing need TEM analysis, and are also addressed in this work.

Technical advance: an automated device for cryofixation of specimens of electron microscopy using liquid helium.

Metal-contact rapid freezing using liquid helium is theoretically the best method for preserving the fine structure of living cells with high temporal resolution in preparation of tissue samples for electron microscopy. However, this method is not commonly used, because of its technical difficulty and low reproducibility. We have designed and constructed an automatic device which allows simple, rapid and reproducible preparation of high-quality electron microscopic specimens by the non-specialist. We assessed the quality of cryofixation in samples prepared using this device by examining the preservation of cellular ultrastructure in relation to distance from the freezing block, and found that the region within 10 microm of the metal-contact plane was fixed with the highest quality. We applied this device, in combination with freeze-substitution methods and immunocytochemical techniques, to two phenomena involving rapid movement of subcellular components: (1) active movement of subcellular structures in the papillar cells of stigma and (2) light-induced rapid subcellular translocation of phytochrome A. Considering the importance of understanding subcellular processes of living cells for molecular and cell biology, this device will be a useful tool for diverse biological applications in the near future.

Biochemical, electron microscopic and immunohistological observations of cationic detergent-extracted cells: detection and improved preservation of microextensions and ultramicroextensions

BackgroundFilopodia, retraction fibers and microvilli, are fragile microextensions of the plasma membrane that are easily damaged by mechanical force during specimen preparation for microscopy. To preserve these structures for electron microscopy glutaraldehyde is generally used, but it often causes antigen masking. By contrast, formaldehyde is generally used for immunofluorescence light microscopy, but few studies have been concerned with the loss of microextensions.ResultsWe demonstrate in biochemical experiments that cultured cells needed to be kept in 4% formaldehyde for at least 60 min at room temperature or for 20 min at 37°C to irreversibly crosslink most of the polypeptides. Also, fragmentation of fragile microextensions was observed after Triton X-100 extraction depending on concentration and extent of crosslinking. We also report on a novel fixation procedure that includes the cationic detergent dodecyltrimethylammonium chloride (DOTMAC). Treatment of NIH3T3 cells with DOTMAC resulted in complete removal of membrane lipids and in good preservation of the cytoskeleton in microextensions as well as preservation of ultramicroextensions of <0.05μm in diameter that have not been observed previously unless glutaraldehyde was used. Stress fibers and microextensions of DOTMAC-extracted cells were readily stained with anti-β-actin antibodies, and antibodies to vinculin and moesin stained focal contacts and microextensions, respectively.ConclusionsSome microextensions were fragmented by the standard Triton X-100 permeabilization method. By contrast, DOTMAC completely extracted membrane lipids while maintaining the cytoskeleton of microextensions. Thus, DOTMAC treatment may provide a valuable new tool for the reliable visualization of previously undetectable or poorly detectable antigens while preserving the actin cytoskeleton of microextensions.

Biological specimen preparation for transmission electron microscopy

Biological specimen preparation for transmission electron microscopy

Method for Cross-sectional Transmission Electron Microscopy Specimen Preparation of Composite Materials Using a Dedicated Focused Ion Beam System.

: A new method for transmission electron microscope (TEM) specimen preparation using a focused ion beam (FIB) system that results in a lower rate of gallium (Ga) implantation has been developed. The method was applied to structural and analytical studies of composite materials such as silicon (Si)-devices and magneto-optical disk. To protect the specimens against Ga ion irradiation, amorphous tungsten (W) was deposited on the surface of the specimen prior to FIB milling. The deposition was quite effective in reducing the Ga implantation rate, and energy-dispersive X-ray (EDX) analysis of these specimens detected 0.3-1.5% Ga incorporated in the thinned area. FIB milling times for these specimens were 1.5-2 hr, with a starting thickness of about 50 µm. Although the milling rate was high, all the materials were properly prepared for TEM study, and clear crystal lattice images were observed on all specimens.

Book Review, The Science of Biological Specimen Preparation for Microscopy by M. Malecki and G. M. Roomans

Advance in our knowledge of the molecular organization of living material rests on two kinds of technological development: (1) microscope instrumentation and (2) specimen preparation. This includes new techniques appropriate for a specific mode of microscopy, and information on any structural alterations produced during such specimen preparation.

Transmission electron microscope specimen preparation of Zn powders using the focused ion beam lift-out technique

Particles of Zn powder have been studied to show that high-quality scanning electron microscope (SEM) and transmission electron microscope (TEM) specimens can be rapidly produced from a site-specific region on a chosen particle by the focused ion beam (FIB) lift-out technique. A TEM specimen approximately 20-µm long by 5-µm wide was milled to electron transparency, extracted from the bulk particle, and micromanipulated onto a carbon coated copper mesh TEM grid. Using the FIB lift-out method, we were able to prepare a site-specific TEM specimen from a difficult material in under 3 hours. The TEM analysis of the lift-out specimen revealed a large amount of thin area free from characteristic signs of damage that may be observed as a result of conventional argon ion milling. The overall microstructure of the specimen prepared by the FIB lift-out method was consistent with samples prepared by conventional metallographic methods. A grain size of ∼10 to 20 µm was observed in all specimens by both TEM and SEM analysis. Light optical microscopy revealed the presence of internal voids in ∼10 to 20 pct of all particles. The SEM analysis showed the voids to extend over ∼70 pct of the particle volume in some cases.

Quantitative analysis of chondroitin sulphate retention by tannic acid during preparation of specimens for electron microscopy

Quantitative analysis of chondroitin sulphate retention by tannic acid during preparation of specimens for electron microscopy