Sample preparation procedures for biological atomic force microscopy

Since the late 1980s, atomic force microscopy (AFM) has been increasingly used in biological sciences and it is now established as a versatile tool to address the structure, properties and functions of biological specimens. AFM is unique in that it provides three-dimensional images of biological structures, including biomolecules, lipid films, 2D protein crystals and cells, under physiological conditions and with unprecedented resolution. A crucial prerequisite for successful, reliable biological AFM is that the samples need to be well attached to a solid substrate using appropriate, nondestructive methods. In this review, we discuss common techniques for immobilizing biological specimens for AFM studies.     

The preparation of fragile protoplasts for electron microscopy

A method is described whereby highly fragile protoplasts may be successfully prepared for electron microscopy.     

Rapid preparation of plant tissues for electron microscopy

Various plant tissues were prepared for electron microscopy using a rapid process similar to that of Hayat & Giaquinta (1970) for animal tissue. The entire preparation took less than 4 h instead of the several days taken for the usual method and gave very good results. Root, leaf and fruit tissues were used.     

Chitosan utilized for bacterial preparation for scanning electron microscopy

Bacterial sample preparation is crucial for its observation by scanning electron microscopy (SEM). However, the current polylysine (PLL) method leads to bacterial morphological changes. To overcome this problem, we employed chitosan (CS) to coat coverslips to prepare bacteria for SEM and compared it with the PLL method. Coverslips coated with 0.025% (w/v) CS showed satisfactory bacterial binding ability. Within 30 min of binding time, the number of bacteria on CS-coated and PLL-coated coverslips exhibited no differences. Four bacteria strains were employed to compare the differences in SEM images between the two methods. Most of the bacteria showed irregular surface or sticky substances after settling on PLL-coated coverslips, while bacteria with clear surface texture were observed on CS-coated coverslips. Transmission electron microscopy (TEM) images showed deformed bacterial envelope on PLL-coated coverslips; meanwhile, similar intact envelope was observed from the bacteria on CS-coated coverslips and the bacteria without any treatment. The TEM results verified the morphological differences of SEM between the two methods. Except for morphology, the length of the rod-shaped bacteria was longer on CS-coated coverslips than that on PLL-coated coverslips, less shrinkage of the sample was observed, and CS could preserve the length of the rod-shaped bacteria better than PLL in its preparation for SEM. It is demonstrated that the low-cost CS could be utilized in bacterial preparation for SEM to acquire preferable images. Bacterial suspension with optical density at 600 nm of about 0.5, deposited on 0.025% CS-coated coverslips for 30 min, and followed by routine fixation, dehydration, and drying are optimal parameters.     

The preparation of cross-section specimens for transmission electron microscopy

The structure and chemistry of thin solid films are best studied by transmission electron microscopy (TEM) when they are viewed in cross-section—that is, when the surface normal of the film is made perpendicular to the electron beam. In this orientation, the substrate, the thin film layers, and the interfaces between them can be imaged either simultaneously or individually. Further, information from each of these regions remains distinct from that obtained from the others, eliminating the problems of superimposition that are a consequence of viewing a layered structure in the conventional manner (i.e., parallel to the surface normal). A technique for fabricating TEM specimens that can be viewed in cross-section is described here. Although the majority of our work is with silicon-based materials, the technique can be readily adapted to the study of other systems.     

Sectionable microcarrier beads: An improved method for the preparation of cell monolayers for electron microscopy

Cross-linked dextran beads provide an excellent surface for tissue-cultured cell monolayers, and can be processed for transmission (TEM) and scanning (SEM) electron microscopy, as well as light microscopy (LM). Cells are grown to confluency on the surface of the microcarriers, where at any point aliquots can be removed and experimentally treated as desired (e.g. immunocytochemistry) providing a representative sample. Sample preparation for TEM follows standard procedures for any cell monolayer, but infiltration times must be at least doubled to allow penetration of the beads. The polymerized blocks can then be sectioned for TEM or LM with no additional steps required. SEM sample preparation involves attaching the fixed bead/cell suspension to a glass coverslip with poly-1-lysine, dehydration, critical point drying, and coating for conductivity. The fixed and dried sample can also be attached directly to the SEM stub as free beads and subsequently gold coated. These beads provide (1) an increased surface area of cells visible per area of thin section, (2) eliminates the careful orientation required for flat substrate methods of embedding, (3) decreases the amount of sample manipulation in the forms of re-embedding and gluing, and (4) decreases the amount of drying artifact seen as cracking in SEM monolayer preparations.     

A simple method for whole-cell preparation in electron microscopy

A simple method for whole-cell preparation without using gold or platinum grids as substrata for culture is described. Cells were cultured on formvar film over round pores, each 3 mm in diameter, of Thermonox coverslips. The cells on the formvar coated coverslip are fixed, stained, dehydrated in situ, and introduced into a critical point drying apparatus. A small quantity of 0.2% mesh-cement is applied to slot grids, and they are laid onto the formvar film over the pores of the coverslip. After the grids are removed from the plastic substratum, they are ready for observation under the electron microscope.     

Removal of serum from virus suspensions in preparation for immune electron microscopy.

Serum from viral suspensions is precipitated by shaking with Freon 112, and after concentration the virus is observed by immune electron microscopy.     

Microwave-assisted rapid plant sample preparation for transmission electron microscopy

The preparation of plant leaf material for transmission electron microscopical investigations can be a very time- and labour-consuming task as the reagents infiltrate the samples quite slowly and as usually most steps have to be performed manually. Fixation, buffer washes, dehydration, resin infiltration and polymerization of the resin-infiltrated leaf samples can take several days before the specimen can be cut ultrathin and used for ultrastructural investigations. In this study, we present a microwave-assisted automated sample preparation procedure that reduces preparation time from at least 3 days to about 5 h – with only a few steps that have to be performed manually – until the plant sample can be ultrathin sectioned and observed with the transmission electron microscope. For studying the efficiency of this method we have compared the ultrastructure of different leaf material ( Arabidopsis thaliana , Nicotiana tabacum and Picea abies ) which was prepared with a conventional, well-established chemical fixation and embedding protocol and a commercially available automated microwave tissue processor. Despite the massive reduction in sample preparation time no negative effects on cutting properties of the blocks, stability of the sections in the electron beam, contrast and ultrastructure of the cells were observed under the transmission electron microscope when samples were prepared with the microwave-assisted protocol. Additionally, no negative effects were detected on the dimensions of fine structures of grana stacks (including membranes, inter- and intrathylakoidal spaces), the nuclear envelope and the plasma membrane as the diameter of these structural components did not differ between leaf samples (of the same species) that were processed with the automated microwave tissue processor or by conventional fixation and embedding at room temperature.     

The preparation of human articular cartilage for scanning electron microscopy

This paper compares sixteen preparative techniques thought to be of advantage in the study by scanning electron microscopy (SEM) of human articular cartilage surfaces. The adequacy of surface preservation obtained with the techniques, was judged subjectively, first, by the reproducibility of secondary electron images of normal cartilage, and second, by comparing the results with those obtained by reflected light microscopy of the fresh unfixed cartilage surface over a magnification range of × 20 – × 240. Adequate surface preservation was confirmed when cartilage surfaces had been dehydrated through ethanol to propylene oxide and vacuum dried; dehydrated through amyl acetate and quenched in Freon before freeze-drying; dehydrated and passed through amyl acetate at low temperature before freeze drying. Valuable information can be obtained from different specimens by varying the technique of preparation. At different ages, different surface features are best preserved. In a systematic study it has been found essential to adopt a uniform preparative method and to control the results by reflected light microscopy. Even with the most perfect preparation, the surface appearances cannot be identical with those that function under load in vivo.     

A specimen preparation technique for transmission electron microscopy of surface layers

A TEM specimen preparation method is described, with the aid of which electron transparent foils can be obtained across the external surface of a specimen. After careful pre-treatment, steel specimens have been electrolytically coated with nickel. Conventional thinning in a plane cutting the substrate-coating interface, gave thin foils displaying the internal structure as a function of depth under the initial free surface. The method has also been applied to minute metal particles, of dimensions too small to allow manipulating and foil preparation by conventional methods. Image examples are shown, and the applicability of the method is discussed.     

Sample preparation for the quick sizing of metal nanoparticles by atomic force microscopy

Two alternative pretreatment methods for depositing metal nanoparticles on mica for atomic force microscopy (AFM) imaging are presented. The treated substrates are flat and clean, thus they are amenable of use to characterize very small nanoparticles. The methods do not require any instrumentation or particular expertise. As they are also very quick, the need for storage of the prepared substrates is avoided altogether. These proposed methods, which are compared with the results of transmission electron microscopy analysis, allow the quick sizing and characterization of nanoparticles with the atomic force microscope and could thus help expanding the user community of nanoparticle researchers who could use the AFM for their characterization needs.     

Surface structure imaging by electron microscopy

Reconstructed structures at monolayer level on ‘clean and well-defined’ surfaces can be imaged by transmission electron microscopy in fixed beam illumination mode. The specimens are cleaned in-situ in the electron microscope in ultra high vacuum. Transmission electron diffraction pattern intensities can give useful information for determining the surface unit cell size of the structure, and the atom positions (geometric arrangement of atoms in the unit cell) especially those with a large unit cell, since the diffraction intensities are interpreted kinematically. High resolution surface imaging which gives directly the atom positions is tested here for a single monolayer terrace on Ag (111) surface. The result shows the value of HREM for studies of surface crystallography.