Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples

A new device (NTEGRA Tomo) that is based on the integration of the scanning probe microscope (SPM) (NT‐MDT NTEGRA SPM) and the Ultramicrotome (Leica UC6NT) is presented. This integration enables the direct monitoring of a block face surface immediately following each sectioning cycle of ultramicrotome sectioning procedure. Consequently, this device can be applied for a serial section tomography of the wide range of biological and polymer materials. The automation of the sectioning/scanning cycle allows one to acquire up to 10 consecutive sectioned layer images per hour. It also permits to build a 3‐D nanotomography image reconstructed from several tens of layer images within one measurement session. The thickness of the layers can be varied from 20 to 2000 nm, and can be controlled directly by its interference colour in water. Additionally, the NTEGRA Tomo with its nanometer resolution is a valid instrument narrowing and highlighting an area of special interest within volume of the sample. For embedded biological objects the ultimate resolution of SPM mostly depends on the quality of macromolecular preservation of the biomaterial during sample preparation procedure. For most polymer materials it is comparable to transmission electron microscopy (TEM). The NTEGRA Tomo can routinely collect complementary AFM and TEM images. The block face of biological or polymer sample is investigated by AFM, whereas the last ultrathin section is analyzed with TEM after a staining procedure. Using the combination of both of these ultrastructural methods for the analysis of the same particular organelle or polymer constituent leads to a breakthrough in AFM/TEM image interpretation. Finally, new complementary aspects of the object's ultrastructure can be revealed.     

Atomic force microscopy of histological sections using a chemical etching method

Physiology and pathology have a big deal on tissue morphology, and the intrinsic spatial resolution of an atomic force microscope (AFM) is able to observe ultrastructural details. In order to investigate cellular and subcellular structures in histological sections with the AFM, we used a new simple method for sample preparation, i.e. chemical etching of semithin sections from epoxy resin-embedded specimens: such treatment appears to melt the upper layers of the embedding resin; thus, removing the superficial roughness caused by the edge of the microtome knife and bringing into high relief the biological structures hidden in the bulk. Consecutive ultrathin sections embedded in epoxy resin were observed with a transmission electron microscope (TEM) to compare the different imaging properties on the same specimen sample. In this paper we report, as an example, our AFM and TEM images of two different tissue specimens, rat pancreas and skeletal muscle fibres, showing that most of the inner details are visible with the AFM. These results suggest that chemical etching of histological sections may be a simple, fast and cost-effective method for AFM imaging with ultrastructural resolution.     

The cutting of ultrathin sections with the thickness less than 20 nm from biological specimens embedded in resin blocks

Low voltage electron microscopes working in transmission mode, like LVEM5 (Delong Instruments, Czech Republic) working at accelerating voltage 5 kV or scanning electron microscope working in transmission mode with accelerating voltage below 1 kV, require ultrathin sections with the thickness below 20 nm. Decreasing of the primary electron energy leads to enhancement of image contrast, which is especially useful in the case of biological samples composed of elements with low atomic numbers. As a result treatments with heavy metals, like post‐fixation with osmium tetroxide or ultrathin section staining, can by omitted. The disadvantage is reduced penetration ability of incident electrons influencing the usable thickness of the specimen resulting in the need of ultrathin sections of under 20 nm thickness. In this study we want to answer basic questions concerning the cutting of extremely ultrathin sections: Is it possible routinely and reproducibly to cut extremely thin sections of biological specimens embedded in commonly used resins with contemporary ultramicrotome techniques and under what conditions? Microsc. Res. Tech. 79:512–517, 2016. © 2016 Wiley Periodicals, Inc.     

The precise measurement of the thickness of ultrathin sections by a ‘re-sectioned’ section technique

The thickness of ultrathin tissue sections embedded in Epon-Araldite and cut with a diamond knife was measured by re-sectioning and electron microscopic examination of the section profiles. A secondary section mounted on a Formvar-coated slot grid provided enough normally cut segments (seven to seventeen) for measurements giving a precise estimate of mean thickness, comparable to that obtainable by interference microscopy (±2.3% or less for grey to dark gold sections). The standard deviation of section thickness within sections was never more than 5 nm, corresponding to a coefficient of variation of 6.5% or less for sections more than 48 nm thick. This suggests that variation in section thickness, within sections, may be less than has been supposed, so that quantitative work may be based on thickness measurements made over a limited representative area. A silver interference colour was associated with sections 49–60 nm thick.     

X-ray microanalysis of freeze-dried and frozen-hydrated cryosections

The elemental composition and the ultrastructure of biological cells were studied by scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray microanalysis. The preparation technique involves cryofixation, cryoultramicrotomy, cryotransfer, and freeze-drying of samples. Freeze-dried cryosections 100-nm thick appeared to be appropriate for measuring the distribution of diffusible elements and water in different compartments of the cells. The lateral analytical resolution was less than 50 nm, depending on ice crystal damage and section thickness. The detection limit was in the range of 10 mmol/kg dry weight for all elements with an atomic number higher than 12; for sodium and magnesium the detection limits were about 30 and 20 mmol/kg dry weight, respectively. The darkfield intensity in STEM is linearly related to the mass thickness. Thus, it becomes possible to measure the water content in intracellular compartments by using the darkfield signal of the dry mass remaining after freeze-drying. By combining the X-ray microanalytical data expressed as dry weight concentrations with the measurements of the water content, physiologically more meaningful wet weight concentrations of elements were determined. In comparison to freeze-dried cryosections frozen-hydrated sections showed poor contrast and were very sensitive against radiation damage, resulting in mass loss. The high electron exposure required for recording X-ray spectra made reproducible microanalysis of ultrathin (about 100-nm thick) frozen-hydrated sections impossible. The mass loss could be reduced by carbon coating; however, the improvement achieved thus far is still insufficient for applications in X-ray microanalysis. Therefore, at present only bulk specimens or at least 1-μm thick sections can be used for X-ray microanalysis of frozen-hydrated biological samples.     

Preparation of high-quality ultrathin transmission electron microscopy specimens of a nanocrystalline metallic powder

This article explores the achievable transmission electron microscopy specimen thickness and quality by using three different preparation methods in the case of a high-strength nanocrystalline Cu-Nb powder alloy. Low specimen thickness is essential for spatially resolved analyses of the grains in nanocrystalline materials. We have found that single-sided as well as double-sided low-angle Ar ion milling of the Cu-Nb powders embedded into epoxy resin produced wedge-shaped particles of very low thickness (<10 nm) near the edge. By means of a modified focused ion beam lift-out technique generating holes in the lamella interior large micrometer-sized electron-transparent regions were obtained. However, this lamella displayed a higher thickness at the rim of ≥30 nm. Limiting factors for the observed thicknesses are discussed including ion damage depths, backscattering, and surface roughness, which depend on ion type, energy, current density, and specimen motion. Finally, sections cut by ultramicrotomy at low stroke rate and low set thickness offered vast, several tens of square micrometers uniformly thin regions of ～10-nm minimum thickness. As major drawbacks, we have detected a thin coating on the sections consisting of epoxy deployed as the embedding material and considerable nanoscale thickness variations.     

Atomic force microscopy of histological sections using a new electron beam etching method

In order to examine histological sections of the rat vomeronasal epithelium with the atomic force microscope (AFM), we developed an electron beam etching method that improves the resolution of AFM images. This method results in AFM images comparable to those obtained with the transmission electron microscope (TEM). Ultrathin tissue sections embedded in epoxy resin were observed before and after the treatment with electron beam radiation. Before electron beam treatment, epithelial structures such as the microvilli surface, dendritic processes, the supporting cell layers and the neuronal cell layers were all visible using the AFM. However, only a few subcellular structures could also be resolved. The AFM images were not as clear as those obtained with the TEM. After electron beam treatment, however, the resolution of AFM images was greatly improved. Most of the subcellular structures observed in TEM images, including the inner membrane of mitochondria, ciliary-structure precursor body, junctional complexes between the neurons and supporting cells, and individual microvilli were now visible in the AFM images. The electron beam treatment appeared to melt the embedding resin, bringing subcellular structures into high relief. The result of this study suggests that electron beam etching of histological samples may provide a new method for the study of subcellular structure using the AFM.     

Coating sections for electron microscopic autoradiography: a stripping technique using liquid emulsion.

A coating technique for electron microscope autoradiography is described which combines the advantage of forming an emulsion film by a dipping method with the ease of coating sections already on grids. Sections are coated so that a formvar support film separates the section and the emulsion crystals. This intermediate layer of formvar ensures a random distribution of the emulsion crystals. Using light gold sections, Ilford L-4 emulsion and Microdol-X development, the resolution of this technique, as determined by the half distance method, was 150 nm. The additional layer of formvar slightly reduced the image quality with biological samples in the electron microscope. This technique has a minimal loss of resolution and image quality for moderate resolution electron microscope autoradiography.     

Contrast and quantitation in uniform regions of thin sections using transmission electron microscopy

A direct approach to quantitative measurements of uniform regions in thin sections is described. Accelerating voltages around 80 kV and objective aperture angles of about 9·3 mrad will provide conditions where contrast is directly proportional to specimen mass thickness. An extensive treatment of electron scattering in Formvar films for wide ranges of electron microscopic operating conditions is summarized in a simple, empirical equation. The extent to which Formvar results may be generalized to other materials, both embedding media and structures within the thin section, is treated. Using these results, precise measurements of local section thickness and of specimen density and/or dry mass of regions which penetrate the entire section thickness are possible, with the accuracy dependent upon irradiation effects and specimen makeup.     

Cross-sectional atomic force microscopy imaging of polycrystalline thin films

Atomic force microscopy (AFM) can be used to image cross-sections of thin-film samples. So far, however, it has mainly been used to study cross-sections of epitaxial systems or integrated circuits on crystalline substrates. In this paper, we show that AFM is a powerful tool to image fractured cross-sections of polycrystalline thin films deposited on crystalline and non-crystalline substrates, yielding unique information on the three-dimensional properties of the cross-sections, with a spatial resolution in the nm range. Original images of three different heterostructure systems are presented: Si(wafer)/SnO2/CdS/CdTe, glass/Mo/Cu(In,Ga)Se2,/CdS/ZnO, and glass/SnO2/WO3. We discuss the results by comparing AFM and scanning electron microscopy (SEM) images, and explain, for the different materials, why the AFM provides useful additional information.     

Dynamic force microscopy imaging of native membranes

We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.     

A determination of thickness and surface relief in reembedded sections of an epoxy- and a melamine-resin containing ferritin as size standard

Chemical and physical data of two electron microscopic embedding media (the non-polar epoxy resin Epon 812 and the polar melamine resin Nanoplast FB 101) suggest that less kinetic energy must be applied for cutting a section from a Nanoplast block than from an Epon block of the same hardness and that, consequently, the cutting qualities of Nanoplast are better. To test this hypothesis, normal and extremely thin sections of Epon- and Nanoplast-embedded horse spleen ferritin micropellets were reembedded and resectioned for a determination of thickness and surface roughness. The ease with which extremely thin sections can be cut from the Nanoplast resin (8 nm versus 15 nm in Epon) and the smooth surface of these sections support the hypothesis that the cutting quality of an embedding material is determined primarily by its energy balance, i.e. by the kinetic energy which must be introduced for sectioning and the bonding energy which is released exothermically from a polymer while being sectioned.     

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.     

生物大分子超微结构研究中的原子力显微术

原子力显微镜作为第三代显微探测工具,具有原子级的空间分辨率,其样品制备方法简单易行,可在离体的近生理条件下直接观测生物样品及其动态变化过程,能够对样品进行力学操纵,在观察生物大分子的结构和生物力学特性上具有显著的优势。本文尝试从蛋白质、核酸、多糖的超微结构和力学特性的研究角度入手,期望向读者展现出原子力显微镜在大分子生物学研究中的应用前景。     

Biological X-ray microanalysis.

By means of X-ray microanalysis it is now practical to detect approximately 10(-19) g of an element in a static-probe analysis within an ultrathin section, with analytical spatial resolution in the range 20--30 nm. The main difficulties for biological microanalysis are connected not with sensitivity but with specimen preparation and beam damage. Careful cryopreparation, beginning with the quench-freezing of a small block of tissue, is essential even for determining the storage sites, or sites of binding in vivo, of physiologically active elements. In frozen-dried or frozen-hydrated sections of quench-frozen tissue, it is now possible to measure local mass fractions of diffusible as well as of bound elements.     

Estimation of section thickness and quantification of iron standards with EELS

The possibilities of using electron energy-loss spectroscopy (EELS) for quantification of elemental concentrations in ultrathin sections are examined. Dynabeads, which are polystyrene beads with a known iron content, are proposed as internal iron standards. The quantity of an element present depends on the thickness of the specimen. A prerequisite for estimation of absolute section thicknesses with EELS is the knowledge of the mean free path λ of electrons in the specimen. This factor is determined for the embedding resins Epon and Nanoplast by comparing EELS data with directly observed thicknesses in re-embedded sections. Dynabeads were found to include iron in a homogeneous distribution and to be stable in the electron beam.     

Factors which influence light microscopic visualization of biological material in sections prepared for electron microscopy

Epoxy-embedded biological material, sectioned for conventional or high-voltage electron microscopy, can be visualized within the section with good contrast and detail by phase-contrast or dark-field light microscopy. The (phase) contrast of such material is not substantially influenced by the type of embedding resin or section support substrate. It is, however, influenced by the type of fixation, by heavy metal (uranyl and lead) staining and by the section thickness. After screening ultrathin and semithin sections for content with the light microscope, one need stain and examine only those grids containing sections of interest. This approach eliminates the need to screen sections with the electron microscope and, in some cases, the need to stain non-useful sections. This time-saving procedure is particularly useful for studies requiring ultrastructural examination of a selected area or structure which is large enough to be visualized with the light microscope but which comprises only a small volume of the embedded material.     

Combining Ar ion milling with FIB lift-out techniques to prepare high quality site-specific TEM samples

Focused ion beam (FIB) techniques can prepare site‐specific transmission electron microscopy (TEM) cross‐section samples very quickly but they suffer from beam damage by the high energy Ga⁺ ion beam. An amorphous layer about 20–30 nm thick on each side of the TEM lamella and the supporting carbon film makes FIB‐prepared samples inferior to the traditional Ar⁺ thinned samples for some investigations such as high resolution transmission electron microscopy (HRTEM) and electron energy loss spectroscopy (EELS). We have developed techniques to combine broad argon ion milling with focused ion beam lift‐out methods to prepare high‐quality site‐specific TEM cross‐section samples. Site‐specific TEM cross‐sections were prepared by FIB and lifted out using a Narishige micromanipulator onto a half copper‐grid coated with carbon film. Pt deposition by FIB was used to bond the lamellae to the Cu grid, then the coating carbon film was removed and the sample on the bare Cu grid was polished by the usual broad beam Ar⁺ milling. By doing so, the thickness of the surface amorphous layers is reduced substantially and the sample quality for TEM observation is as good as the traditional Ar⁺ milled samples.     

X-ray imaging of various biological samples using a phase-contrast hard X-ray microscope

In this study, we visualized the internal structures of various bio-samples and found the optimum conditions of test samples for the 7 keV hard X-ray microscope of the Pohang light source. From the captured X-ray images, we could observe the intercellular and intracellular structures of dehydrated human cells and mouse tumor tissues without using any staining materials in a spatial resolution better than 100 nm. The metastasized lung tissue, which was several tens of micrometers in thickness, was found to be very well suited to this hard X-ray microscope system, because it is nearly impossible to observe such a nontransparent and thick sample with a high spatial resolution better than 100 nm using any microscopes such as a soft X-ray microscope, an optical microscope, or an electron microscope.     

Embedding in Spurr's resin is a good choice for immunolabelling after freeze drying as shown with chemically unfixed dendritic cells

Immunocytochemical reactions on biological specimens depend on many factors, the most crucial one being the maintenance of antigenicity. Antigens are vulnerable at each stage during preparation for electron microscopy. One of the least traumatic methods of preparing biological tissues for post‐embedding immunolabelling includes the following steps: (1) physical stabilization of the native biological material by rapid freezing (cryofixation) and keeping the immobilized biological sample at low temperature, thereby avoiding any movements of water, ions and macromolecules; (2) dehydrating the frozen biological material by freeze‐drying at low temperature; (3) embedding of the dehydrated specimen. Here we show that embedding of chemically unfixed dendritic cells in Spurr's resin after cryofixation and freeze‐drying enables the conservation of fine ultrastructure without cell distortion or shrinkage. Furthermore, we demonstrate the feasibility of protein localization in ultrathin sections by immunolabelling of the major histocompatibility class II molecules.