The correlation of light and electron microscope observations

A technique is described to allow electron microscopic investigation of a specific feature of a section on a glass slide. A section on a glass slide (previously treated with a silicone release agent) is processed as required for light microscopy. The section is then impregnated with Araldite and cured with an epoxy resin block on top of it. The section and block are removed from the slide and viewed with a light microscope. The selected area for ultrastructural study remains under continuous observation while the block is trimmed. Semi-thin (1 μm) sections retain the original staining for light microscopy and ultra-thin sections are stained with heavy metals in the normal manner. We show how an inflammatory lesion in a large area of muscle in a case of polymyositis may be quickly located and studied at the ultrastructural level.     

NanoSIMS imaging of Bacillus spores sectioned by focused ion beam

Preparation and sectioning of bacterial spores by focused ion beam and subsequent high resolution secondary ion mass spectrometry analytical imaging is demonstrated. Scanning transmission electron microscopy mode imaging in a scanning electron microscope is used to show that the internal structure of the bacterial spore can be preserved during focused ion beam sectioning and can be imaged without contrast staining. Ion images of the sections show that the internal elemental distributions of the sectioned spores are preserved. A rapid focused ion beam top‐sectioning method is demonstrated to yield comparable ion images without the need for sample trenching and section lift‐out. The lift‐out and thinning method enable correlated transmission electron microscopy and high resolution secondary ion mass spectrometry analyses. The top‐cutting method is preferable if only secondary ion mass spectrometry analyses are performed because this method is faster and yields more sample material for analysis; depth of useful sample material is ～300 nm for top‐cut sections versus ～100 nm for electron‐transparent sections.     

High resolution optical microscopy of animal tissues by the use of sub-micrometer thick sections and a new stain

The introduction of 1 micron-thick sections from plastic embedded material represents a great technical improvement for the study of tissues under the optical microscope. However, even sections of this thickness appear too thick when observed with oil immersion objectives of high numerical aperture. The extremely shallow depth of field of these lenses allows them to differentiate several focal planes within a one micron thick section. This in turn results in ghost images being formed from out of focus structures, a problem particularly vexing when photomicrography is attempted. To circumvent this difficulty, we reduced the thickness of the sections down to an optimum of 0.4 micron. These thinner sections do require a very energetic stain to give enough contrast to the cellular structures; Stevenel Blue, a stain recently adapted for plastic sections [del Cerro et al., Microsc. Acta 83, (2), 117--121 (1980)] proved to be the most suitable for this purpose of several stains tested. In summary, submicrometer thick sections stained with Stevenel Blue allow to reach the limits of visibility permitted by the best available objectives and effectively merge the realm of optical microscopy with that of low power electron microscopy.     

En bloc optical sectioning of resin-embedded specimens using a confocal laser scanning microscope

Reconstruction of 3D structures of specimens embedded for light or electron microscopy is usually achieved by cutting serial sections through the tissues, then assembling the images from each section to reconstruct the original structure or feature. This is both time-consuming and destructive, and may lead to areas of particular interest being missed. This paper describes a method of examining specimens which have been fixed in glutaraldehyde and embedded in epoxy resin, by utilising the autofluorescence preserved or enhanced by aldehyde fixation, and by using a confocal laser scanning microscope to section optically such specimens in the block down to a depth of about 200 μm. In this way, the accurate estimation of the depth of particular features could be used to facilitate subsequent sectioning at the light microscope or electron microscope level for more detailed studies, and 3D images of tissues/structures within the block could be easily prepared if required.     

Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens

Stain density is an important parameter for optimising the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue and liver tissue.     

An electron-optical lens effect as a possible source of contrast in biological preparations

Heavy-metal stain aggregates on the surface of thin sections of biological material have higher contrast than those embedded within the sections and both have greater contrast than can be accounted for by the amplitude image. Disturbances of the incident illumination by a specimen in both light- and electron-optical systems and their possible contribution to image contrast are considered. The hypothesis is proposed that a lens effect produced by the stain aggregates may account for their contrast in the electron microscope in a similar manner to the contrast of glass beads in the light microscope with a low numerical aperture.     

Capsule-supporting ring: a new device for resin embedding of glass-mounted specimens

The goal of specimen preparation for transmission electron microscopy is to obtain high-quality ultra-thin sections with which we can correlate cellular structure to physiological function. In this study, we newly developed a capsule-supporting ring that can be useful for resin embedding of glass-mounted specimens. The present device allowed us to re-embed a semi-thin section on a microscope slide into a resin block not only for efficient ultra-thin sectioning but also for a correlative light and electron microscopy. Similar to epoxy resins for morphological observations, semi-thin sections of low-viscosity hydrophilic resins, such as Lowicryl series, can be re-embedded into the resin, which can be useful for cytochemical gold labelling. A further application of the present device improved flat embedding of cultured cells on glass cover slips for electron microscopy, preserving in situ sub-cellular structures close to their native state. We practically describe the use of capsule-supporting ring and demonstrate representative micrographs as results.     

Backscattered electron SEM imaging of cells and determination of the information depth

Backscattered electron imaging of HT29 colon carcinoma cells in a scanning electron microscope was studied. Thin cell sections were placed on indium‐tin‐oxide‐coated glass slides, which is a promising substrate material for correlative light and electron microscopy. The ultrastructure of HT29 colon carcinoma cells was imaged without poststaining by exploiting the high chemical sensitivity of backscattered electrons. Optimum primary electron energies for backscattered electron imaging were determined which depend on the section thickness. Charging effects in the vicinity of the SiO₂ nanoparticles contained in cell sections could be clarified by placing cell sections on different substrates. Moreover, a method is presented for information depth determination of backscattered electrons which is based on the imaging of subsurface nanoparticles embedded by the cells.     

Silicon nitride windows for electron microscopy of whole cells

Silicon microchips with thin, electron transparent silicon nitride windows provide a sample support that accommodates both light‐, and electron microscopy of whole eukaryotic cells in vacuum or liquid, with minimum sample preparation steps. The windows are robust enough that cellular samples can be cultured directly onto them, with no addition of a supporting film, and there is no need to embed or section the sample, as is typically required in electron microscopy. By combining two microchips, a microfluidic chamber can be constructed for the imaging of samples in liquid in the electron microscope. We provide microchip design specifications, a fabrication outline, instructions on how to prepare the microchips for biological samples, and examples of images obtained using different light and electron microscopy modalities. The use of these microchips is particularly advantageous for correlative light and electron microscopy.     

A hybrid scanning force and light microscope for surface imaging and three-dimensional optical sectioning in differential interference contrast

The design of a scanned-cantilever-type force microscope is presented which is fully integrated into an inverted high-resolution video-enhanced light microscope. This set-up allows us to acquire thin optical sections in differential interference contrast (DIC) or polarization while the force microscope is in place. Such a hybrid microscope provides a unique platform to study how cell surface properties determine, or are affected by, the three-dimensional dynamic organization inside the living cell. The hybrid microscope presented in this paper has proven reliable and versatile for biological applications. It is the only instrument that can image a specimen by force microscopy and high-power DIC without having either to translate the specimen or to remove the force microscope. Adaptation of the design features could greatly enhance the suitability of other force microscopes for biological work.     

A technique for studying one and the same section of a cell in sequence with the light and electron microscope

Methods for mounting and staining relatively thin sections on electron microscope grids, in order that one and the same section of a cell can be photographed in sequence with the light and electron microscope are described. Toluidine blue is used as a stain and hexachlorabuta-1,3 diene as a medium which enables the grid carrying the stained sections to be temporarily mounted under a coverslip and examined with an oil-immersion lens. Results obtained with pollen mother cells of Fritillaria lanceolata at zygotene are illustrated.     

Rapid method for resectioning of light microscopy sections for electron microscopy

A rapid method is described for obtaining ultrathin sections from light microscopy sections. Five-micrometre epoxy sections, heat-flattened to slides, were affixed to the tips of plastic blocks by light-curable dental bond, and cured while still on the microscope stage by illumination with blue light for 2 min. Sections were detached from the slides by rapid cooling and then resectioned for electron microscopy.     

Correlative light and backscattered electron microscopy of bone--Part I: Specimen preparation methods

A method for preparing nondecalcified bone and tooth specimens for imaging by both light microscopy (LM) and backscattered electron microscopy in the scanning electron microscope (BSE-SEM) is presented. Bone blocks are embedded in a polymethylmethacrylate (PMMA) mixture and mounted on glass slides using components of a light-cured dental adhesive system. This method of slide preparation allows correlative studies to be carried out between different microscopy modes, using the same histologic section. It also represents a large time savings relative to other mounting methods whose media require long cure times.     

Transmission electron microscopy applied to the study of works of art: sample preparation methodology and possible techniques

Technical examination of a work of art is a necessary preliminary stage both for proper conservation/restoration of the work and for purposes of dating and/or authentication. There is a wide variety of methods and procedures, and of these a particularly valuable technique is stratigraphic analysis in view of the data that it furnishes on the composition of the pictorial layers of which a painting is composed. The techniques utilized in this type of analysis to date have been essentially light microscopy and scanning electron microscopy. Transmission electron microscopy can provide new data for characterization of pictorial layers, thanks to the possibility of individually using ultrathin sections of paint sample. This study provides morphological analysis and microanalysis by X-ray energy dispersion, with determination of the crystalline structure of each particle by electron diffraction. The sample preparation method for producing thin sections from the pictorial layers for examination in the TEM is described. This allows the stratigraphic section to be preserved exactly as applied by the artist. The first results from the examination of three microsamples from actual old works of art are presented. The individual components of each strata were successfully identified in all cases.     

Identification of bacteria by studying one section under light microscopy,scanning, and transmission electron microscopy

A method for bacterial identification has been developed by means of studying the same histological sections through several types of microscopy. With this method, one section was processed and analyzed respectively for light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Sections of gingival biopsies were Gram stained and bacteria tentatively identified by LM. Photographs of the sections were taken and presketched transparent acetate sheets (PTAS) were made from the photos. The same section was later prepared for SEM, areas previously thought to contain bacteria were localized by placing the PTAS onto the SEM monitoring screen. The SEM specimens were subsequently processed for TEM, bacteria were located, and micrographs obtained. The results showed that out of ten diseased gingival biopsies observed under the LM, bacteria were found to be present in all the specimens and were identified as both Gram positive and Gram negative. By transferring the section from LM to SEM, the bacteria could be relocated and their morphotype (cocci, rods, etc.) clearly identified in most of the cases. Since cocci may resemble other biological granular structures under SEM, they require further analysis under TEM for additional positive identification. This study demonstrated that the method described here is a useful tool for assessing the presence and identifying bacteria within the gingival tissues.     

Electron microscopy of semithick sections: Advantages for biomedical research

Many transmission electron microscopes are available which can be used to examine biological material in 0.25–0.50-μm-thick sections. When compared to the traditional thin section, these “semithick” sections possess a number of inherent advantages: They can be screened for content with the phase contrast light microscope, they facilitate many types of studies requiring an analysis of serial sections, and they are frequently the optimum thickness for stereomicroscopy. Structures such as microtubule-associated components, as well as structural relationships between cellular constituents, may also be clearly visible in semithick sections which are not visible, or go unnoticed, in thin sections. Together these advantages enable an investigator to obtain a more complete three-dimensional picture of a cell or cell component in a significantly (i.e., up to 90%) shorter period of time than would be required if thin sections were used. Semithick sections may, therefore, make a study feasible which is not approachable, or which is approachable only with great difficulty, by conventional thin sectioning techniques.     

A routine flat embedding method for electron microscopy of microorganisms allowing selection and precisely orientated sectioning of single cells by light microscopy

A simple method is described to embed material in resin, in the form of microscope slides, to observe it with high resolution light microscopy, to select, orient and section it for TEM. This method can be applied to many kinds of material but is particularly useful for the study of rare or tiny plant or animal microorganisms from field or culture. A diamond scriber, translucent hydrosoluble resin release agent, translucent and smooth resin stubs and a longitudinally perforated block-holder for ultramicrotome are the specific tools of this method.     

Video microscopy of dynamic plant cell organelles: principles of the technique and practical application

Video microscopy, including video-enhanced contrast, ultraviolet and video-intensified fluorescence microscopy, was applied to the visualization and analysis of organelles and cytoskeletal elements at the border of resolution of the light microscope. We describe the principles of video microscopy and the necessary technical equipment, and discuss the advantages and limitations with the example of three selected plant cells. In characean internodal cells we observed the motility and disappearance of Golgi secretory vesicles during wound wall formation by video-enhanced contrast microscopy. In Byblis gland hairs we investigated the movement of different organelles along bundles of actin microfilaments by ultraviolet microscopy, and in onion inner epidermal cells we visualized the arrangement of actin microfilaments during different stages of plasmolysis with video-intensified fluorescence microscopy.     

Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events

Biological studies have relied on two complementary microscope technologies – light (fluorescence) microscopy and electron microscopy. Light microscopy is used to study phenomena at a global scale to look for unique or rare events, and it also provides an opportunity for live imaging, whereas the forte of electron microscopy is the high resolution. Traditionally light and electron microscopy observations are carried out in different populations of cells/tissues and a 'correlative' inference is drawn. The advent of true correlative light-electron microscopy has allowed high-resolution imaging by electron microscopy of the same structure observed by light microscopy, and in advanced cases by video microscopy. Thus a rare event captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by electron microscopy. Here, the potential and difficulties of this approach, along with the most impressive breakthroughs obtained by these methods, are discussed.     

Enhancement of contrast in living and fixed specimens by the use of fibre optics

A method is described which enhances the contrast of living and fixed specimens examined with the stereomicroscope. It consists of immersing the ends of flexible fibre optic light sources together with the specimen in the fluid used for examination. It is reported that not only does this method increase the contrast of living specimens but that it may also be applied to specimens being prepared as thin sections or freeze fracture surfaces for examination with the transmission electron microscope. A further method of enhancement of contrast is suggested which involves the fitting of light filters of complementary colours, one to each of the fibre optic light sources, before immersion with the specimen.