Towards native-state imaging in biological context in the electron microscope

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context.     

Simultaneous visualization of myeloperoxidase reactivity and immunogold labeling by backscattered electron imaging with the scanning electron microscope

We describe a method to label circulating human granulocytes with an immunogold marker and then incubate them for demonstration of myeloperoxidase activity. The cells were observed in the backscattered electron imaging (BEI) mode of the scanning electron microscope. This permits simultaneous visualization of cell surface morphology, the immunological surface marker, and the cytochemical reactivity of each individual cell. Enhanced identification of the various cell types and more precise characterization of cell surface features in the different steps of leukocyte differentiation are expected to result from application of this technique.     

The primary cilium of telocytes in the vasculature: electron microscope imaging

Blood vessels are highly organized and complex structure, which are far more than simple tubes conducting the blood to almost any tissue of the body. The fine structure of the wall of blood vessels has been studied previously using the electron microscope, but the presence the telocytes associated with vasculature, a specific new cellular entity, has not been studied in depth. Interestingly, telocytes have been recently found in the epicardium, myocardium, endocardium, human term placenta, duodenal lamina propria and pleura. We show the presence of telocytes located on the extracellular matrix of blood vessels (arterioles, venules and capillaries) by immunohistochemistry and transmission electron microscopy. Also, we demonstrated the first evidence of a primary cilium in telocytes. Several functions have been proposed for these cells. Here, the telocyte-blood vessels cell proximity, the relationship between telocytes, exosomes and nervous trunks may have a special significance.     

Electron spectroscopic imaging: parallel energy filtering and microanalysis in the fixed-beam electron microscope

A new imaging modality in electron microscopy uses energy filtration to produce micrographs with elastically scattered electrons or with electrons that have lost a specific, often characteristic amount of energy in interacting with the specimen. No deleterious effects on microscope performance are encountered. Instead, microanalysis of specimens is made possible with a spatial resolution of 3 to 5 A and a sensitivity of detection of 2 X 10(-21) g corresponding to about 50 atoms of phosphorus. Elements detected range from hydrogen (Z = 1) to uranium (Z = 92). Examples of elemental mapping show membrane structure, DNA within nucleosomes, and RNA within ribosomal particles.     

Scanning electron microscope imaging of bacteria based on DNA sequence

Aims:  To develop a scanning electron microscopic approach using in situ hybridization (SEM–ISH) for gaining both genetic and morphological information about target bacteria.
Methods and Results:  Target cells were hybridized with DNA-targeted polynucleotide probes, and a tyramide signal amplification system was used to increase the sensitivity. The protocol of SEM–ISH enabled to detect low copy number target DNA sequences in individual cells.
Conclusions:  SEM–ISH allowed the in situ detection of bacteria carrying a specific gene.
Significance and Impact of the Study:  Combining morphological study with SEM and ISH techniques appears to be a valuable tool to understand the spatial distribution of target cells in complex microbial communities on various materials.     

Elemental characterization of Daphnia resting eggs by X-ray analytical microscopy

Resting eggs of Daphnia, a key crustacean zooplankton of freshwater food chains, can remain viable for more than a century. These eggs are able to withstand freezing and drying, and can survive the harsh environment of a predator's digestive system. Until recently little was known about the chemical composition, microanatomy, and physical properties of the resting eggs. The current study utilized a physical technique, the X-ray analytical microscope, to identify and localize component elements of the Daphnia resting egg. The analysis demonstrated that phosphorus, sulfur, potassium, and calcium were detected as elemental components of the resting egg, and detection intensities of the four elements differed according to the position of the eggs. Phosphorus and calcium were mostly detected in regions of the eggshell that surrounded the two embryos. In addition, sulfur was distributed throughout the eggshell whereas potassium was localized to the areas that corresponded to where the embryos were encased. Through the use of X-ray analytical microscopy, the current study identifies elemental characteristics in relationship to the structure of the Daphnia resting eggs.     

Charge contrast imaging of biomaterials in a variable pressure scanning electron microscope

Charge contrast imaging (CCI) is a dynamic phenomenon recently reported in insulating and semiconducting materials imaged with low vacuum or variable pressure scanning electron microscopes (SEM). Data presented in this paper illustrates that CCI can also be applied to biominerals and biological soft-tissues and that useful and unique structural information can be obtained from routine samples. Various resin-embedded samples were considered and example images from several different biomaterials are presented. Due to the diverse nature of samples that appear to exhibit charge contrast, this imaging technique has prospective application in a wide range of biological and biomedical research. This work represents the first application of CCI to biomaterials and in particular, highlights a new method for investigating the formation, structure and growth of biominerals.     

Changes in iodine mapping in rat thyroid during the course of iodine deficiency: imaging and relative quantitation by analytical ion microscope

The analytical ion microscope (AIM) makes possible imaging and relative quantitation of multiple stable or labeled elements on an even tissue section, according to their mass. The purpose of this work was to follow at the rat thyroid follicle level the changes in 127I mapping during low iodine diet (LID) in relation to the ability of thyroid to pick up radioiodine (129I) and to synthesize Tg from its precursor, 2H-labeled leucine. The overall picture of images and countings of 127I shows a progressive decrease of the luminal iodine concentration which on day 80 was 10-fold lower than that of control value. In control rat thyroid cell, concentration was 10-fold lower than that of follicular lumina and was unchanged until 35 days, but the size of the cytoplasmic compartment increased, suggesting a redistribution of iodine stores between thyroid cells and follicular lumina. 129I was always found in colloid as well as in cells at all stages. After 35 days of LID, cytoplasmic and luminal radioiodine concentrations decreased. In control rats, [2H]leucine was found mainly in the cells. During LID its localization was evidenced progressively in most of the lumina. The most striking fact was the presence up to 35 days of some large residual follicles with high 127I concentration and low 129I and 2H incorporation. These data demonstrate the follicular heterogeneity of thyroid response to progressive chronic TSH stimulation induced by LID.     

Use of the analytical electron microscope (AEM) in cytochemical studies of the central nervous system.

Central nervous tissues (median eminence and arcuate nucleus) were studied by means of energy dispersive x-ray analysis using electron optical systems (analytical electron microscopy). These studies were conducted after the tissue had been treated specifically for localized biogenic amines (BA). The results indicate that not only is the BA cytochemical reaction highly specific, and that BAs can be localized intraneuronally in areas not previously identified, but also that the analytical electron microscope is a very valuable and potentially powerful tool in the studies of inclusion bodies and organelles in the central nervous system and other tissues. Thus, the nonspecific density production by osmium tetroxide can be elucidated from the specific BA reaction plus other areas of the nervous system containing density producing heavy metals, i.e., iron are readily identifiable.     

Ultrastructural study of polyspermy during early embryo development in pigs, observed by scanning electron microscope and transmission electron microscope

Polyspermy is generally considered a pathological phenomenon in mammals. Incidence of polyspermy in porcine eggs in vivo is extremely high (30-40%) compared with other species, and polyspermy rate in the in vitro fertilized eggs in pigs can reach 65%. It is still unknown whether polyspermy to a certain degree is a physiological condition in pigs, and whether porcine eggs have any capability with which to remove the accessory sperm in the cytoplasm. The objectives in the present study are to observe the ultrastructural changes of accessory sperm during early embryonic development in pigs. A total of 58 normal, early embryos at one-, two, three-, and four-cell and morular stages were collected from gilts and were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The surface ultrastructure showed that sperm fusion with the zona pellucida was a continuous process during one-, two-, three-, and four-cell and morular stages, as observed by the SEM. Accessory sperm were present in the cytoplasm of cleaved embryos. The sperm heads in the cytoplasm of cleaved embryos did not decondense. TEM revealed the presence of a condensed sperm head within a lysosome (or phagolysosome) in a three-cell embryo. These observations suggest that polyspermy may be a physiological condition in pigs and that early embryos may develop to term if accessory sperm do not interrupt the embryo genome. Furthermore, lysosome activity could be another physiological mechanism for removing accessory sperm in the cytoplasm of fertilized eggs and cleaved embryos after fertilization in pigs.     

Sequence organization in Xenopus DNA studied by the electron microscope.

Xenopus laevis DNA was extracted from red blood cells and sheared to a mean length of 2780 nucleotides. The DNA was stripped of foldback-containing fragments and incubated to C₀t 10 (mol · s · l⁻¹), allowing most repetitive sequences to form duplex structures. Duplex-containing fragments were eluted from an hydroxylapatite column and visualized for electron microscopy by spreading from 57% formamide according to the modified Kleinschmidt technique of Davis et al. (1971). The mean length of the fragments observed was 2445 nucleotides. A total of 1700 DNA strands were photographed and studied. Less than 5% of the total strand length was in uninterpretable structures. Every molecule falling within the confines of the plates was included in the sample. Over 50% of the total strand length in the sample was found in structures bearing at least one interspersed repetitive sequence duplex terminated by four single-strand regions. The fraction of DNA present in duplex regions was almost exactly that predicted if the duplex regions represent all the interspersed middle repetitive sequence in the Xenopus genome. Direct measurement of visualized duplexes shows that the mean length of interspersed repetitive sequence elements in this genome is 345 nucleotides. Duplex length was shown to be independent of the length of the strands bearing the duplexes. These observations provide direct confirmation of the length of approximately 300 nucleotides indicated for interspersed repetitive sequences by earlier physical-chemical studies 011 Xenopus DNA. In strands carrying two duplexes terminated by single-strand regions the interduplex, or single-copy sequence element length could be measured. Sequence interspersion curves generated from these data are roughly consistent with those derived earlier from measurements of hydroxylapatite binding as a function of fragment length.