Intracellular imaging of Qdots‐labeled DNA in cyanobacteria

In this contribution, they have attempted to develop a labeling technique for in vivo imaging of functionally active plasmid DNA in cyanobacterial cells through its decoration with semiconductor quantum dots (Qdots) as fluorescent nanoprobes. For that purpose biotinylated plasmid slr2060 DNA was conjugated with Qdots‐streptavidine. The intact DNA was visualized in a single green color by light microscopy. These Qdots‐DNA conjugates were capable of expressing the acyltransferase enzyme. Qdots‐DNA conjugates and confocal microscope imaging technique were adopted to visualize the gene transport across the membrane of the live cyanobacteria cell in real time. Long‐term kinetic study enabled to reveal the steps of extracellular and intracellular microenvironment for plasmid transportation into the live cell. To confirm these processes a confocal microscope and indicator plate assay test were applied in tandem. In this contribution, Qdots‐labeled plasmid DNA was utilized for the first time for long‐term intracellular imaging studies in cyanobacteria species PCC6803. The results showed that the Qdots‐labeled plasmid DNA detection could be used as a powerful labeling technique for visualization of exogenous DNA entry and tracking into living cells by confocal microscopy. Microsc. Res. Tech. 79:447–452, 2016. © 2016 Wiley Periodicals, Inc.     

Metabolic labeling of Escherichia coli genomic DNA with erythrosine‐11‐dUTP for functional imaging via correlative microscopy

The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice‐embedded proliferating cells of Escherichia coli K‐12 (thymidine‐requiring mutant, strain N4316) via correlative light‐electron microscopy. For that purpose, erythrosine‐11‐dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice‐embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high‐resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.     

Decreased uptake of bodipy-labelled compounds in the presence of the nuclear stain, DRAQ5

We have found the nuclear stain DRAQ5 to decrease the cellular uptake of a series of boron dipyrromethane (bodipy)‐labelled compounds. This phenomenon is consistent between Lysotracker Green DND 26, Lysotracker Red DND 99 and bodipy‐labelled mycolactone. Although DRAQ5 uptake was not prevented, DRAQ5 was in significant excess in each case. As the effect is consistent among two cell types, RAW 264.7 monocyte/macrophages and Bend 3 endothelial cells, we hypothesize that it may be a result of the two dyes complexing in solution into a form that is not taken up by the cells. This hypothesis is confirmed by fluorescence resonance energy transfer analysis in solution.     

Morphological and functional characterization of circulating hemocytes using microscopy techniques

In the present study, Microscopy studies were performed to characterize the blood cells of the mangrove crab Episesarma tetragonum. Three types of hemocytes were observed: granulocytes, semi‐granulocytes, and hyalinocytes or agranulocytes. Hyalinocytes have a distinguished nucleus surrounded by the cytoplasm, and a peculiar cell type was present throughout the cytosol, lysosomes with hemocyte types (granules) stained red (pink). Giemsa staining was used to differentiate between the large and small hemocytes. Ehrlich's staining was used to differentiate granule‐containing cells in acidophils (55%), basophils (44%), and neutrophils (<1%). Periodic acid–Schiff staining was used to identify the sugar molecules in the cytoplasm. Cell‐mediated immune reactions including phagocytosis, encapsulation, agglutination, and peroxidase‐mediated cell adhesion are the functions of hemocytes. Agglutination reaction involves both kind of cells involved in yeast and heme‐agglutination responses in invertebrates. The beta glucan outer layer of yeast cells was recognized by hemocyte receptors. Human RBC cells were agglutinated via granulocytes. E. tetragonum hemocytes are an important animal model for studying both ultrastructural and functional activity of circulating cells. In addition, E. tetragonum hemocytes exhibited excellent antibacterial and antibiofilm activities were studied through plating and microplate assays. Biofilm inhibition was also visualized through changes in biochemical assays and morphological variations were visualized through levels in in situ microscopy analysis.     

MRT letter: Localization of endogenous hydrogen peroxide by modified processes of sample preparation for transmissionelectron microscope in Escherichia coli

The bacterial endogenous hydrogen peroxide (H₂O₂) was detected cytochemically by its reaction with cerium chloride (CeCl₃) to produce electron‐dense deposits of cerium perhydroxides. The sequence of fixation and CeCl₃ staining of H₂O₂ in the processing of transmission electron microscope (TEM) sample preparation is crucial to the localization of endogenous H₂O₂ in Escherichia coli. In this study, results confirmed that the process that fixation simultaneously with CeCl₃ staining provided optimum effects for H₂O₂ localization in E. coli. The modified process of TEM provides very efficient protection for H₂O₂ localization and more accurate quantization for the H₂O₂ accumulation in bacterial cells. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy

Endothelial glycocalyx (GCX) is located on the apical surface of vascular endothelial cells and is composed of a negatively‐charged network of proteoglycans and glycoproteins. The GCX plays an important role in maintaining the integrity of vascular walls and preventing leakage of plasma. Therefore, degradation of the GCX is believed to lead to pathological leakage of plasma. Because the GCX is a very thin layer, its ultrastructural image has been demonstrated on electron microscope. To explore the function of the GCX, it should be visualized by a microscope in vivo. Thus, we developed in vivo visualization technique of the GCX under fluorescence microscopy using a mouse dorsal skinfold chamber (DSC) model. To label and visualize the GCX, we used fluorescein isothiocyanate (FITC)‐labeled lectin, which has a high specificity for sugar moieties. We examined the affinity of the different lectins to epivascular regions under an intravital fluorescent microscope. Among seven different lectins we examined, FITC labeled Triticum vulgaris (wheat germ) agglutinin (WGA) delineated the GCX most clearly. Binding of WGA to the GCX was inhibited by chitin hydrolysate, which contained WGA‐binding polysaccharide chains. Furthermore, the septic condition attenuated this structure, suggesting structural degradation of endothelial GCX layer. In conclusion, FITC‐labeled WGA lectin enabled visualization of endothelial GCX under in vivo fluorescence microscopy. Microsc. Res. Tech. 79:31–37, 2016. © 2015 Wiley Periodicals, Inc.     

Ultrastructural aspects of mouse nerve‐muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX‐I and Bbil‐TX: Unknown myotoxic effects

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve‐muscle preparations exposed to Bothrops venoms and their phospholipase A₂ toxins (PLA₂) can exhibit a neurotoxic pattern as increase in frequency of miniature end‐plate potentials (MEPPs) as well as in amplitude of end‐plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA₂ toxins (BthTX‐I and Bbil‐TX) in mouse isolated nerve‐phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX‐I and Bbil‐TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic‐like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.     

Quantitative evaluation of boron neutron capture therapy (BNCT) drugs for boron delivery and retention at subcellular‐scale resolution in human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS)

Boron neutron capture therapy (BNCT) of cancer depends on the selective delivery of a sufficient number of boron‐10 (¹⁰B) atoms to individual tumour cells. Cell killing results from the ¹⁰B (n, α)⁷Li neutron capture and fission reactions that occur if a sufficient number of ¹⁰B atoms are localized in the tumour cells. Intranuclear ¹⁰B localization enhances the efficiency of cell killing via damage to the DNA. The net cellular content of ¹⁰B atoms reflects both bound and free pools of boron in individual tumour cells. The assessment of these pools, delivered by a boron delivery agent, currently cannot be made at subcellular‐scale resolution by clinically applicable techniques such as positron emission tomography and magnetic resonance imaging. In this study, a secondary ion mass spectrometry based imaging instrument, a CAMECA IMS 3f ion microscope, capable of 500 nm spatial resolution was employed. Cryogenically prepared cultured human T98G glioblastoma cells were evaluated for boron uptake and retention of two delivery agents. The first, L‐p‐boronophenylalanine (BPA), has been used clinically for BNCT of high‐grade gliomas, recurrent tumours of the head and neck region and melanomas. The second, a boron analogue of an unnatural amino acid, 1‐amino‐3‐borono‐cyclopentanecarboxylic acid (cis‐ABCPC), has been studied in rodent glioma and melanoma models by quantification of boron in the nucleus and cytoplasm of individual tumour cells. The bound and free pools of boron were assessed by exposure of cells to boron‐free nutrient medium. Both BPA and cis‐ABCPC delivered almost 70% of the pool of boron in the free or loosely bound form to the nucleus and cytoplasm of human glioblastoma cells. This free pool of boron could be easily mobilized out of the cell and was in some sort of equilibrium with extracellular boron. In the case of BPA, the intracellular free pool of boron also was affected by the presence of phenylalanine in the nutrient medium. This suggests that it might be advantageous if patients were placed on a low phenylalanine diet prior to the initiation of BNCT. Since BPA currently is used clinically for BNCT, our observations may have direct relevance to future clinical studies utilizing this agent and provides support for individualized treatment planning regimens rather than the use of fixed BPA infusion protocols.     

Defense reactions of <i>Dermatobia hominis</i> (Diptera: Cuterebridae) larval hemocytes

The defense reactions against biological (Histoplasma capsulatum and Escherichia coli) and non-biological materials (China ink and nylon thread) were tested in vivo in third instar larvae of Dermatobia hominis. The cellular defense performed by larval hemocytes was observed under electron microscopy. China ink particles were phagocytosed by granular cells 5 h after injection. E. coli cells were internalized by granular cells as early as 5 min after injection and totally cleared 180 min post-injection, when many hemocytes appeared disintegrated and others in process of recovering. H. capsulatum yeasts provoked, 24 h after being injected, the beginning of nodule formation. Nylon thread was encapsulated 24 h after the introduction into the hemocoel. Our results suggest that granular cells were the phagocytic cells and also the responsible for the triggering of nodule and capsule formation. In the presence of yeasts cells and nylon thread, they released their granules that chemotactically attracted the plasmatocytes that on their turn, flattened to surround and isolate the foreign material.     

Visualization of BrdU-labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy

We have attempted to observe the native shape of DNA in rapidly frozen whole cyanobacterial cells through 5-bromo-2-deoxyuridine (BrdU) incorporation and visualization with a Hilbert differential contrast transmission electron microscopy (HDC TEM). The incorporation of BrdU into the DNA of Synechococcus elongatus PCC 7942 was confirmed with fluorescently labelled anti-BrdU antibodies and through EDX analysis of ultra-thin sections. HDC TEM observed cells that had incorporated BrdU into their DNA exhibited electron dense areas at the location corresponding to fluorescently labelled BrdU. Since various strings and strands were observed in high contrast with the HDC TEM, we conclude that the method promises to allow us to identify and understand bulk structural changes of the in vivo DNA and the nucleoid through observation at high resolution.     

Pre-screening for antigen detectability in cells: a TEM-based solid phase digital immunogold detection method utilizing ultra low volumes of reagents

Rapid and sensitive pre‐screening for the presence of antigens in cell samples and confirmation of reactivity of antibodies, before proceeding with electron microscopy, is highly desirable. Most of the methods developed for this purpose are generally not very efficient and suitable for dealing with very small volumes of sample and reagents. In this work we present a simple, sensitive and rapid solid phase transmission electron microscope (TEM) based method for the detection of picogram (pg) levels of soluble antigens using as little as 10 µL of reagents. Protein was adsorbed onto grids coated with polystyrene films to form the solid phase. The presence of antigen was detected using immunogold labelling. Gold particles adhering to the film were visualized and counted in a TEM providing a digital signal. This method was 100‐fold more sensitive than dot blot in detection of rabbit IgG. We have demonstrated the utility of this technique by screening for Vitreoscilla haemoglobin (VHb) antigen in cell lysates and confirming the results directly with immunogold labelling transmission electron microscopy of cell sections.     

Quantitative subcellular imaging of boron compounds in individual mitotic and interphase human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS)

Boron measurements at subcellular scale are essential in boron neutron capture therapy (BNCT) of cancer as the nuclear localization of boron‐10 atoms can enhance the effectiveness of killing individual tumour cells. Since tumours contain a heterogeneous population of cells in interphase as well as in the M phase (mitotic division) of the cell cycle, it is important to evaluate the subcellular distribution of boron in both phases. In this work, the secondary ion mass spectrometry (SIMS) based imaging technique of ion microscopy was used to quantitatively image boron from two BNCT agents, clinically used p‐boronophenylalanine (BPA) and 3‐[4‐(o‐carboran‐1‐yl)butyl]thymidine (N4), in mitotic metaphase and interphase human glioblastoma T98G cells. N4 belongs to a class of experimental BNCT agents, designated 3‐carboranyl thymidine analogues (3CTAs), which presumably accumulate selectively in cancer cells due to a process referred to as kinase‐mediated trapping (KMT). The cells were exposed to BPA for 1 h and N4 for 2 h. A CAMECA IMS‐3f SIMS ion microscope instrument capable of producing isotopic images with 500 nm spatial resolution was used in the study. Observations were made in cryogenically prepared fast frozen, and freeze‐fractured, freeze‐dried cells. Three discernible subcellular regions were studied: the nucleus, a characteristic mitochondria‐rich perinuclear cytoplasmic region, and the remaining cytoplasm in interphase T98G cells. In metaphase cells, the chromosomes and the cytoplasm were studied for boron localization. Intracellular concentrations of potassium and sodium also were measured in each cell in which the subcellular boron concentrations were imaged. Since the healthy cells maintain a K/Na ratio of approximately 10 due to the presence of Na‐K‐ATPase in the plasma membrane of mammalian cells, these measurements provided validation for cryogenic sample preparation and indicated the analysis healthy, well preserved cells. The BPA‐treated interphase cells revealed significantly lower concentrations of boron in the perinuclear mitochondria‐rich cytoplasmic region as compared to the remaining cytoplasm and the nucleus, which were not significantly different from each other. In contrast, the BPA‐treated metaphase cells revealed significantly lower concentration of boron in their chromosomes than cytoplasm. In addition, the cytoplasm of metaphase cells contained significantly less boron than the cytoplasm of interphase cells. These observations provide valuable information on the reduced uptake of boron from BPA in mitotic cells for BPA‐mediated BNCT. SIMS observations on N4 revealed that boron was distributed throughout the interphase and mitotic cells, including the chromosomes. The presence of boron in chromosomes of metaphase cells treated with N4 is indicative of a possible incorporation of this thymidine analogue into DNA. The 3‐D SIMS imaging approach for the analysis of mitotic cells shown in this work should be equally feasible to the evaluation of other BNCT agents.     

Electron microscopic visualization of complementary labeled DNA with platinum‐containing guanine derivative

The object of the present report is to provide a method for a visualization of DNA in TEM by complementary labeling of cytosine with guanine derivative, which contains platinum as contrast‐enhanced heavy element. The stretched single‐chain DNA was obtained by modifying double‐stranded DNA. The labeling method comprises the following steps: (i) stretching and adsorption of DNA on the support film of an electron microscope grid (the hydrophobic carbon film holding negative charged DNA); (ii) complementary labeling of the cytosine bases from the stretched single‐stranded DNA pieces on the support film with platinum containing guanine derivative to form base‐specific hydrogen bond; and (iii) producing a magnified image of the base‐specific labeled DNA. Stretched single‐stranded DNA on a support film is obtained by a rapid elongation of DNA pieces on the surface between air and aqueous buffer solution. The attached platinum‐containing guanine derivative serves as a high‐dense marker and it can be discriminated from the surrounding background of support carbon film and visualized by use of conventional TEM observation at 100 kV accelerated voltage. This method allows examination of specific nucleic macromolecules through atom‐by‐atom analysis and it is promising way toward future DNA‐sequencing or molecular diagnostics of nucleic acids by electron microscopic observation. Microsc. Res. Tech. 79:280–284, 2016. © 2016 Wiley Periodicals, Inc.     

The effects of human menstrual blood stem cells‐derived granulosa cells on ovarian follicle formation in a rat model of premature ovarian failure

Many studies have reported that human endometrial mesenchymal stem cells (HuMenSCs) are capable of repairing damaged tissues. The aim of the present study was to investigate the effects of HuMenSCs transplantation as a treatment modality in premature ovarian failure (POF) associated with chemotherapy‐induced ovarian damage. HuMenSCs were isolated from menstrual blood samples of five women. After the in vitro culture of HuMenSCs, purity of the cells was assessed by cytometry using CD44, CD90, CD34, and CD45 FITC conjugate antibody. Twenty‐four female Wistar rats were randomly divided into four groups: negative control, positive control, sham, and treatment groups. The rat models of POF used in our study were established by injecting busulfan intraperitoneally into the rats during the first estrus cycle. HuMenSCs were transplanted by injection via the tail vein into the POF‐induced rats. Four weeks after POF induction, ovaries were collected and the levels of Amh, Fst, and Fshr expression in the granulosa cell (GC) layer, as well as plasma estradiol (E2) and progesterone (P4) levels were evaluated. Moreover, migration and localization of DiI‐labeled HuMenSCs were detected, and the labeled cells were found to be localized in GCs layer of immature follicles. In addition to DiI‐labelled HuMenSCs tracking, increased levels of expression of Amh and Fshr and Fst, and the high plasma levels of E2 and P4 confirmed that HuMenSC transplantation had a significant effect on follicle formation and ovulation in the treatment group compared with the negative control (POF) group.     

Occurrence of fenestral diaphragms and knots in renal glomerular endothelia of diabetic mutant MafA−/−MafK+ mice as revealed in embedment‐free transmission electron microscopy

Using the advantages (high contrast and transparency and efficient 3D viewing) of embedment‐free section transmission electron microscopy (TEM), the occurrence of numerous fenestral diaphragms was clearly shown in 3D en‐face viewing of the renal glomerular capillary endothelium of severe overt diabetes mellitus mice, which were generally MafA‐deficient and simultaneously MafK‐overexpressed specifically in pancreatic β‐cells. This presents another example of nephritis‐induced diaphragmed fenestrae in the renal glomerular endothelium. In addition, knot‐/umbilicus‐like structures discrete from and larger than the central knots of regular diaphragms of fenestrated endothelium were clearly demonstrated to occur randomly in the renal glomerular endothelial fenestrae of mutant mice and wild ones. The knot‐structures were revealed to be protrusions of underlining basement lamina in conventional TEM by section‐tilting observation. Microsc. Res. Tech. 78:207–212, 2015. © 2015 Wiley Periodicals, Inc.     

Light and transmission electron microscopic structure of skin glands and dermal scales of a caecilian amphibian Gegeneophis ramaswamii,with a note on antimicrobial property of skin gland secretion

Amphibian skin secretions contain a variety of bioactive compounds that are involved in diverse roles such as communication, homeostasis, defence against predators, pathogens, and so on. Especially, the caecilian amphibians possess numerous cutaneous glands that produce the secretory material, which facilitate survival in their harsh subterranean environment. Inspite of the fact that India has a fairly abundant distribution of caecilian amphibians, there has hardly been any study on their skin and its secretion. Herein, we describe, using light microscopy and electron microscopy, two types of dermal glands, mucous and granular, in Gegeneophis ramaswamii. The mucous glands are filled with mucous materials. The mucous‐producing cells are located near the periphery. The granular glands are surrounded by myoepithelial cells. A large number of granules of different sizes are present in the lumen of the granular gland. The granule‐producing cells are present near the myoepithelial lining of the gland. There are small flat disk‐like dermal scales in pockets in the transverse ridges of the posterior region of the body. Each pocket contains 1–4 scales of various sizes. Scanning electron microscopic (SEM) study of the skin surface showed numerous funnel‐shaped glandular openings. The antibacterial activity of the skin secretions was revealed in the test against Escherichia coli, Klebsiella pneumoniae, and Aeromonas hydrophila, all gram‐negative bacteria. SEM analyses confirm the membrane damage in bacterial cells on exposure to skin secretions of G. ramaswamii.     

Morphological and ultrastructural characterization of ionoregulatory cells in the teleost oreochromis niloticus following salinity challenge combining complementary confocal scanning laser microscopy and transmission electron microscopy using a novel prefixation immunogold labeling technique

Aspects of ionoregulatory or mitochondria‐rich cell (MRC) differentiation and adaptation in Nile tilapia yolk‐sac larvae following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt are described. Investigations using immunohistochemistry on whole‐mount Nile tilapia larvae using anti‐ Na⁺/K⁺‐ATPase as a primary antibody and Fluoronanogold? (Nanoprobes) as a secondary immunoprobe allowed fluorescent labeling with the high resolution of confocal scanning laser microscopy combined with the detection of immunolabeled target molecules at an ultrastructural level using transmission electron microscopy (TEM). It reports, for the first time, various developmental stages of MRCs within the epithelial layer of the tail of yolk‐sac larvae, corresponding to immature, developing, and mature MRCs, identifiable by their own characteristic ultrastructure and form. Following transfer to hyperosmotic salinities the density of immunogold particles and well as the intricacy of the tubular system appeared to increase. In addition, complementary confocal scanning laser microscopy allowed identification of immunopositive ramifying extensions that appeared to emanate from the basolateral portion of the cell that appeared to be correlated with the localization of subsurface tubular areas displaying immunogold labeled Na⁺/K⁺‐ATPase. This integrated approach describes a reliable and repeatable prefixation immunogold labeling technique allowing precise visualization of NaK within target cells combined with a 3D imaging that offers valuable insights into MRC dynamics at an ultrastructural level. Microsc. Res. Tech., 76:1016–1024, 2013. © 2013 Wiley Periodicals, Inc.     

Exploring the use of soft X‐ray microscopy for imaging subcellular structures of the inner ear1

The soft X‐ray microscope at the Lawrence Berkeley National Laboratory was developed for visualization of biological tissue. Soft X‐ray microscopy provides high‐resolution visualization of hydrated, non‐embedded and non‐sectioned cells and is thus potentially an alternative to transmission electron microscopy. Here we show for the first time soft X‐ray micrographs of structures isolated from the guinea‐pig inner ear. Sensory outer hair cells and supporting pillar cells are readily visualized. In the hair cells, individual stereocilia can easily be identified within the apical hair bundle. The underlying cuticular plate is, however, too densely composed or too thick to be clearly visualized, and thus appears very dark. The cytoplasmic structures protruding from the cuticular plates as well as the fibrillar material surrounding and projecting from the cell nuclei can be seen. In the pillar cells the images reveal individual microtubule bundles. Soft X‐ray images of the acellular tectorial membrane and thin two‐layered Reissner's membrane display a level of resolution comparable to low‐power electron microscopy.     

Labelling quality and chromosome morphology after low temperature FISH analysed by scanning far-field and near-field optical microscopy

A non‐enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context ‘low temperature’ means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 °C. For LTFISH, the DNA probe had to be single‐stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near‐field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH‐procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single‐stranded DNA probes to double‐stranded DNA targets are discussed.     

ESI in situ analyses of extrachromosomal RNP granules

Observation of unstained ultrathin sections of salivary gland cells of Chironomus thummi and C. tentans, by means of electron spectroscopic imaging (ESI), has revealed that phosphorus is distributed in two types of granular structures in the nucleoplasm of these cells. In addition to a specific type of premessenger ribonucleoprotein (RNP) particle, known as the Balbiani ring (Br) granule, ESI revealed a new type of phosphorus-rich small granular component. Examination of unstained sections by energy-filtering transmission electron microscopy (EFTEM) offers the opportunity of obtaining the signal from the specimen alone, thus avoiding the possible contributions of heavy metals present in any staining product.