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PubMed1994

The history of urinary microscopy to the end of the 19th century.

In the 17th and 18th centuries, several authors performed urinary microscopy occasionally and were often unable to give their observations a practical diagnostic application. Such men included De Peiresc, Boerhaave, Ledermüller and Galeazzi. In the 1st half of the 19th century, however, urinary microscopy began to be used systematically. Rayer and Vigla identified for the first time elements other than crystals in urine and contributed to the methodology of handling samples for microscopy. Becquerel described dysmorphic erythrocytes, and Simon and Henle observed casts in urine and in histological preparations. In contrast, Bird mentioned casts only in passing, though he described many other elements and published the first complete book on urinary microscopy. The 2nd half of the 19th century was characterized by further advances, and in the book of Beale tubular cells were distinguished from other epithelial cells. Different types of casts were also linked with different renal diseases. By 1875 the classification of casts was complete. The work of the 19th century microscopists culminated in Rieder's book on clinical microscopy, which described each element of urinary sediment through 36 beautiful chromolithographic plates.

England↗
PubMedDate not supplied

An ultrastructural histochemistry and light microscopy study of the early development of renal proximal tubular vacuolation after a single administration of the contrast enhancement medium "Iotrolan".

The time course of contrast media (CM)-induced renal proximal tubular vacuolation was investigated in rats by light microscopy, transmission electron microscopy (TEM), and ultrastructural histochemistry for acid phosphate activity. Young adult male rats were treated with a single dose of 3.0 g I/kg Iotrolan (Isovist 300 mg I/ml) and sacrificed at 0 min, 5-min, 15-min, 15-min, 2-hr, and 24-hr intervals. Light microscopy of vibratome sections of freshly excised tissue of cryostat and paraffin sections was also performed to allow comparison of the appearance of the vacuoles in the fresh state with light and electron microscopy. The sequence of events seen to occur can be summarized as follows. CM-induced vacuolation occurred at a low level a soon as 5 min after compound administration. The vacuolation was observed by TEM but could not be detected by light microscopy. This was followed by an increase in size and numbers of vacuoles up to the 24-hr timepoint with a sequential increase in the staining for acid phosphatase activity of the vacuoles, most marked at the 24-hr timepoint. At timepoints less than 24 hr there appeared to be no marked increased in the normal complement by lysosomes or in the components of the Golgi-endoplasmic reticulum-lysosome pathway. At 24 hr, the vast majority, but not all, of the CM-induced vacuoles were positive for acid phosphatase activity. The intensity of staining varied, and there was evidence of infusion of small lysosomes with CM-induced vacuoles. These results suggest that formation of CM-induced vacuoles is a 2-stage process, following a normal pathway for the handling of endogenous and exogenous substances.

Animals↗
PubMedDate not supplied

Microscopy of tonsillar smears and sections in tonsillar gonorrhoea.

Microscopy of methylene-blue and Gram-stained smears from the tonsillar surface and an immunofluorescence (IF) test were carried out for 130 patients, 129 with genital and/or anal gonorrhoea, 27 of whom also had tonsillar gonorrhoea. One patient had only tonsillar gonorrhoea. 5 of the 28 patients with tonsillar gonorrhoea had acute tonsillitis and for these, agreement was found between culture, light microscopy and IF test, while only 5 of the remaining 23 patients had positive microscopy. Among the 102 patients who did not have tonsillar gonorrhoea a few false-positive light microscopies and IF tests were found. Microscopy of haematoxylin-eosin stained sections of 8 tonsils from 4 patients with recurrent tonsillitis and tonsillar gonorrhoea showed subacute tonsillitis. Methylene-blue and Gram-stained sections revealed gram-negative diplococci morphologically similar to gonococci. The bacteria were located in the superficial layers of the mucous membrane (frequently intracellularly in leukocytes) and occasionally in cellular debris in the crypts.

Antibodies, Bacterial↗
PubMed1998

Codistribution of procathepsin B and mature cathepsin B forms in human prostate tumors detected by confocal and immunofluorescence microscopy.

Cathepsin B (CB) is involved in invasion and metastasis of a variety of solid organ tumors, including human prostate cancer. The tertiary structures of the proenzyme and mature forms of CB are related closely, as revealed by crystallographic studies. However, the cellular distributions of the CB forms have not been defined in human prostate and its tumors. Our objective was to investigate the distribution and codistribution of CB and procathepsin B (proCB) in human prostate tumors. Human prostate tissue samples that were obtained from 21 prostatectomy and/or cystectomy patients were collected immediately after surgery and processed for this study. We used a rabbit antihuman liver CB immunoglobulin G (IgG) that recognizes both mature CB and proCB and a mouse antipropeptide monoclonal antibody IgG that recognizes only proCB. Fluorescein isothiocyanate (FITC)-conjugated donkey antirabbit IgG and indocarbocyanine (Cy3; rhodamine)-conjugated donkey antimouse IgG were used to differentiate localization of the enzyme forms. Immunofluorescence of FITC and Cy3 was examined in prostate sections by using epifluorescence and confocal laser-scanning microscopy. Because fluorescence is dependent on section thickness, time needed for study and photography, and the antigenic sites of proCB and mature CB localized by antibodies and by fluorescent markers (Cy3 vs. FITC), the cellular distributions and the relative intensity of fluorescence on cryostat sections were assessed qualitatively. Immunofluorescence of Cy3 for localizing proCB and of FITC for localizing mature CB were observed in prostatic epithelial cells and their tumors and in stromal connective tissue cells. By using confocal microscopy, colocalization of the enzyme forms in the same cells was indicated by yellow fluorescence. In stromal cells (such as smooth muscles, fibroblast, and macrophages), the distribution of proCB and relative fluorescence intensity was moderate to predominant in human prostate and its tumors. In neoplastic prostate, the cellular distributions of CB ranged from low to predominant levels. In some neoplastic glands, Cy3 fluorescence for proCB was absent, whereas the mature form of CB localized in cancer cells and in the subjacent extracellular matrix. Confocal microscopy showed a close association of CB with extracellular matrix surrounding neoplastic acini and invasive cells, indicating that the enzyme form was probably involved in degradation of the matrix proteins. The negative control study showed no specific immunofluorescence for proCB or CB in prostate cancer cases. We have shown a differential distribution of proenzyme and mature forms of CB in normal prostate, benign prostatic hyperplasia, and neoplastic prostate. The enzyme forms were assessed by determining the cellular distributions of CB and proCB. Our study indicates that the differential distribution of proCB and CB might provide clues into aggressiveness of prostate cancers within Gleason grades. However, we emphasize that our observation should be evaluated in a larger series of prostate samples before a definitive conclusion can be reached. This is the first report to show codistribution of proenzyme and mature forms of CB by using confocal microscopy.

Animals↗
PubMed1985

Studies of otoconia in the developing chick by polarized light microscopy.

Using polarized light microscopy we were able to observe the mineralization patterns of embryonic and neonatal chick otoconia. We compared preparations of freshly dissected material spread under mineral oil to material that had been treated with various fixatives and dehydration agents. We found that the standard fixation agent, glutaraldehyde, and some immersion oils etched embryonic chick otoconia but that fixation with 70% acetone or 70% alcohol followed by dehydration to 100% acetone or 100% alcohol left the otoconia intact. The size and shape of freshly dissected chick otoconia observed with polarized light microscopy were similar to those of acetone-fixed, critical-point-dried material examined by SEM. Embryonic forms of otoconia were found to have a fluted pattern that was different in morphology from otoconia found in hatched chicks and adults. Embryonic chick otoconia did not exhibit the multifaceted surface morphology seen in embryonic rat otoconia. Comparisons of the same fields of otoconia under phase contrast microscopy and polarized light microscopy indicated that the freshly dissected otoconia of embryos exhibit little or no unmineralized (non-birefringent) material but that glutaraldehyde-fixed otoconia exhibited unmineralized areas where etching had occurred. Size frequency distributions of freshly dissected embryonic and mature otoconia in five ages of embryos and hatched chicks were consistent with a hypothesized developmental sequence of otoconia.

Animals↗
PubMed2002

Surface imaging microscopy, an automated method for visualizing whole embryo samples in three dimensions at high resolution.

Modern biology is faced with the challenge of understanding the specification, generation, and maintenance of structures ranging from cells and tissues to organs and organisms. By acquiring images directly from the block face of an embedded sample, surface imaging microscopy (SIM) generates high-resolution volumetric images of biological specimens across all of these scales. Surface imaging microscopy expands our range of imaging tools by generating three-dimensional reconstructions of embryo samples at high resolution and high contrast. SIM image quality is not limited by depth or the optical properties of overlying tissue, and intrinsic or extrinsic alignment markers are not required for volume reconstruction. These volumes are highly isotropic, enabling them to be virtually sectioned in any direction without loss of image quality. Surface imaging microscopy provided a more accurate three-dimensional representation of a chick embryo than confocal microscopy of the same sample. SIM offers excellent imaging of embryos from three major vertebrate systems in developmental biology: mouse, chicken, and frog. Immediate applications of this technology are in visualizing and understanding complex morphogenetic events and in making detailed comparisons between normal and genetically modified embryos.

Animals↗
PubMed1995

Determination of heat-shock transcription factor 2 stoichiometry at looped DNA complexes using scanning force microscopy.

Gene activation frequently requires an array of proteins bound to sites distal to the transcription start site. The assembly of these protein-bound sites into specialized nucleoprotein complexes is a prerequisite for transcriptional activation. Structural analysis of these higher order complexes will provide crucial information for understanding the mechanisms of gene activation. We have used both electron microscopy and scanning force microscopy to elucidate the structure of complexes formed between DNA and heat-shock transcription factor (HSF) 2, a human heat-shock transcriptional activator that binds DNA as a trimer. Electron microscopy reveals that HSF2 will bring together distant DNA sites to create a loop. We show that this association requires only the DNA binding and trimerization domains of HSF2. Metal shadowing techniques used for electron microscopy obscure details of these nucleoprotein structures. Greatly increased resolution was achieved by directly imaging the complexes in the scanning force microscope, which reveals that at least two trimers are required for the association of HSF2-bound DNA sites.

DNA↗
PubMed2005

Maxillary sinus augmentation with Bio-Oss particles: a light, scanning, and transmission electron microscopy study in man.

Biological interactions occurring at the bone-biomaterial interface are critical for long-term clinical success. Bio-Oss is a deproteinized, sterilized bovine bone that has been extensively used in bone regeneration procedures. The aim of the present study was a comparative light, scanning, and electron microscopy evaluation of the interface between Bio-Oss and bone in specimens retrieved after sinus augmentation procedures. Under light microscopy, most of the particles were surrounded by newly formed bone, while in a few cases, at the interface of some particles it was possible to observe marrow spaces and biological fluids. Under scanning electron microscopy, in most cases, the particle perimeter appeared lined by bone that was tightly adherent to the biomaterial surface. Transmission electron microscopy showed that the bone tissue around the biomaterial showed all the phases of the bone healing process. In some areas, randomly organized collagen fibers were present, while in other areas, newly formed compact bone was present. In the first bone lamella collagen fibers contacting the Bio-Oss surface were oriented at 243.73 +/- 7.12 degrees (mean +/- SD), while in the rest of the lamella they were oriented at 288.05 +/- 4.86 degrees (mean +/- SD) with a statistically significant difference of 44.32 degrees (p < 0.001). In the same areas the intensity of gray value was 172.56 +/- 18.15 (mean +/- SD) near the biomaterial surface and 158.71 +/- 21.95 (mean +/- SD) in the other part of the lamella with an unstatistically significant difference of 13.79 (p = 0.071). At the bone-biomaterial interface there was also an electron-dense layer similar to cement lines. This layer had a variable morphology being, in some areas, a thin line, and in other areas, a thick irregular band. The analyses showed that Bio-Oss particles do not interfere with the normal osseous healing process after sinus lift procedures and promote new bone formation. In conclusion, this study serves as a better understanding of the morphologic characteristics of Bio-Oss and its interaction with the surrounding tissues.

Absorbable Implants↗
PubMed2004

Antecedents of two-photon excitation laser scanning microscopy.

In 1931, Maria Göppert-Mayer published her doctoral dissertation on the theory of two-photon quantum transitions (two-photon absorption and emission) in atoms. This report describes and analyzes the theoretical and experimental work on nonlinear optics, in particular two-photon excitation processes, that occurred between 1931 and the experimental implementation of two-photon excitation microscopy by the group of Webb in 1990. In addition to Maria Göppert-Mayer's theoretical work, the invention of the laser has a key role in the development of two-photon microscopy. Nonlinear effects were previously observed in different frequency domains (low-frequency electric and magnetic fields and magnetization), but the high electric field strength afforded by lasers was necessary to demonstrate many nonlinear effects in the optical frequency range. In 1978, the first high-resolution nonlinear microscope with depth resolution was described by the Oxford group. Sheppard and Kompfner published a study in Applied Optics describing microscopic imaging based on second-harmonic generation. In their report, they further proposed that other nonlinear optical effects, such as two-photon fluorescence, could also be applied. However, the developments in the field of nonlinear optical stalled due to a lack of a suitable laser source. This obstacle was removed with the advent of femtosecond lasers in the 1980s. In 1990, the seminal study of Denk, Strickler, and Webb on two-photon laser scanning fluorescence microscopy was published in Science. Their paper clearly demonstrated the capability of two-photon excitation microscopy for biology, and it served to convince a wide audience of scientists of the potential capability of the technique.

History, 20th Century↗
PubMed1991

The three-dimensional architecture of the mitotic spindle, analyzed by confocal fluorescence and electron microscopy.

Fluorescence microscopy techniques have become important tools in mitosis research. The well-known disadvantages of fluorescence microscopy, rapid bleaching, phototoxicity and out-of-focus contributions blurring the in-focus image are obstacles which still need to be overcome. Confocal fluorescence microscopy has the potential to improve our capabilities of analyzing cells, because of its excellent depth-discrimination and image processing power. We have been using a confocal fluorescence microscope for the study of the mechanism of poleward chromosome movement, and report here 1) a cell preparation technique, which allows labeling of fixation sensitive spindle antigens with acceptable microtubule preservation; 2) the use of image processing methods to represent the spatial distribution of various labeled elements in pseudocolour; 3) a novel immunoelectron microscopic labeling method for microtubules, which allows the visualization of their distribution in semithin sections at low magnification; and 4) a first attempt to study microtubule dynamics with a confocal fluorescence microscope in living cells, microinjected with rhodamine labeled tubulin. Our experience indicates that confocal fluorescence microscopy provides real advantages for the study of spatial colocalization of antigens in the mitotic spindle. It does not, however, overcome the basic limits of resolution of the light microscope. Therefore, it has been necessary to use an electron microscopic method. Our preliminary results with living cells show that it is possible to visualize the entire microtubule network in stereo, but that the sensitivity of the instrument is still too low to perform dynamic time studies. It will be worthwhile to further develop this new type of optical instrumentation and explore its usefulness on both fixed and living cells.

Animals↗
PubMed2004

Microscopy of biological sample through advanced diffractive optics from visible to X-ray wavelength regime.

The aim of this report is to demonstrate a unified version of microscopy through the use of advanced diffractive optics. The unified scheme derives from the technical possibility of realizing front wave engineering in a wide range of electromagnetic spectrum. The unified treatment is realized through the design and nanofabrication of phase diffractive elements (PDE) through which wave front beam shaping is obtained. In particular, we will show applications, by using biological samples, ranging from micromanipulation using optical tweezers to X-ray differential interference contrast (DIC) microscopy combined with X-ray fluorescence. We report some details on the design and physical implementation of diffractive elements that besides focusing also perform other optical functions: beam splitting, beam intensity, and phase redistribution or mode conversion. Laser beam splitting is used for multiple trapping and independent manipulation of micro-beads surrounding a cell as an array of tweezers and for arraying and sorting microscopic size biological samples. Another application is the Gauss to Laguerre-Gauss mode conversion, which allows for trapping and transfering orbital angular momentum of light to micro-particles immersed in a fluid. These experiments are performed in an inverted optical microscope coupled with an infrared laser beam and a spatial light modulator for diffractive optics implementation. High-resolution optics, fabricated by means of e-beam lithography, are demonstrated to control the intensity and the phase of the sheared beams in x-ray DIC microscopy. DIC experiments with phase objects reveal a dramatic increase in image contrast compared to bright-field x-ray microscopy. Besides the topographic information, fluorescence allows detection of certain chemical elements (Cl, P, Sc, K) in the same setup, by changing the photon energy of the x-ray beam.

Escherichia coli↗
PubMed2005

High-resolution simultaneous three-photon fluorescence and third-harmonic-generation microscopy.

In recent years, nonlinear laser scanning microscopy has gained much attention due to its unique ability of deep optical sectioning. Based on our previous studies, a 1,200-1,300-nm femtosecond laser can provide superior penetration capability with minimized photodamage possibility. However, with the longer wavelength excitation, three-photon-fluorescence (3PF) would be necessary for efficient use of intrinsic and extrinsic visible fluorophores. The three-photon process can provide much better spatial resolution than two-photon-fluorescence due to the cubic power dependency. On the other hand, third-harmonic-generation (THG), another intrinsic three-photon process, is interface-sensitive and can be used as a general structural imaging modality to show the exact location of cellular membranes. The virtual-transition characteristic of THG prevents any excess energy from releasing in bio-tissues and, thus, THG acts as a truly noninvasive imaging tool. Here we demonstrated the first combined 3PF and THG microscopy, which can provide three-dimensional high-resolution images with both functional molecule specificity and sub-micrometer structural mapping capability. The simultaneously acquired 3PF and THG images based on a 1,230-nm Cr:forsterite femtosecond laser are shown with a Hoechst-labeled hepatic cell sample. Strong 3PF around 450 nm from DNA-bounded Hoechst-33258 can be observed inside each nucleus while THG reveals the location of plasma membranes and other membrane-based organelles such as mitochondria. Considering that the maximum-allowable laser power in common nonlinear laser microscopy is less than 10 mW at 800 nm, it is remarkable that even with a 100-mW 1,230-nm incident power, there is no observable photo damage on the cells, demonstrating the noninvasiveness of this novel microscopy technique.

Bisbenzimidazole↗
PubMed2005

Electron microscopy of cultured angiogenic endothelial cells.

Angiogenesis is a multi-step process involving migration, proliferation, and a specific spatial arrangement of endothelial cells. On the basis of a model of cultured microvascular endothelial cells derived from the bovine corpus luteum, all stages of in vitro angiogenesis as well as intussusceptive remodeling were characterized by scanning and transmission electron microscopy. To preserve the delicate three-dimensional cellular structures for electron microscopy, modified processing techniques for both transmission and scanning electron microscopy including micro-corrosion casting of cultured cells were established. The detailed results on morphological alterations and cellular interactions confirmed and complemented earlier studies of in vitro angiogenesis. Electron microscopy proved to be an efficient tool for detection and supervision of all major endothelial differentiation processes resembling in vivo conditions that are generally considered important in a realistic in vitro model of angiogenesis: occurrence of function-related cellular junctions; development of specific surface features indicating cellular polarity; production of extracellular matrix material; mechanisms leading to the formation of an internal lumen; specific spatial arrangement of endothelial cells within capillary-like networks; detachment of apoptotic cells as well as intussusception of specific cells within the course of vascular remodeling. The abundance of quickly available information provided by electron microscopic approaches may be useful for subsequent, e.g., biochemical or molecular, studies and thus delivers important controls for further experimental designs.

Animals↗
PubMed1991

Quantitative light and scanning electron microscopy of ferret sperm.

Sperm were obtained via electroejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black-footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black-footed ferret.

Acrosome↗
PubMed1993

The sea urchin egg jelly coat is a three-dimensional fibrous network as seen by intermediate voltage electron microscopy and deep etching analysis.

The egg jelly (EJ) coat which surrounds the unfertilized sea urchin egg undergoes extensive swelling upon contact with sea water, forming a three-dimensional network of interconnected fibers extending nearly 50 microns from the egg surface. Owing to its solubility, this coat has been difficult to visualize by light and electron microscopy. However, Lytechinus pictus EJ coats remain intact, if the fixation medium is maintained at pH 9. The addition of alcian blue during the final dehydration step of sample preparation stains the EJ for visualization of resin embedded eggs by both light and electron microscopy. Stereo pairs taken of thick sections prepared for intermediate voltage electron microscopy (IVEM) produce a three-dimensional image of the EJ network, consisting of interconnected fibers decorated along their length by globular jelly components. Using scanning electron microscopy (SEM), we have shown that before swelling, EJ exists in a tightly bound network of jelly fibers, 50-60 nm in diameter. In contrast, swollen EJ consists of a greatly extended network whose fibrous components measure 10 to 30 nm in diameter. High resolution stereo images of hydrated jelly produced by the quick-freeze/deep-etch/rotary-shadowing technique (QF/DE/RS) show nearly identical EJ networks, suggesting that dehydration does not markedly alter the structure of this extracellular matrix.

Alcian Blue↗
PubMed1992

Direct detection of immunogold reactions by real-time video microscopy.

Video-enhanced microscopy allows the detection and tracking of individual colloidal gold particles. The analysis of immunogold reactions can also be conducted as a function of time and thus allows the study of dynamic events in living cells. The direct visualization in real time is reported of the reaction of immunogold particles with a surface antigen. This time-resolved immunocytochemistry was achieved by continuous observation of living cells infected with a virus (respiratory syncytial virus) following their incubation with colloidal gold (30 nm) coated with antiviral antibodies. The progress of the immunoreaction was visualized as a sequential deposition of individual gold granules on the viral particles until saturation was reached after 60 min. Binding of colloidal gold was an irreversible event as no elution or dislocation of surface-bound granules took place. Comparative imaging of colloidal gold particles by electron microscopy and by video microscopy demonstrated that the video-imaged immunoreactions represented events involving single gold particles; their signal was sometimes clearly enhanced by secondary depositions taking place in close proximity, i.e. at a distance below the lateral resolution of the light microscope. Our experiments demonstrate that video-enhanced microscopy provides a powerful tool for studying antibody-antigen reactions with a high spatial and temporal resolution.

Animals↗
PubMed1982

Studies on the development of extra-endothelial and intra-endothelial pigment deposits by means of direct and indirect contact specular microscopy of the cornea.

Retro-corneal pigmentation as a result of primary (degenerative) or secondary (inflammatory, traumatic) pigment dispersion represents one of the most frequent findings in specular microscopy. The analysis of the pigment distribution patterns of dense deposits like those in Krukenberg spindle (KS) permits the postulation of a hypothesis concerning the development of such typical corneal pigmentations and the pigmentation of the posterior surface of the cornea in general, such as diffuse pigment dispersion (Vogt 1930), which is seen frequently. In a particular case of double KS with extreme myopia and cataract, endothelial morphological findings were documented over a period of 1 year, both before and after cataract extraction. This was done by means of contact and non-contact specular microscopy in various spindle areas. The morpho-metric studies (cell density/mm2; cell perimeter; largest smallest and average cell diameter; specific surface; horizontal and vertical cell orientation) were carried out automatically using a computerised video image analyser. The changes in the area of densest pigmentation in the KS centre confirm the histologically proven capacity of pigment phagocytosis by human corneal endothelium. These findings indicate that in specular microscopy, both endothelial cellular surface modifications and intra-cellular changes are documentable. The extracellular pigment deposits can also be shown by means of 'indirect' specular microscopy, the optical principle of which is described.

Aged↗
PubMed2003

Loss of lamin A/C expression revealed by immuno-electron microscopy in dilated cardiomyopathy with atrioventricular block caused by LMNA gene defects.

Mutations of the LMNA gene encoding the lamin A and C nuclear envelope proteins cause an autosomal dominant form of dilated cardiomyopathy (DCM) with atrioventricular block (AVB). The aim of this study was to investigate ultrastructural nuclear membrane changes by conventional electron microscopy and protein expression by immuno-electron microscopy in the heart of patients with DCM and AVB due to LMNA gene mutations. Four immunohistochemical techniques were used: pre-embedding and post-embedding in Epon-Araldite resin and London Resin White (LRW), with and without silver enhancement. Parallel light microscopy immunohistochemistry studies were performed. Conventional electron microscopy showed a loss of integrity of the myocyte nuclei with blebs of the nuclear membrane, herniations and delamination of the nuclear lamina and nuclear pore clustering. Post-embedding LRW was the most informative technique for morphology and immuno-labelling. Immuno-labelling was almost absent in the nuclear envelope of patients with LMNA gene mutations, but intensely present in controls. The loss of labelling selectively affected myocyte nuclei; the endothelial cell nuclei were immunostained in patients and controls. Light immunohistochemistry confirmed the results. These findings confirm the hypothesis that LMNA gene defects are associated with a loss of protein expression in the selective compartment of non-cycling myocyte nuclei.

Adult↗