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PubMed2005

Use of confocal microscopy in comparative studies of vertebrate morphology.

Laser scanning confocal microscopy provides a means to acquire and analyze images of complex morphological structures and to help place molecules or cells of interest in their proper morphological context. Confocal microscopy is a form of fluorescence microscopy that sharpens the images collected by visualizing the light from only one plane of focus. This allows for the collection of multiple focal planes in what is called a z-stack, which provides three-dimensional data. Five steps that any investigator using a confocal microscope should follow are described: (1) labeling and (2) mounting of specimens for viewing, (3) optimizing the image on the confocal, and (4) collecting and (5) analyzing of confocal image data. We describe three specific protocols incorporating these steps from our work on vertebrate inner ear development. The first two describe a collection of z-stacks in living, fluorescently labeled, and intact embryos. The second protocol is for time-lapse imaging of multiple focal planes at each time point. The third protocol describes confocal imaging of preserved material double labeled with antibodies and by retrograde labeling of neurons via axonal uptake. Finally, three alternative or complementary approaches to standard confocal microscopy are described and discussed.

Anatomy, Comparative↗

PubMed2003

Confocal laser scanning microscopy of urinary bladder after intravesical instillation of a fluorescent dye.

OBJECTIVES: To assess the potential of confocal laser scanning microscopy for imaging of the urinary bladder after intravesical instillation of a fluorescent dye. METHODS: The study was performed on the bladder of male Copenhagen rats. For confocal fluorescence microscopy (CFM), a standard confocal laser scanning microscope (Zeiss LSM 410) was used. Before measuring, the fluorescent marker SYTO 17 was instilled intravesically. After 2 hours of incubation, the rat was killed, the bladder excised and opened, and CFM was performed starting from the surface going through the urothelium and superficial layers of the lamina propria. Except for the opening incision, the bladder was left intact and no biopsies were taken. After imaging, the bladder was sent for conventional histologic studies. RESULTS: CFM allows imaging of cellular details of the entire urothelium (superficial umbrella cells, intermediate, and basal urothelial cells) and superficial layers of the lamina propria. CFM images are close to those obtained by standard microscopy after conventional hematoxylin-eosin staining. Cell structure (eg, shape, size, chromatin texture, nucleoli, mitotic figures, nuclear/cytoplasmic ratio), as well as the structure of the connective tissue (eg, collagen fibers, blood vessels, erythrocytes), can be studied, allowing a standard histologic evaluation. Furthermore, in contrast to conventional histologic evaluation, CFM provides three-dimensional information and allows the study of intact tissue representing the true in vivo situation. CONCLUSIONS: CFM enables the study of the microscopic anatomy of bladder mucosa in its in vivo state. In combination with optical fiber bundles, endoscopic microscopy of the bladder may be possible in the future.

Administration, Intravesical↗

PubMed1983

Posterior internal ophthalmomyiasis. Identification of a surgically removed Cuterebra larva by scanning electron microscopy.

The clinical presentation of an 11-year-old boy with unilateral posterior internal ophthalmomyiasis is described. Ophthalmoscopy of the affected eye in this individual disclosed characteristic subretinal depigmented linear tracks and a larva within the vitreous. The intravitreal larva was retrieved successfully by pars plana vitrectomy and identified as the first instar of Cuterebra sp. (rodent botfly) by scanning electron microscopy. This was done by examining the external features of the larva by scanning electron microscopy and comparing them to scanning electron micrographs that were prepared on other first instar larvae of flies known to be implicated in internal ophthalmomyiasis. The study demonstrates the usefulness of scanning electron microscopy for taxonomic identification of fly larvae. Since even a small fragment of these organisms may contain characteristic features of external morphology, scanning electron microscopy may be useful in cases where the specimen is damaged or incomplete.

Child↗

PubMed1985

Electron microscopy of Merkel cell carcinoma from formalin-fixed tissue.

Merkel cell carcinomas have characteristic, but not pathognomonic, histomorphologic features. The diagnosis can be confirmed by immunohistochemistry studies and electron microscopy. However, differential diagnostic problems often occur only after all tumor material has been fixed in formalin and embedded in paraffin, which gives poor tissue preservation for electron microscopy. Therefore, in addition to histologic and immunohistochemical studies, parallel ultrastructural investigations were performed on tumor specimens fixed in glutaraldehyde and routinely processed, for electron microscopy, on formalin-fixed tissue and on formalin-fixed paraffin blocks. Formalin fixation led to an almost complete loss of neurosecretory granules and cell membranes; however, the characteristic paranuclear whorls of intermediate filaments were retained in all tumors. Merkel cell carcinomas are immunoreactive for neuron-specific enolase (NSE), but NSE-antisera have only recently become commercially available. Cytokeratin is demonstrable as a paranuclear clump, and this feature was found in four out of six tumors. Thus histologic, immunohistochemical, and electron microscopy studies of formalin-fixed tissue are able to confirm the diagnosis of neuroendocrine Merkel cell carcinoma.

Adenocarcinoma↗

PubMed1987

In vivo epiluminescence microscopy of pigmented skin lesions. I. Pattern analysis of pigmented skin lesions.

The importance of recognizing early melanoma is generally accepted. Because not all pigmented skin lesions can be diagnosed correctly by their clinical appearance, additional criteria are required for the clinical diagnosis of such lesions. In vivo epiluminescence microscopy provides for a more detailed inspection of the surface of pigmented skin lesions, and, by using the oil immersion technic, which renders the epidermis translucent, opens a new dimension of skin morphology by including the dermoepidermal junction into the macroscopic evaluation of a lesion. In an epiluminescence microscopy study of more than 3000 pigmented skin lesions we have defined morphologic criteria that are not readily apparent to the naked eye but that are detected easily by epiluminescence microscopy and represent relatively reliable markers of benign and malignant pigmented skin lesions. These features include specific patterns, colors, and intensities of pigmentation, as well as the configuration, regularity, and other characteristics of both the margin and the surface of pigmented skin lesions. Pattern analysis of these features permits a distinction between different types of pigmented skin lesions and, in particular, between benign and malignant growth patterns. Epiluminescence microscopy is thus a valuable addition to the diagnostic armamentarium of pigmented skin lesions at a clinical level.

Basal Cell Carcinoma↗

PubMed1987

In vivo epiluminescence microscopy of pigmented skin lesions. II. Diagnosis of small pigmented skin lesions and early detection of malignant melanoma.

Pattern analysis by epiluminescence microscopy of pigmented skin lesions was tested in a study of 318 small pigmented skin lesions that were diagnostically equivocal when examined with the naked eye. An improvement of clinical diagnosis was achieved by epiluminescence microscopy for practically all lesions, both benign and malignant, and was equally impressive for melanocytic and nonmelanocytic lesions. Improvement in diagnostic accuracy was as follows: for small nodular melanomas, from 50% to 70%; for superficial spreading melanoma in situ, from 46% to 80%; for invasive superficial spreading melanoma, from 64% to 90%; and for early lentigo maligna and lentigo maligna melanoma, from 67% to 88%. Conversely, the diagnosis of pigmented Spitz nevi improved from 46% to 93% and of pigmented basal cell carcinomas from 60% to 90%, which appears equally important because most of these lesions had clinically been considered to represent melanomas. The use of epiluminescence microscopy also resulted in considerable improvement in the diagnosis of dysplastic nevi, which was particularly helpful in making therapeutic decisions. Epiluminescence microscopy greatly expands the diagnostic armamentarium available for pigmented skin lesions at a clinical level and thus increases the chances of detecting or ruling out melanoma in its earliest stages.

Basal Cell Carcinoma↗

PubMed2000

Three-dimensional characterization of interior structures of exocytotic apertures of nerve cells using atomic force microscopy.

We examined the interior structure of exocytotic apertures in synaptic vesicles of neuroblastoma x glioma hybrid cells using atomic force microscopy. The atomic force microscopy detected apertures of 50-100nm in diameter at various depths within the varicosities of these cells. We were also able to image a regular radial pattern on the wall and lump-like structures at the bottom of these apertures. In contrast, scanning electron microscopy could only detect the apertures but not the fine details of their interior. The cells examined here exhibited the same electrophysiological properties and expression of synaptophysin and syntaxin 1 as presynaptic terminals, as studied by various electrophysiological and imaging techniques. Our results indicate that atomic force microscopy allows three-dimensional viewing of the fine structures located inside exocytotic apertures in nerve cells.

Calcium↗

PubMed2003

Superiority of 3D wavelet-packet denoising in MR microscopy.

Three dimensional Magnetic Resonance Imaging (MRI) datasets are becoming increasingly important in clinical and research applications because of their inherent signal to noise (SNR) advantages, high resolution and isotropic voxels. Despite SNR advantages, some 3D acquisitions may be SNR-limited, particularly in MR microscopy. Historically, both classic filtering and wavelet-based denoising techniques have been performed on a slice-by-slice basis. In principle, adaptive techniques such as best- basis wavelet-packet denoising might offer inherent advantages when performed in 3D, instead of 2D, by tracking through plane "structure" and suppressing noise "pseudostructure." This hypothesis was tested in 10 volumetric MR microscopy datasets from several different MR microscopy atlas projects. 3D wavelet-packet denoised images consistently yielded lower minimum mean-square error and subjectively perceived noise power than corresponding 2D denoised images using otherwise identical algorithms and parameters. MR microscopy researchers preferred the denoised images to the unprocessed images for their atlas projects.

Animals↗

PubMed2003

Stromal haze after laser in situ keratomileusis: clinical and confocal microscopy findings.

PURPOSE: To report clinical and confocal microscopy characteristics of haze-like opacities in corneas after laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany. METHODS: Eighteen eyes of 11 patients with clinically apparent corneal clouding were examined by slitlamp and confocal microscopy (Confoscan P4, Tomey) 1 to 9 months after primary LASIK or LASIK retreatment. RESULTS: Postoperative slitlamp examination showed faint, white, snowflake-like clouding at the interface level in all patients. One patient had folds and rather diffuse haze-like opacities. Confocal microscopy revealed highly reflective structures in the flap stroma and at the interface level in all patients, probably due to numerous activated keratocytes and their processes. The confocal microscopy appearance was similar to that of photorefractive keratectomy haze. CONCLUSION: Focal wound-healing reactions in the central flap stroma and interface resulting in significant keratocyte activation could be observed after LASIK.

Adult↗

PubMed2003

Saturated patterned excitation microscopy with two-dimensional excitation patterns.

The techniques of patterned excitation microscopy (PEM, also referred to in the literature as structured illumination, harmonic excitation light microscopy, or laterally modulated excitation microscopy), has recently been extended to the non-linear regime, permitting a further increase in resolution breaking the Abbe diffraction limit (saturated PEM, saturated patterned excitation microscopy (SPEM)). Fluorescence saturation was suggested as the non-linear effect employed to achieve this aim. Here a two-dimensional extension of the linear and the non-linear patterned excitation technique is introduced and simulations of the expected resolution improvement are presented. The simulations account for photon statistics, a sub-optimal degree of modulation and a high amount of background fluorescence in the sample. The resulting point-spread-functions achieve a full width at half maximum of 215 nm (widefield), 118 nm (linear PEM), and 57 nm (saturated PEM, 9x9 orders). For higher resolution, an increased number of detected photons and of raw data images are required. A potential method for substantially decreasing the required number of raw images in PEM and SPEM is discussed.

Fluorescence↗

PubMedDate not supplied

Low energy loss electron microscopy of chromophores.

A novel prism-mirror-prism imaging electron spectrometer with 1 eV energy resolution for a transmission electron microscope permits imaging with spectral energies corresponding to light-optical colour absorptions. The instrument selects the molecular orbital excitations of natural chromophores or of specific dyes normally used in biological light microscopy for delineation and chemical identification, but images them with electron microscopic detail. Heavy atom contrast agents customarily used in electron microscopy are not required. The first results exploit the intrinsic red colour of hematin molecules to demonstrate the potential of the technique and address its spatial resolution. Glycosaminoglycans in cartilage stained with Alcian blue are selectively depicted in situ by means of the electron-induced molecular absorption of this chromophore. Thus, with the use of specific colours the direct or indirect analysis of local chemistry by electron microscopy is possible, and can be carried out with a depiction of spatial detail as small as 16 A, or at least 100-fold finer than observed by light microscopy.

Alcian Blue↗

PubMed2000

Recent structural insight into mitochondria gained by microscopy.

Novel applications of microscopy have recently provided new insights into mitochondrial structures. Diverse techniques such as high resolution scanning electron microscopy, transmission electron microscopy, electron microscope tomography and light microscopy have contributed a better understanding of mitochondrial compartmentalization, dynamic networks of mitochondria, intermembrane bridges, segregation of mitochondrial DNA and contacts with the endoplasmic reticulum among other aspects. This review focuses on advances reported in the last five years concerning aspects of mitochondrial substructure or dynamics gained through new techniques, whether they be novel microscope methods or new ways to prepare or label specimens. Sometimes these advances have produced surprising results and more often than not, they have challenged current conceptions of how mitochondria work.

Animals↗

PubMed2003

Electron microscopy and the investigation of new infectious diseases.

OBJECTIVES: To review and assess the role of electron microscopy in the investigation of new infectious diseases. DESIGN: To design a screening strategy to maximize the likelihood of detecting new or emerging pathogens in clinical samples. RESULTS: Electron microscopy remains a useful method of investigating some viral infections (infantile gastroenteritis, virus-induced outbreaks of gastroenteritis and skin lesions) using the negative staining technique. In addition, it remains an essential technique for the investigation of new and emerging parasitic protozoan infections in the immunocompromised patients from resin-embedded tissue biopsies. Electron microscopy can also have a useful role in the investigation of certain bacterial infections. CONCLUSIONS: Electron microscopy still has much to contribute to the investigation of new and emerging pathogens, and should be perceived as capable of producing different, but equally relevant, information compared to other investigative techniques. It is the application of a combined investigative approach using several different techniques that will further our understanding of new infectious diseases.

Animals↗

PubMed2006

Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review.

In low-income and middle-income countries, direct (unconcentrated) sputum smear microscopy is the primary method for diagnosing pulmonary tuberculosis. The method is fast, inexpensive, and specific for Mycobacterium tuberculosis in high incidence areas. The main limitations of direct microscopy are its relatively low sensitivity, especially in individuals co-infected with HIV, and variable quality of the test in programme conditions. Thus, there is a need to identify methods to improve the sensitivity of microscopy. Physical and chemical sputum processing methods, including centrifugation, sedimentation, and bleach, have been studied and found to show promise. We did a systematic review to assess the ability of different processing methods to improve the sensitivity of microscopy. By searching many sources, we identified 83 studies. Overall, by comparison with direct smears, the results suggested that centrifugation with any of several chemical methods (including bleach) is more sensitive, that overnight sedimentation preceded by chemical processing is more sensitive, and that specificity is similar. There were insufficient data to determine the value of sputum processing methods in patients with HIV infection. Operational studies are needed to determine whether the increased sensitivity provided by processing methods is sufficient to offset their increased cost, complexity, and potential biohazards, and to examine their feasibility.

Bacteriological Techniques↗

PubMed2006

In vivo confocal microscopy of the ocular surface.

Over the past two decades, the applications of in vivo confocal microscopy to the investigation of ocular surface diseases in the living eye have been greatly extended. Confocal microscopy enables detailed investigation of tarsal and palpebral conjunctiva, central and peripheral cornea, tear film, and lids, and it allows evaluation of the ocular surface at the cellular level. High-quality imaging in both contact and noncontact modes has allowed new understanding of the functions of the ocular surface system, and in the coming years, such knowledge will become increasingly comprehensive and specific. Confocal microscopy may provide a link between well-established ex vivo histology and in vivo study of ocular pathology, not only in clinical science but also in clinical practice. The purpose of this review is to summarize the current knowledge about in vivo confocal microscopy of the ocular surface.

Conjunctiva↗

PubMed1976

Determination of the mass of viruses by quantitative electron microscopy.

The photometric method of quantitative determination of dry mass by electron microscopy has been applied to the study of various types of viruses: animal, plant, insect, and bacterial. The method is applicable to all viruses having a mass of 1 x 10-18g or greater. The molecular weight of viruses can be calculated from the mass value by multiplying it by Avogadro's number. In comparison to other methods of determining the molecular weight of viruses, sedimentation and diffusion, sedimentation equilibrium, light scattering, and electron microscopy counting, the method of quantitative electron microscopy is competitive. In some ways quantitative electron microscopy is superior to other methods for the determination of molecular weight: There is no limitation to the size of the virus, no experimental time involved and no concentration and purity of virus preparations required, and finally it is independent of the geometry of the virion. In one important aspect it is unique when compared to other methods; namely, it affords one the capacity to analyse individual virus particles.

Biophysical Phenomena↗

PubMed2002

Intracellular processing of poly(ethylene imine)/ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments.

PURPOSE: Critical steps in the subcellular processing of poly(ethylene imine)/nucleic acid complexes, especially endosomal/lysosomal escape, were visualized by using living cell confocal laser scanning microscopy (CSLM) to obtain an insight into their mechanism. METHODS: Living cell confocal microscopy was used to examine the intracellular fate of poly(ethylene imine)/ribozyme and poly(L-lysine)/ribozyme complexes over time, in the presence of and without bafilomycin Al, a selective inhibitor of endosomal/lysosomal acidification. The compartment of complex accumulation was identified by confocal microscopy with a fluorescent acidotropic dye. To confirm microscopic data, luciferase reporter gene expression was determined under similar experimental conditions. RESULTS: Poly(ethylene imine)/ribozyme complexes accumulate in acidic vesicles, most probably lysosomes. Release of complexes occurs in a sudden event, very likely due to bursting of these organelles. After release, poly(ethylene imine) and ribozyme spread throughout the cell, during which slight differences in distribution between cytosol and nucleus are visible. No lysosomal escape was observed with poly(L-lysine)/ribozyme complexes or when poly(ethylene imine)/ ribozyme complexes were applied together with bafilomycin A1. Poly(ethylene imine)/plasmid complexes exhibited a high luciferase expression, which was reduced approximately 200-fold when lysosomal acidification was suppressed with bafilomycin A1. CONCLUSIONS: Our data provide, for the first time, direct experimental evidence for the escape of poly(ethylene imine)/nucleic acid complexes from the endosomal/lysosomal compartment. CLSM, in conjunction with living cell microscopy, is a promising tool for studying the subcellular fate of polyplexes in nucleic acid/gene delivery.

Animals↗

PubMed1995

Analysis of drug distribution in hydrogels using fourier transform infrared microscopy.

PURPOSE: The purpose of this work was to study solute (drug and protein)/polymer interactions that affect solute diffusion in and subsequent release from swellable dosage forms based on environmentally responsive, pH-sensitive polymer networks. METHODS: Ionizable pH-sensitive hydrogels were synthesized by free-radical polymerization of acrylic acid (AA) and 2-hydroxyethyl methacrylate (HEMA) with ethylene glycol dimethacrylate as crosslinking agent. The degree of crosslinking and degree of hydrophilicity were controlled by varying the relative concentration of crosslinking agent and comonomer, respectively. The role of solute distribution within the hydrogels as related to the transport behavior was investigated by Fourier transform infrared (FTIR) microscopy. RESULTS: The solute-loaded hydrogels were cryotomed into 4 microns thin sections. The concentration profile of the solute was constructed from the two dimensional intensities measured by monitoring the infrared vibrational band indicative of that compound in the x and y direction. These studies indicated that the model solute, oxprenolol HCl, was evenly distributed throughout the bulk and surface of the hydrogel samples. Solute/polymer interactions were investigated as a function of the polymer composition and swelling media pH. The concentration profiles of oxprenolol HCl solution loaded into PAA and P(AA-co-HEMA) hydrogels were analyzed by scanning electron microscopy by FTIR microscopy. For hydrophilic polymers (hydrogels) containing ionizable pendant groups, the molecular weight between crosslinks, the degree of swelling, and the degree of ionization were altered by local changes in pH and ionic strength. CONCLUSIONS: The data demonstrated that it is possible to evaluate the polymer/solute interactions by using FTIR microscopy.

Diffusion↗