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PubMed1980

Electron microscopy in the diagnosis of neuroblastoma.

I review the accumulated knowledge regarding the electron microscopy of neuroblastic tumors using a retrospective study of 13 primarily small cell neoplasms for which the diagnosis of a neuroblastic tumor (neuroblastoma, ganglioneuroblastoma, ganglioneuroma) was either considered or eventually established. A fine fibrillary background, even if focally distributed by light microscopy and seemingly not recovered in the thick sections, correlated with the presence ultrastructurally of neuritic processes bearing dense core ("neurosecretory") granules and lucent vesicles. In three cases without any fibrillary background by light microscopy, granule-bearing processes were not found by electron microscopy, and different diagnoses were subsequently established for two of those cases. From the standpoint of diagnosis, the sampling error normally introduced in ultrastructural examinations has not been reported to be a significant problem, nor was it in the present cases. Therefore, in supporting a light microscopic diagnosis of neuroblastoma, neuritic processes containing dense granules with or without lucent vesicles should be documented. Neuroblastic tumors with evidence of maturity, ie, ganglion cell differentiation, had the best developed morphologic features by both forms of microscopy. Schwann cells were observed in four cases, including one neuroblastoma. The presence of these cells by electron microscopy, although absent by light microscopy, may reflect an early phase of tumor maturation.

Adolescent↗

PubMed2002

MR microscopy and high resolution small animal MRI: applications in neuroscience research.

The application of magnetic resonance (MR) imaging in the study of human disease using small animals has steadily evolved over the past two decades and strongly established the fields of "small animal MR imaging" and "MR microscopy." An increasing number of neuroscience related investigations now implement MR microscopy in their experiments. Research areas of growth pertaining to MR microscopy studies are focused on (1). phenotyping of genetically engineered mice models of human neurological diseases and (2). rodent brain atlases. MR microscopy can be performed in vitro on tissue specimens, ex vivo on brain slice preparations and in vivo (typically on rodents). Like most new imaging technologies, MR microscopy is technologically demanding and requires broad expertise. Uniform guidelines or "standards" of a given MR microscopy experiment are non-existent. The main focus therefore of this review will be on biological applications of MR microscopy and the experimental requirements. We also take a critical look at the biological information that small animal (rodent) MR imaging has provided in neuroscience research.

Animals↗

PubMed2002

Time-resolved line focus acoustic microscopy of layered anisotropic media: application to composites.

This paper presents theoretical and experimental studies of the time-domain response of line focus acoustic microscopy from a layered anisotropic medium. A method for elastic constant reconstruction from acoustic microscopy signatures also is presented. The microscopy response is complicated by multiple reflections in the layers and by the anisotropic nature of the material. The model is based on a new, stable recursive stiffness matrix algorithm developed for a multilayered anisotropic medium, which is applied to the interpretation of the time-resolved acoustic microscopy signature. Specific examples are given for unidirectional and multidirectional graphite epoxy composites. It is shown that the fluid load has a significant effect on the leaky surface waves in these composites, increasing surface wave speed above that for the slow transverse wave. This results in its absence from the microscopy signature of the surface wave. The theoretical results are compared with experiments carried out using a line focus PVDF transducer developed at National Institute of Standards and Technology (NIST). Time-resolved acoustic microscopy has been applied to the determination of elastic constants of a unidirectional composite or of one lamina in a cross-ply composite. The lateral waves and multiple reflections of bulk waves appearing in the microscopy signatures are used for the elastic properties reconstruction. The reconstruction results are compared to data obtained by the self-reference double-through-transmission ultrasonic bulk wave method.

Journal Article↗

PubMed1999

Does the availability of blood slide microscopy for malaria at health centers improve the management of persons with fever in Zambia?

Some Ministries of Health in Africa plan to make blood slide microscopy available in peripheral health centers to improve malaria diagnosis over the current practice, which relies solely on clinical findings. To assess whether microscopy improves the management of febrile persons in health centers, we prospectively reviewed medical records of all outpatients visiting six health centers with laboratories in Zambia during a 2-3-day period. Staff interviews and a blinded review of a series of blood slides from each facility by two expert microscopists were also conducted. Of 1,442 outpatients, 655 (45%) reported fevers or had a temperature > or = 37.5 degrees C. Blood slide microscopy was ordered in 28-93% of patients with fever (mean = 46%). Eighty-eight (35%) patients without parasitemia were prescribed an antimalarial drug. Antimalarial drugs were prescribed with equal frequency to those who were referred for a blood slide (56%) and those not referred (58%). The sensitivity of microscopy was 88% and the specificity was 91%. Use of malaria microscopy varied widely, indicating that clinicians are not using standard criteria for ordering this test. Although diagnosis by microscopy was generally accurate, it appeared to have had little impact on the treatment of persons with fever. Guidelines for using blood slide microscopy are needed and prescription of antimalarial drugs should be discouraged when slide results are negative.

Ambulatory Care Facilities↗

PubMed1994

Ion microscopy in biology.

Ion microscopy, a mass spectrometry based isotopic imaging technique, is uniquely suited for ion transport related problems in biological systems. Due to its high sensitivity, it can image the transport and distribution of both major and minor elements (isotopes) at subcellular resolutions. The images of major elements such as K, Na, CI, etc., can be viewed directly and recorded in real-time from the microchannel plate-fluorescent screen detector of the instrument. The low concentration physiologically important elements, such as Ca, need about one minute of integration for good quality imaging. The isotopic imaging capability of ion microscopy provides a unique approach for the use of stable isotopes as tracers. In this way, one can image both the endogenous and the transported isotopes independently. Strict cryogenic sample preparations are essential for ion transport studies. Correlative imaging of the same cell with laser scanning confocal microscopy and ion microscopy can positively identify smaller cytoplasmic compartments such as the Golgi apparatus in calcium images. We have identified the Golgi apparatus as a calcium storing organelle. Another unique application of ion microscopy is the imaging of boron from boronated drugs used in Boron Neutron Capture Therapy (BNCT) of cancer. Ion microscopy is capable of rapid screening of potential drugs for BNCT. This critical information is essential for the fundamental understanding of BNCT. Ion microscopy is now at the stage where it can provide previously unattainable answers to important biomedical questions.

Animals↗

PubMed1998

Combined fluorescent and gold immunoprobes: reagents and methods for correlative light and electron microscopy.

Immunoprobes which incorporate both a fluorescent label and a 1.4 nm gold cluster compound were prepared by covalent conjugation to Fab' antibody fragments of the Nanogold cluster label followed by a fluorescent moiety. These new immunoconjugates allow the collection of two complementary sets of data, from fluorescence and electron microscopy, from a single labeling experiment. By using Fab' fragments, the entire probe is smaller than a whole IgG molecule. A simple fluorescence assay was used to investigate the fluorescence properties of the new probes. They were used to localize the pre-mRNA splicing factor SC35 in the HeLa cell nucleus by both fluorescence and electron microscopy, and also for labeling leukocyte microtubules; labeling was imaged using fluorescence microscopy and, after silver enhancement, by a variety of optical methods and by electron microscopy. Combined Nanogold and Texas Red, Cy3, Lissamine Rhodamine B, and AMCA probes were also prepared, and in preliminary experiments show similar properties to the combined fluorescein and gold cluster probes. The fluorescent and gold cluster probes enable a new degree of correlation between fluorescence and electron microscopy, and may also be used to check labeling of specimens before processing for electron microscopy.

Cell Nucleus↗

PubMed1991

Diagnosis of urinary tract infection in children: fresh urine microscopy or culture?

Fresh unspun and unstained urine specimens from 342 children with previous urinary tract infection (UTI) or symptoms compatible with a UTI were examined by microscopy at a magnification of x 400 in a mirrored counting chamber by a clinician, and sent for culture in a microbiology laboratory; 200 samples were also plated onto dip-slides. When microscopy and culture results were discrepant, further urine samples were collected until a diagnosis of UTI (24) or sterile urine (318) could be confirmed. Initial microscopy correctly identified 23 of 24 UTIs and 286 of 318 sterile urines; 1 false-positive result was caused by vaginal contamination with lactobacilli. 32 specimens (9%) gave an equivocal result on microscopy; the 1 other true-positive result was identified correctly on microscopy of the next urine specimen obtained. Culture of the initial urines correctly identified all 24 UTIs, but only 82% of the negative samples. Of the samples from uninfected children, 35 (11%) showed a mixed growth which was sterile on repeat sampling, and 21 (6.6%) initially grew a false-positive pure growth of more than 10(5) colony-forming units/ml of one organism. True UTIs were associated with bacterial counts above 10(7)/ml. Microscopy by a clinician represents a cheaper, quicker, and more reliable screening test for UTI in children than does routine culture in a microbiology laboratory.

Adolescent↗

PubMed2006

Principles of two-photon excitation microscopy and its applications to neuroscience.

The brain is complex and dynamic. The spatial scales of interest to the neurobiologist range from individual synapses (approximately 1 microm) to neural circuits (centimeters); the timescales range from the flickering of channels (less than a millisecond) to long-term memory (years). Remarkably, fluorescence microscopy has the potential to revolutionize research on all of these spatial and temporal scales. Two-photon excitation (2PE) laser scanning microscopy allows high-resolution and high-sensitivity fluorescence microscopy in intact neural tissue, which is hostile to traditional forms of microscopy. Over the last 10 years, applications of 2PE, including microscopy and photostimulation, have contributed to our understanding of a broad array of neurobiological phenomena, including the dynamics of single channels in individual synapses and the functional organization of cortical maps. Here we review the principles of 2PE microscopy, highlight recent applications, discuss its limitations, and point to areas for future research and development.

Animals↗

PubMed1997

Light and scanning electron microscopy of rabbit lens capsules with intraocular lenses.

PURPOSE: To examine postoperative changes in the lens capsules of rabbit eyes after phacoemulsification and aspiration of the crystalline lens and implantation of posterior chamber intraocular lenses (IOLs) using light and scanning electron microscopy. SETTING: Research Laboratory, Department of Ophthalmology, Wakayama Medical College, Japan. METHODS: The crystalline lens was emulsified and aspirated and an IOL implanted in the capsular bag or ciliary sulcus of each eye in adult albino rabbits under general anesthesia. Animals were killed after 4 weeks, and the lens capsules were removed. The specimens were observed under phase-contrast microscopy and processed for light and scanning electron microscopy. RESULTS: Phase-contrast microscopy revealed presumed lens epithelial cells (LECs) on the central posterior capsules in association with regenerating lenticular fibers and Elschnig pearls in the peripheral capsules. Scanning electron microscopy showed the accumulation of fibrous extracellular matrix on the surface of the posterior capsule in eyes in which the IOL was implanted in the ciliary sulcus. Deposition of packed material attached to the surface of IOLs and of Soemmering's ring were observed in eyes with in-the-bag IOL fixation. At a higher magnification, a parallel arrangement of lenticular fibers was seen in the regenerated lens structure on posterior capsules. An identical structure was observed under light microscopy. Outgrowth of presumed LECs from residual anterior lens capsules and adhesion of macrophages and giant cells were observed on the IOL surface. CONCLUSION: Two types of postoperative changes were observed in lens capsules after implantation of IOLs: accumulation of fibrous extracellular matrix and newly formed lenticular fibers. These changes are attributed to the proliferation of LECs and can induce posterior capsule opacification after IOL implantation.

Animals↗

PubMed2006

[Two-photon laser scanning fluorescence microscopy for functional cellular imaging: Advantages and challenges or One photon is good... but two is better!].

One of the main challenges of modern biochemistry and cell biology is to be able to observe molecular dynamics in their functional context, i.e. in live cells in situ. Thus, being able to track ongoing molecular events with maximal spatial and temporal resolution (within subcellular compartments), while minimizing interference with tissue biology, is key to future developments for in situ imaging. The recent use of non-linear optics approaches in tissue microscopy, made possible in large part by the availability of femtosecond pulse lasers, has allowed major advances on this front that would not have been possible with conventional linear microscopy techniques. Of these approaches, the one that has generated most advances to date is two-photon laser scanning fluorescence microscopy. While this approach does not really provide improved resolution over linear microscopy in non absorbing media, it allows us to exploit a window of low absorbance in live tissue in the near infrared range. The end result is much improved tissue penetration, minimizing unwanted excitation outside the focal area, which yields an effective improvement in resolution and sensitivity. The optical system is also simplified and, more importantly, phototoxicity is reduced. These advantages are at the source of the success of two-photon microscopy for functional cellular imaging in situ. Yet, we still face further challenges, reaching the limits of resolution that conventional optics can offer. Here we review some recent advances in optics/photonics approaches that hold promises to improve our ability to probe the tissue in finer areas, at faster speed, and deeper into the tissue. These include super-resolution techniques, introduction of non paraxial optics in microscopy and use of amplified femtosecond lasers, yielding enhanced spatial and temporal resolution as well as tissue penetration.

Cells↗

PubMed2006

The unique pollen morphology of Duparquetia (Leguminosae: Caesalpinioideae): developmental evidence of aperture orientation using confocal microscopy.

BACKGROUND AND AIMS: The phylogenetic affinities of the aberrant monotypic genus Duparquetia (subfamily Caesalpinioideae) are at present unresolved. Preliminary results from molecular analyses suggest a basal, isolated position among legumes. A study of Duparquetia pollen was carried out to provide further morphological characters to contribute to multi-data set analyses. Understanding the development of Duparquetia pollen was necessary to clarify the orientation of the apertures. METHODS: Pollen grains and developing microspores were examined using light microscopy, confocal microscopy and scanning electron microscopy. Evidence for the orientation of the apertures was provided by the examination of microspores within developing tetrads, using (a) confocal microscopy to locate the position of the ectoapertures, and (b) light microscopy and Alcian blue stain to locate the position of the endoapertures. KEY RESULTS: Confocal microscopy has been used for the first time to examine developing microspores in order to obtain information on ectoapertures that was unavailable using other techniques. Pollen in Duparquetia develops in tetrahedral tetrads as in other eudicots, with the apertures arranged in a modified pattern following Fischer's rule. Pollen grains are asymmetrical and have one equatorial-encircling ectoaperture with two equatorial endoapertures, a unique feature in Leguminosae, and in eudicots. CONCLUSIONS: The pollen morphology of Duparquetia is so unusual that it provides little information to help determine its closest relatives. However, it does fit with a pattern of greater pollen morphological diversity in the first-branching caesalpinioid legume groups than in the more derived clades. The latitudinal ectoaperture of Duparquetia is unique within the Fabales and eudicot clades, resembling more closely the monosulcate pollen found in monocots and basal angiosperms; however, developmental patterns are recognizably similar to those of all other legume pollen types.

Fabaceae↗

PubMed2004

Quantitative phase microscopy: a new tool for investigating the structure and function of unstained live cells.

1. The optical transparency of unstained live cell specimens limits the extent to which information can be recovered from bright-field microscopic images because these specimens generally lack visible amplitude-modulating components. However, visualization of the phase modulation that occurs when light traverses these specimens can provide additional information. 2. Optical phase microscopy and derivatives of this technique, such as differential interference contrast (DIC) and Hoffman modulation contrast (HMC), have been used widely in the study of cellular materials. With these techniques, enhanced contrast is achieved, which is useful in viewing specimens, but does not allow quantitative information to be extracted from the phase content available in the images. 3. An innovative computational approach to phase microscopy, which provides mathematically derived information about specimen phase-modulating characteristics, has been described recently. Known as quantitative phase microscopy (QPM), this method derives quantitative phase measurements from images captured using a bright-field microscope without phase- or interference-contrast optics. 4. The phase map generated from the bright-field images by the QPM method can be used to emulate other contrast image modes (including DIC and HMC) for qualitative viewing. Quantitative phase microscopy achieves improved discrimination of cellular detail, which permits more rigorous image analysis procedures to be undertaken compared with conventional optical methods. 5. The phase map contains information about cell thickness and refractive index and can allow quantification of cellular morphology under experimental conditions. As an example, the proliferative properties of smooth muscle cells have been evaluated using QPM to track growth and confluency of cell cultures. Quantitative phase microscopy has also been used to investigate erythrocyte cell volume and morphology in different osmotic environments. 6. Quantitative phase microscopy is a valuable, new, non-destructive, non-interventional experimental tool for structural and functional cellular investigations.

Animals↗

PubMed1968

Interference contrast and phase contrast microscopy of sporulation and germination of Bacillus megaterium.

The techniques of Nomarski interference contrast microscopy and phase-contrast microscopy were compared for their utility in monitoring sporulation and germination in Bacillus megaterium. The Nomarski technique permitted rapid and easy delineation of septation and engulfment during sporulation, whereas with phase contrast microscopy these stages were not detected at all. The later stages of sporulation were easily seen by either technique. Thus, of the seven stages of sporulation as recognized by the electron microscopy of thin sections, five can now be routinely detected quantitatively by optical microscopy: septation (stage II), engulfment (stage III), phase-dark forespore (corresponding to cortex formation, stage IV), phase-bright spore in a sporangium (corresponding to coat formation, stage V), and the free spore (stage VII). This means that now only stage I (axial filament) and stage VI (maturation of the refractile spore) require electron microscopy for routine detection. There was no advantage in using Nomarski optics for germination studies.

Bacillus megaterium↗

PubMed1975

Conditions critical for optimal visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron microscopy.

The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of infected cells which were fixed with glutaraldehyde followed by postfixation in OsO4 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively smooth surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of resolving viral particles on cell surfaces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used.

Adsorption↗

PubMed2003

Intravital imaging of the kidney using multiparameter multiphoton microscopy.

Intravital optical microscopy provides a powerful means of studying the cell biology in the most physiologically relevant setting. The ability of multiphoton microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of multiple organs. Presented here are examples of how two-photon microscopy can be applied to intravital studies of kidney physiology and the study of disease processes. These include studies of cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters. Two-photon microscopy can now provide a level of investigation previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution. Therefore, application of this technology will provide direct visualization of organ-specific and cell-specific responses to an array of stimuli and therapeutic approaches, enhancing our understanding and treatment of disease processes.

Animals↗

PubMed2001

Correlative fluorescence and electron microscopy on ultrathin cryosections: bridging the resolution gap.

Microscopy has become increasingly important for analysis of cells and cell function in recent years. This is due in large part to advances in light microscopy that facilitate quantitative studies and improve imaging of living cells. Analysis of fluorescence signals has often been a key feature in these advances. Such studies involve a number of techniques, including imaging of fluorescently labeled proteins in living cells, single-cell physiological experiments using fluorescent indicator probes, and immunofluorescence localization. The importance of fluorescence microscopy notwithstanding, there are instances in which electron microscopy provides unique information about cell structure and function. Correlative microscopy in which a fluorescence signal is reconciled with a signal from the electron microscope is an additional tool that can provide powerful information for cellular analysis. Here we review two different methodologies for correlative fluorescence and electron microscopy using ultrathin cryosections and the advantages attendant on this approach. (J Histochem Cytochem 49:803-808, 2001)

Animals↗

PubMed2006

The use of light- and electron microscopy for studies on the cell- and molecular biology of parasites and parasitic diseases.

Lightmicroscopical (LM) and electron microscopi cal (EM) techniques, have had a major influence on the development and direction of cell biology, and particularly also on the investigation of complex host-parasite relationships. Earlier, microscopy has been rather descriptive, but new technical and scientific advances have changed the situation. Microscopy has now become analytical, quantitative and three-dimensional, with greater emphasis on analysis of live cells with fluorescent markers. The new or improved techniques that have become available include immunocytochemistry using immunogold labeling techniques or fluorescent probes, cryopreservation and cryosectioning, in situ hybridization, fluorescent reporters for subcellular localization, micro-analytical methods for elemental distribution, confocal laser scanning microscopy, scanning tunneling microscopy and live-imaging. Taken together, these tools are providing both researchers and students with a novel and multidimensional view of the intricate biological processes during parasite development in the host.

Animals↗

PubMed2006

Static magnetic fields affect cell size, shape, orientation, and membrane surface of human glioblastoma cells, as demonstrated by electron, optic, and atomic force microscopy.

BACKGROUND: It is common knowledge that static magnetic fields (SMF) do not interact with living cells; thus, fewer studies of SMF compared with variable magnetic fields are carried out. However, evidence demonstrated that SMF affect cellular structures. To investigate the effect of exposure to increasing doses of SMF on cell morphology, human glioblastoma cells were exposed to SMF ranging between 80 and 3,000 G (8 and 300 mT). METHODS: Cell morphology of human glioblastoma cells, derived from a primary culture, was studied by electron and optic microscopy. FITC-phalloidin staining of actin filaments was also investigated. Finally, cell surface structure changes were detected by atomic force microscopy. RESULTS: Scanning electron microscopy demonstrated a dose-dependent cell shape modification, progressive cell detachment, loss of the long villi, and appearance of membrane roughness and blebs. FITC-phalloidin staining confirmed the villi retention and cell dimension decrease. At 3,000 G, the appearance of apoptotic morphology was also observed by transmission electron microscopy. Cell exposed to SMF showed different orientation and alignment when compared with nonexposed cells. The atomic force microscopy of the exposed cells' membrane surfaces demonstrated the disappearance of the ordered surface ripples and furrows typical of the unexposed cells, and the occurrence of surface membrane corrugation at increasing dose exposure CONCLUSIONS: Our experimental procedures demonstrated that exposure to SMF affects not only cell size, shape, and orientation but also human glioblastoma cells' membrane surfaces.

Apoptosis↗