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PubMed1998

Microwave procedures for electron microscopy and resin-embedded sections.

Microwaves now have well-established applications in routine light microscopy. They are employed in tissue fixation and to accelerate a wide spectrum of staining procedures. Besides producing superior preservation of cellular antigens through microwave fixation, this form of irradiation has been employed for antigen retrieval, a procedure that has been a major factor in the optimization of immunolabelling in paraffin sections and cytological preparations. A commercial tissue processor has recently been developed which employs microwaves in a markedly accelerated, one-step processing of tissue blocks, completing the procedure within a fraction of conventional times. Microwaves have also been successfully applied in a variety of molecular techniques such as in situ hybridization and polymerase chain reaction. The adoption of microwaves in electron microscopic procedures has been slower, largely because the requirement for speed in processing is not as great, except in diagnostic samples. However, as this review will show, there are equally as many innovative applications of microwaves in electron microscopy. Microwaves have been employed for rapid processing of fine needle aspiration biopsy samples, in keeping with the requirement for speed in this method of diagnosis. Ultrafast fixation of tissue samples has resulted in the better demonstration of cellular enzymes and proteins. It has been clearly shown that microwave-stimulated on grid staining in uranyl acetate and lead citrate produces more consistent results and without background precipitation. Microwaves can be used to hasten resin polymerization and exposure to microwaves results in antigen retrieval in both resin-embedded thick sections and for immuno-electron microscopy. Immunolabelling shows enhanced sensitivity and the technique is anticipated to contribute greatly to the optimization of immuno-electron microscopy. The potential for greatly accelerated preparation of samples for electron microscopy exists but is yet to be fully realized.

Image Enhancement↗
PubMed2006

In situ single-molecule imaging with attoliter detection using objective total internal reflection confocal microscopy.

Confocal microscopy is widely used for acquiring high spatial resolution tissue sample images of interesting fluorescent molecules inside cells. The fluorescent molecules are often tagged proteins participating in a biological function. The high spatial resolution of confocal microscopy compared to wide field imaging comes from an ability to optically isolate and image exceedingly small volume elements made up of the lateral (focal plane) and depth dimensions. Confocal microscopy at the optical diffraction limit images volumes on the order of approximately 0.5 femtoliter (10(-15) L). Further resolution enhancement can be achieved with total internal reflection microscopy (TIRM). With TIRM, an exponentially decaying electromagnetic field (near-field) established on the surface of the sample defines a subdiffraction limit dimension that, when combined with conventional confocal microscopy, permits image formation from <7 attoL (10(-18) L) volumes [Borejdo et al. (2006) Biochim. Biophys. Acta, in press]. Demonstrated here is a new variation of TIRM, focused TIRM (fTIRM) that decreases the volume element to approximately 3 attoL. These estimates were verified experimentally by measuring characteristic times for Brownian motion of fluorescent nanospheres through the volume elements. A novel application for TIRM is in situ single-molecule fluorescence spectroscopy. Single-molecule studies of protein structure and function are well-known to avoid the ambiguities introduced by ensemble averaging. In situ, proteins are subjected to the native forces of the crowded environment in the cell that are not present in vitro. The attoL fluorescence detection volume of TIRM permits isolation of single proteins in situ. Muscle tissue contains myosin at a approximately 120 microM concentration. Evidence is provided that >75% of the bleachable fluorescence detected with fTIRM is emitted by five chromophore-labeled myosins in a muscle fiber.

Animals↗
PubMed2001

Quantitative analysis of angiogenesis using confocal laser scanning microscopy.

Angiogenesis is essential for tumor growth and metastasis. Angiogenesis is commonly quantified by measuring microvessel density (MVD) within tumors. In this report, we compared light microscopy with confocal laser scanning microscopy (CLSM) in the qualitative and quantitative analysis of angiogenesis. MVDs were determined manually in a lung tumor xenograft and a normal skeletal muscle using CD31 immunohistochemical staining and light microscopy. Area of three-dimensional representation of microvessels, detected as CD31 immunofluorescence, was measured automatically using computer-assisted CLSM. By manual counting under light microscopy, the relative level of MVD of the lung tumor vs. skeletal muscle was 0.8. However, the corresponding relative level of microvessels was 3.4 as determined by computer-assisted CLSM. Furthermore, the architecture of microvessels was better delineated with CLSM than with light microscopy. We have applied this CLSM method for analyzing the antiangiogenic effect of an anticancer drug, paclitaxel, in the lung tumor xenograft model. We conclude that CLSM is an appropriate method for quantitative and qualitative analysis of microvasculature in normal and tumor tissues.

Angiogenesis Inhibitors↗
PubMed2005

Deep tissue two-photon microscopy.

With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional-including confocal-fluorescence microscopy. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.

Animals↗
PubMed1991

Applications of confocal scanning optical microscopy to dentistry.

Confocal optical microscopy is now a well recognised technique in the fields of biological and materials science. This type of light microscope can be considered as being midway between optical and electron microscopy. Confocal or scanning optical microscopes can make high resolution, thin, optical sections within semitransparent samples such as biological tissues. Surface images of samples can be produced which are similar in character to those of the SEM, but without many of the problems of specimen preparation. The improved resolution and removal of out-of-focus blur allows much more information to be gained from fluorescence microscopy techniques, with the images capable of 3-D reconstruction of the sample. There are basically two types of confocal optical microscope: the laser scanning type (CLSM) and the real-time direct view of tandem scanning microscopes (TSM). The former are best suited to immunofluorescence microscopy, whilst the latter are more appropriate for high-speed reflection imaging, having originally been developed for in vivo microscopy.

Dentistry, Operative↗
PubMed2002

Clinical relevance of hair microscopy in alopecia.

Hair microscopy can clarify the cause of hair loss in a range of diagnoses. Most of these are associated with hair breakage, the rest are related to lack of growth. Hair breakage may be due to excessive trauma or underlying susceptibility, where structural clues may be present. Lack of growth reflects follicular dynamics and represents the central mechanism of most common causes of alopecia. In such conditions, microscopy only reveals nonspecific confirmation of short anagen. Although this may assist clinical diagnosis, microscopy in alopecia only allows exclusion of diagnoses related to hair breakage. Confidence in the outcome of hair microscopy is based on the size of the sample of hairs, the length of the hair, the characteristics of the observations and the experience of the person undertaking the microscopy.

Alopecia↗
PubMed2003

Seasonal changes in the inputs to gonadotropin-releasing hormone neurones in the ewe brain: an assessment by conventional fluorescence and confocal microscopy.

The seasonal pattern of breeding in sheep offers an opportunity to examine plasticity of neuronal inputs to gonadotropin-releasing hormone (GnRH) neurones. We used conventional fluorescence microscopy and confocal microscopy to compare the extent of input to GnRH neurones from various neuropeptide/neurotransmitter systems in ewes during the breeding and anestrous seasons. Using double-labelling immunohistochemistry, we counted close appositions between GnRH cells and varicosities that were immunoreactive for either glutamic acid decarboxylase (GAD; for gamma-amino butyric acid-GABA-neurones), dopamine beta hydroxylase (DBH; for noradrenergic neurones), vesicular glutamate transporter-1 (VGluT-1, for glutamatergic neurones), neuropeptide Y (NPY) and tyrosine hydroxylase (TH; for dopaminergic/noradrenergic neurones). The percentage of GnRH cells displaying close appositions to GABA-ergic varicosities was higher (P < 0.02) in anestrus than in the breeding season. The percentage of GnRH cells receiving input from varicosities that were positive for TH, DBH and VGluT-1 was similar in both seasons. Approximately 26-49% of GnRH neurones were seen to receive inputs from NPY, TH, GABAergic or noradrenergic neurones, while a larger number of GnRH cells (72-75%) received input from glutamatergic neurones. Conventional microscopy consistently overestimated the number of close contacts on GnRH neurones compared to confocal microscopy. For TH-immunoreactive varicosities in the preoptic area, only 16-35% were also immunoreactive for DBH, suggesting that the remainder are dopaminergic. Approximately half of the noradrenergic inputs in the preoptic area were also immunoreactive for NPY. In conclusion, we present numerical data on the consensus between light and confocal microscopy and the level of input of various neuronal systems to GnRH cells; the data indicate a seasonal change in the GABAergic input to GnRH neurones.

Anestrus↗
PubMed1992

Absence of ovicidal effects of fenoxycarb in the tick Ixodes dammini as observed by light, scanning, and transmission electron microscopy.

Freshly oviposited eggs from fed females of the northern deer tick Ixodes dammini were divided into two groups--untreated controls and fenoxycarb-exposed ova. The level of fenoxycarb exposure in the experimental group was equal to a concentration that proved 100% lethal in the cat flea, Ctenocephalides felis (Marchiondo et al. 1990). Both groups were allowed to develop with samples taken at 48 and 144 h for examination by light microscopy, scanning electron microscopy, and transmission electron microscopy. No disruption of the stages of tick embryological development was observed when specimens were examined by these methods. Eggs not sampled for light microscopy or scanning or transmission electron microscopy during development were allowed to hatch. The resulting larvae were fully developed and motile in both the control and experimental conditions, further supporting the conclusion that embryological development of I. dammini is not disrupted by concentrations of fenoxycarb which would be practical in the field.

Animals↗
PubMed1983

Comparison between direct methods for determination of microbial cell volume: electron microscopy and electronic particle sizing.

Size frequency distributions of different phototrophic and heterotrophic microorganisms were determined by means of scanning and transmission electron microscopy and electronic particle sizing. Statistically significant differences existed among the three techniques used in this study. Cells processed for electron microscopy showed lower mean cellular volumes than those processed for electronic particle sizing, reflecting a shrinkage by factors ranging from 1.1 to 6.2 (mean, 2.3). Processing of cells for scanning electron microscopy caused higher shrinkage than processing for transmission electron microscopy. Shrinkage was dependent neither on the size nor on the cell wall type of the microorganism. When processed for scanning electron microscopy, phototrophic bacteria were strongly shrunken, whereas heterotrophic microorganisms were less affected. A direct relationship existed among phototrophic bacteria between percentage of shrinkage and specific pigment content. This was probably a consequence of the pigment extraction by organic solvents during the dehydration process, previous to the critical point drying, necessary to examine the specimens under the scanning electron microscope.

Bacteria↗
PubMed2000

Analysis of antiphotobleaching reagents for use with FluoroNanogold in correlative microscopy.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence and electron microscopy. We have employed FluoroNanogold (FNG) as the detection system in these types of studies. This immunoprobe consists of a gold cluster compound to which a fluorochrome-labeled antibody is covalently linked. In these preparations, the fluorescence signal from FNG is first recorded then the gold cluster compound is subjected to a silver enhancement reaction before examination by electron microscopy. Potential complications are those associated with photochemical reactions that occur during fluorescence microscopy. We have evaluated this and some anti-photobleaching agents (i.e., 1,4-diazabicyclo[2.2.2]octane [DABCO],p-phenylenediamine [PPD], and N-propyl gallate [NPG]) for their utility with FNG in correlative microscopy. When DABCO was employed, the gold signal from FNG was dramatically diminished but the fluorescence signal was unaffected. The gold signal of DABCO-treated samples decreased to approximately 30% of that of the other samples. On the other hand, PPD and NPG did not adversely affect the FNG labeling. We recommend that either PPD or NPG be used and that DABCO be avoided as an antiphotobleaching reagent for this technique.

Frozen Sections↗
PubMed1992

Preparation of muscle samples for comparative electron microscopy.

The interpretation of muscle structure by scanning electron microscopy (SEM) has not been consistent among various studies. Consequently, the literature is confusing with respect to the identity of T-tubules, transverse ridges, Z-disks, and intermyofibrillar connections. The objective of this research was to evaluate the effects of different methods of sample preparation and imaging on ultrastructural details of previously identified transverse structures and intermyofibrillar connections and to verify or disprove the commonality of these structures under different viewing conditions. Scanning electron microscopy coupled with a cold stage, SEM at room temperature, and transmission electron microscopy (TEM) of thin sections were most appropriate for exposing detail of inter- and intracellular structures and for measuring sarcomere length and spacing of intermyofibrillar connections. Scanning electron microscopy of samples mounted on a cold stage, fractured, and sublimed provided excellent images of meat and muscle ultrastructure and may be used in correlative microscopy. Sarcomere length and spacing between intermyofibrillar connections were similar among most specimen preparation techniques and were affected similarly by heat treatments. Results indicate that the regularly spaced transverse structures viewed by conventional SEM and the intermyofibrillar connections viewed by low-temperature SEM are Z-disks.

Animals↗
PubMedDate not supplied

Role of electron microscopy in transplant renal pathology.

The crucial role that electron microscopy plays in diagnostic renal pathology is undisputed. By allowing recognition of findings not identifiable by light microscopic evaluation, electron microscopy has contributed significantly to the understanding of renal diseases and has proven to be of unquestionable value in many diagnostic situations. However, the percentage of cases in which electron microscopic examination adds important information that is either key for establishing or confirming a diagnosis or provides valuable data that influence patient's management remains controversial. This figure depends on the renal biopsy service that is surveyed, but it is reported that on the average ultrastructural evaluation is of value in approximately 30 to 45% of the cases. Correct interpretation of a renal biopsy depends on the ability to correlate light, immunofluorescence, and ultrastructural findings. In contrast, the role of electron microscopy in the examination of renal transplant specimens remains controversial. Many centers do not use routine electron microscopy to examine these specimens and insist that there are only a few specific indications that require ultrastructural evaluation. There is general agreement among renal pathologists that electron microscopy is of importance in the evaluation of renal specimens from patients with proteinuria to distinguish between transplant glomerulopathy, recurrent or de novo glomerulonephritis in order to correctly manage these patients and predict survival of the graft. The other possible indications are much more controversial. This paper summarizes and critically reviews the literature available on this subject and defines recommendations based on the information available at the current time.

Biomarkers↗
PubMed1999

Long-term microstructural analyses of hydroxyapatite implanted in rats using laser-Raman spectrometry and scanning electron microscopy.

To investigate the long-term surface microstructure of a synthetic auditory ossicle (Apaceram) composed of dense hydroxyapatite (HA), thin HA disks were implanted subcutaneously into the interscapular regions of 12 rats. After 6, 14 and 20 months, implanted HA surfaces were observed using stereoscopic microscopy, scanning electron microscopy (SEM) and laser-Raman spectrometry. Visual observation by SEM at 6 months and by stereoscopic microscopy at 14 months indicated a progressive degradation of the HA disk surfaces implanted in the subcutaneous tissue. Visual observation by SEM at 14 and 20 months and by stereoscopic microscopy at 20 months indicated a progressive redeposition on the surfaces of the implants. Raman spectra compared half-peak breadths of v1 signal (PO4(3-), 960 cm(-1)) on the gray and white surface areas of implanted HA disks observed by stereoscopic microscopy. Analysis demonstrates that demineralization at 14 months and remineralization at 20 months occur on the gray areas; demineralization at 6 months and remineralization at 14 months occur on the white areas.

Animals↗
PubMed2000

Correlative microscopy of cerebellar Golgi cells.

The cerebellar Golgi cells of mouse, teleost fish, primate and human species have been studied by means of light and Golgi light microscopic techniques, confocal laser scanning microscopy, slicing technique, ethanol-cryofracturing and freeze-fracture methods for scanning electron microscopy and ultrathin sectioning and freeze-etching replicas for transmission electron microscopy. The Golgi cells appeared in the granular layer as polygonal, stellate, round or fusiform macroneurons surrounded by the granule cell groups. They exhibited ascending dendrites toward the molecular layer and horizontal dendrites and a short beaded axonal plexus confined to the granular layer. Scanning electron microscopy revealed their three-dimensional neuronal geometry and smooth outer surfaces. Freeze-fracture method for SEM showed the stereospatial cytoplasmic arrangement of endoplasmic reticulum, cell organelles and nuclear envelope. By means of transmission electron microscopy the asymmetric synaptic connections of Golgi cell horizontal dendrites--with mossy fiber rosettes at the cerebellar glomerulus--and of Golgi cell axons--with granule cell dendrites at the periphery of glomerular region--were identified. At the molecular layer, Golgi cell ascending dendrites exhibited short neckless spines establishing asymmetric contacts with granule cell axons or parallel fibers. Shaft asymmetric axodendritic and axospinodendritic contacts between Golgi cell dendrites and climbing fibers were also found in the molecular layer.

Animals↗
PubMed1983

Analytical scanning and transmission electron microscopy and x-ray microdiffractometry of calcium pyrophosphate dihydrate crystal deposits in tissues.

This paper reviews current methods and applications for the detection and identification of calcium pyrophosphate dihydrate (CPPD) crystals. Methods reviewed include compensated polarized light microscopy, scanning electron microscopy, transmission electron microscopy, x-ray energy-dispersive elemental analysis, x-ray powder microdiffractometry, and selected area electron diffractometry. Although compensated polarized light microscopy is the best technique for crystal detection in pathologic fluids and tissues, because of crystal size this technique cannot discriminate easily between CPPD(M) and CPPD(T) polymorphs. To determine the presence and relative concentration of CPPD(M) and CPPD(T), x-ray or electron microdiffractometry is required. Analytical scanning and transmission electron microscopy are needed to localize crystals ultrastructurally and to determine the relationship of the crystal deposits to tissue components. The correlative application of multiple techniques on the same sample or adjacent samples provides more precise localization and identification than any single technique.

Calcium Pyrophosphate↗
PubMed1994

Changes in the distribution of F-actin in the fission yeast Schizosaccharomyces pombe by arresting growth in distilled water: correlative studies with fluorescence and electron microscopy.

Freeze-substitution electron microscopy of Schizosaccharomyces pombe cells starved in distilled water was conducted to define ultrastructural counterparts of actin visualized by fluorescence microscopy using rhodamine-conjugated phalloidin (Rh-ph). Starvation in distilled water caused remarkable changes in actin distribution and ultrastructural changes in S. pombe. Fluorescence microscopy of the starved cells showed that the dots of actin at the growing ends became thick actin cables via an enlarged patched form of actin. These changes were reversible, and growth-arrested cells resumed their original pattern of actin distribution upon return to growth medium. Electron microscopy of starved cells showed bundles of thin filaments and clusters of filamentous balls in the cytoplasm, which corresponded to the actin cables and enlarged actin dots, respectively, as seen by fluorescence microscopy. Vesicles polarized at the growing cell ends were dispersed in the cytoplasm by distilled water treatment, indicating that actin organization plays a role in directing vesicle location.

Actin Cytoskeleton↗
PubMed1993

BCG cell imaging using scanning probe microscopy.

Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) were used to obtain images of the surface of whole, intact BCG (bacille Calmette Guerin, a mycobacterium) cells in air and under solution by immobilizing the cells onto glass slides (AFM only) or highly oriented pyrolytic graphite. The technique used for AFM imaging involved depositing a submonolayer of cells under a centrifugal force followed by fixation/dehydration using polar organic solvents. AFM images agree well with images from light and electron microscopy and showed large numbers of BCG cells in their distinctive cord arrangement. The AFM also proved useful for identifying extracellular microgranules which cannot be seen with light microscopy. For STM imaging, the hydrophobicity of BCG enabled strong adhesion from aqueous solution onto graphite. STM images of BCG could only be obtained while scanning in aqueous solution, and the cells showed a large variation in contrast when different samples were imaged. The STM provided greater detail of surface features than the AFM and was able to produce images of periodic layers corroborating observations made by transmission electron microscopy.

Microscopy↗
PubMed1993

Ultrastructural changes of extracellular matrices in diabetic nephropathy revealed by high resolution scanning and immunoelectron microscopy.

BACKGROUND: Diabetic nephropathy is invariably associated with proteinuria. EXPERIMENTAL DESIGN: To delineate the mechanism(s) of proteinuria in diabetic nephropathy, ultrastructural changes of the glomerular basement membranes (GBMs) were studied by high resolution scanning and immunoelectron microscopy. Acellular glomeruli from diabetic and age-matched control human subjects were prepared by detergent method and subjected to conductive staining, the technique in which the tissues are impregnated with metals rather than surface-coated with metallic alloys for visualization by electron microscopy. Subsequent to conductive staining, the tissues were examined by in-lens field emission scanning electron microscopy. RESULTS: Thirty glomeruli, each from the control and diabetic groups, were examined by scanning microscopy. In diabetic GBMs, a loose meshwork structure consisting of numerous pores of approximately 8 nm in diameter and distinct strands was observed. In contrast, meshwork structure was not readily discernible in controls and a few pores were observed. Five glomerular capillary loops, each from control and diabetic groups, were examined by immunoelectron microscopy. In controls, heparan sulfate-proteoglycan was localized in the lamina rara interna and externa, and type-IV collagen was distributed throughout the whole width of the GBM. In diabetic GBMs, a relative loss of staining of heparan sulfate-proteoglycan, both in the lamina rara interna and externa of the GBM, was observed. Type IV collagen was distributed in all layers of the thickened GBM, and the absolute number of the immunogold particles was increased. However, immunogold particle density of type IV collagen per unit area was decreased as compared with the control. CONCLUSIONS: These ultrastructural and immunoelectron microscopic changes in the GBM may explain the loss of charge as well as size selectivities of the glomerulus, as observed in diabetic nephropathy associated with proteinuria.

Aged↗