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PubMed1997

Evaluation of infectious crystalline keratitis with confocal microscopy in a case series.

We sought to determine whether there are unique findings in infections crystalline keratitis (ICK) examined by confocal microscopy and if confocal microscopy is predictive for bacteriology in ICK. A retrospective review of consecutive patients with a presumed diagnosis of ICK by slit-lamp examination was performed. These patients were then examined with confocal microscope and cultured. Sixteen patients were identified by biomicroscopy. Average age was 71 years; 12 of 16 patients were women; 10 of 16 had prior penetrating keratoplasty; and 12 of 16 were taking topical steroids. Confocal microscopy revealed a variable appearance to the crystals in the corneal stroma. Eight of 16 patients had distinct needle-like deposits at varying depths in the stroma, and eight had amorphous deposits grouped at different levels of the stroma. The results of confocal microscopic examination resembled the reported histopathology with clusters of deposits, but its current resolution does not allow identification of bacterial morphology. There was no correlation of morphology with culture results. Organisms were recovered in 12 of 16 patients by culture. In 10 of 16 patients, the infection was successfully treated with topical antibiotics, usually cefazolin. Crystal morphology of ICK can be observed by confocal microscopy. No pathognomonic, single pattern for this disease is seen with the confocal microscope. The latter may be an aid in determining the clinical response to treatment.

Adult↗

PubMedDate not supplied

Scanning electron microscopy and profilometer evaluation of glazed and polished dental porcelain.

The surface roughness of Vita VMK porcelain following oven glazing and eight grinding/polishing treatments was analyzed qualitatively using scanning electron microscopy and quantitatively using a profilometer. Techniques were selected to simulate clinical practice. Scanning electron microscopy evaluation found oven glazing produced a better surface than other polishing methods. On the basis of the profilometric examination, the best roughness average value was obtained using diamond instruments with progressively smaller particle sizes (30, 15, and 8 microns). Scanning electron microscopy analysis showed that all the treatments left the surfaces partially porous and cracked; however, the glazed surface yielded the best result. Although no significant differences were detected for the different treatments, the use of a 30-microns diamond instrument produced a rougher surface. No correlation was found between scanning electron microscopy and profilometer results. This study suggests that several procedures may be used to effectively finish ceramic surfaces.

Analysis of Variance↗

PubMed1997

[New possibilities of optic microscopy in studies of blood cells].

Modern biomedical tasks put forward new requirements to the instrumental base of microscopy. This paper outlines the prospective trends in microscopy which are the most interesting for medicine, on the one hand, and analyzes the probability of meeting the requirements of medicine by modern methods of optic electronics and computer processing of optic information. The authors analyze the following trends: microscopy of three-dimensional objects, super-resolution, microscopic measurements, and automation of microscopes. Progress in optic electronics and computer processing of images permits considering these trends as a priority task of modern microscopy.

Blood Cells↗

PubMed1998

Atomic force microscopy in effusion cytology.

OBJECTIVE: To investigate whether atomic force microscopy (AFM) in combination with classical light microscopy allows simple identification of surface structures of cells from pleural and ascitic fluids for diagnostic purposes in place of scanning electron microscopy (SEM). STUDY DESIGN: We examined a total of 180 cells obtained from 9 reactive pleural or peritoneal effusions, 14 associated with carcinomatosis from histologically confirmed tumors and 5 from mesotheliomas. Cells of interest were selected in air-dried, uncovered, May-Grünwald-Giemsa (MGG)-stained smears and subsequently investigated by AFM. Incorporation of a very compact AFM scanner into the nose piece of a conventional Axioscope light microscope allowed alternating application of both techniques. RESULTS: AFM was able to detect cell surface structures, such as microvilli, phagocytic pits, secretory blebs and lytic holes. The image resolution was sufficient but not as good as that with SEM. We found differences in number, length and diameter of microvilli between cells from mesotheliomas and from metastatic adenocarcinomas. CONCLUSION: As AFM can be carried out in combination with light microscopy quickly and easily on uncovered, MGG-stained smears, we propose this method as a suitable tool for obtaining additional useful information in routine cytologic diagnosis of effusions.

Adenocarcinoma↗

PubMed1996

Multiple labeling in electron microscopy: its application in cardiovascular research.

The heart is a muscular pump kept together by a network of extracellular matrix components. An increase in collagens, as in chronic congestive heart failure (CHF), is thought to have a negative effect on cardiac compliance and, thus, on the clinical condition. Conventional electron microscopy allows for the study of cellular and extracellular components and scanning electron microscopy (SEM) can put these structures in three-dimensional perspective. However, in order to study extracellular matrix components in relation to cells, immunoelectron microscopy is superior. We have used this technique in our studies on heart failure. Heart specimens were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in sodium cacodylate buffer, dehydrated by the method of progressive lowering of temperature and embedded in LR Gold plastic. Immunolabeling could be achieved with different sized gold-conjugated secondary antibodies or protein-A gold conjugates. Depending on the objective, ultra small gold (USG) conjugates or a regular probe size can be used. Labeling efficiency could be increased by bridging antibodies. The double and triple staining procedures were based on single staining methods using one- and two-face labeling. The choice of antibodies and gold conjugates depended on the objectives. Immunoelectron microscopy, using multiple labeling, allowed a detailed study of the organization of the extracellular matrix and its relationship with cardiac myocytes. This may prove to be a useful tool for the study of chronic heart failure.

Actins↗

PubMed1998

Acousto-optic random-access laser scanning microscopy: fundamentals and applications to optical recording of neuronal activity.

A novel approach to laser scanning microscopy is presented that utilizes diffraction-based scanning principles to achieve fast random-access positioning of a focused laser beam. This non-imaging approach overcomes the speed limitation of present reflection-based scanning microscopes while maintaining high spatial resolution. The presented system combines conventional video microscopy with fast non-imaging scanning microscopy. Together with readily available optical indicators of neuronal activity, this system permits multi-site optical recording from living brain tissue. In this paper, we will review the underlying principles of laser scanning microscopy and the steps in development that led to the current acousto-optic scanning system. We will present typical signals recorded with the current system, and we will outline ongoing extensions of the system. We will also discuss the present limitation of this instrumentation and look into directions of future development.

Acoustics↗

PubMed1999

Differential diagnosis of linear corneal images on confocal microscopy.

PURPOSE: This study aimed to detect corneal conditions presenting with linear images on white light confocal microscopy and to analyze their distinguishing characteristics. METHODS: In 1996 and 1997, 153 eyes of 110 patients with various corneal conditions were examined. In vivo examination of the cornea was performed by using a white-light tandem scanning confocal microscope. Images were captured by using a video camera and stored on S-VHS video tapes. In this retrospective study, patient charts and confocal microscopic video records were reviewed. Conditions with linear images were looked for, and the images were analyzed and compared. RESULTS: The only structures presenting as linear images on confocal microscopy in normal subjects consisted of corneal nerves. The following pathologic conditions also had linear images on confocal microscopy: corneal vascularization, mycotic keratitis, lattice corneal dystrophy, and posterior polymorphous dystrophy. Each condition could be identified based on its reflectivity, delineation, size, branching pattern, and location in the cornea. CONCLUSION: Different corneal conditions present with linear images on confocal microscopy. Correct identification is critical to avoid misdiagnosis.

Cornea↗

PubMed2005

From micro to nano: recent advances in high-resolution microscopy.

Improving the spatial resolution of optical microscopes is important for a vast number of applications in the life sciences. Optical microscopy allows intact samples and living cells to be studied in their natural environment, tasks that are not possible with other microscopy methods (e.g. electron microscopy). Major advances in the past two decades have significantly improved microscope resolution. By using interference and structured light methods microscope resolution has been improved to approximately 100 nm, and with non-linear methods a ten times improvement has been demonstrated to a current resolution limit of approximately 30 nm. These methods bring together old theoretical concepts such as interference with novel non-linear methods that improve spatial resolution beyond the limits that were previously assumed to be unreachable.

Image Enhancement↗

PubMed1999

Effects of heat shock on the functional morphology of cell organelles observed by video-enhanced microscopy.

In living astrocytes and MDCK cells we observed morphological phenomena during and after heat shock (HS) utilizing our new perfusable microchamber system, which monitors pH, pO(2), pCO(2), and temperature. By means of electronic light microscopy and confocal laser scanning microscopy, mitochondria were demonstrated to swell and to reduce their motility. The specific fluorescent probe MitoTracker Green revealed that the mitochondrial morphology changed from a rodlike into an annular shape with a central vacuole-findings which were corroborated by transmission electron microscopy. After HS (shift from 37 degrees C to 45 degrees C for 15 min) the mitochondrial membrane potential (DeltaPsi(m)) was depressed in most but not all mitochondria as monitored with the fluorescent probe JC-1. The dual emission images of JC-1 illustrated a heterogeneous red staining of distinct areas of single mitochondria. The shape changes as well as the drop of the membrane potential of the mitochondria indicated severe cellular stress and a direct intervention on the mitochondrial permeability transition.

Animals↗

PubMed1995

Immunofluorescence and confocal laser scanning microscopy studies of osteoblast growth and phenotypic expression in three-dimensional degradable synthetic matrices.

In the development of three-dimensional cell-polymer synthetic matrices for tissue regeneration, visualization of cells growing in these porous structures can be difficult. The focus of this study was the development and use of a novel method that would allow for visualization of osteoblasts inside opaque matrices. The morphologic responses and phenotypic characterization of osteoblasts as they attach, spread, and migrate through a porous three-dimensional biodegradable polymer-ceramic matrix in vitro were studied using immunofluorescence and confocal laser scanning microscopy (CLSM). CLSM offers several advantages over the most commonly used imaging methods [traditional light microscopy and scanning electron microscopy (SEM)]. CLSM filters out-of-focus background and provides more structural details of cells. In addition, CLSM does not require extensive sample preparation as does SEM. When used in conjunction with fluorescence-labeled antibodies to identify cells and their products, it can characterize morphology of growing cells and successfully determine phenotypic function. Using monoclonal antibody to osteocalcin, a bone cell-specific protein, cells throughout the matrix were found to have preserved osteoblast-like phenotype with growth. The morphology of cells throughout the matrix was found to be similar to osteoblast cells grown on tissue culture polystyrene and consisted of spread polygonal forms. Using the technique of CLSM with immunofluorescent antibodies, we have demonstrated for the first time that these three-dimensional degradable polymer matrices can support osteoblast growth and phenotypic expression throughout its structure.

Alkaline Phosphatase↗

PubMed1990

Effects of bisphosphonates on isolated rat osteoclasts as examined by reflected light microscopy.

The bisphosphonates 4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid (ABP), 3-amino-1,1-hydroxypropane-1,1-diphosphonic acid (APD), and 1-hydroxyethane-1,1-diphosphonic acid (EHDP) were compared for their ability to inhibit the osteoclastic resorption of bone in culture. This was achieved by measuring the effect of bisphosphonate concentration on the number of resorption pits formed and the total area of resorption. During this analysis, a new application of reflected polarized light microscopy was developed that has advantages over other microscopy techniques, including scanning electron microscopy (SEM), as applied to the analysis of resorbed bone surfaces. Based on area analysis, the bisphosphonates were effective for the range 10(-7)-10(-8) M, with ABP about two to five times more effective than EHDP or APD. Similar data were obtained by counting the number of resorption pits but with EC50 10 times higher. At lower concentrations (10(-9) M), bisphosphonates may enhance the formation of resorption pits. APD was found to be more toxic (10 times) than ABP or EHDP to osteoclasts and mononuclear cells, but toxic concentrations were at least 10(2) times higher than the resorption EC50. These data plus immunofluorescence, SEM, and transient incubation experiments show that it is the bisphosphonate-bone complex that directly inhibits the excavation of resorption pits by mature osteoclasts. The mechanism of action does not appear to require inhibition of osteoclast differentiation or toxic elimination of osteoclasts. Bisphosphonates, however, subtly affect the cytoskeleton of osteoclasts.

Alendronate↗

PubMed2005

Live cell spinning disk microscopy.

In vivo microscopy of dynamic processes in cells and organisms requires very fast and sensitive acquisition methods. Confocal laser scanning microscopy is inherently speed-limited by the requirement of beam scanning movements. In contrast to single beam scanning systems, the parallelized approach of multi-beam scanning is much faster. Spinning disk confocal microscopes are therefore very suited for fast in vivo imaging. The principles of spinning disk microscopy will be explained in this chapter and a thorough comparison of the performance of single beam and multi-beam scanning systems is made and illustrated with an example of in vivo imaging in Dictyostelium discoideum.

Animals↗

PubMed2006

Identification of elastic fibers and lamellae in porcine pericardium and aorta by confocal, fluorescence and polarized light microscopy.

Pericardial connective tissue has been used to construct bioprostheses to repair various types of injuries, including aortic wall repairs. The arrangement and the distribution of elastic and collagen staple fibers are related directly to the biomechanical properties of the tissue and thus determine the choice tissue for the construction of bioprostheses. Although elastic fibers can be visualized using several histochemical methods, the specificity and mechanism of binding involved remain to be clarified. In this work, we compared the elastic net of the porcine pericardial matrix with that of the aortic wall using 1-anilino-8-naphthalene sulfonate (ANS) and dansyl chloride (DCl) as fluorescent probes and the permanganate-bisulfite-toluidine (PBT) method. Polarized light and fluorescence microscopy were simultaneously used to analyze the tissues. Some samples also were examined using confocal microscopy. Aorta and pericardium treated with ANS and DCl showed elastic fibers and lamellae with an intense blue fluorescence. When stained with the PBT method, the aortic elastic lamellae were clearly metachromatic and, under polarized light, they showed a greenish birefringence. DCl provided clearer fluorescent labelling of elastic fibers when examined using confocal microscopy.

Anilino Naphthalenesulfonates↗

PubMed2003

The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy.

The molecular deposition of starch extracted from normal plants and transgenically modified potato lines was investigated using a combination of light microscopy, environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). ESEM permitted the detailed (10 nm) topographical analysis of starch granules in their hydrated state. CLSM could reveal internal molar deposition patterns of starch molecules. This was achieved by equimolar labelling of each starch molecule using the aminofluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). Starch extracted from tubers with low amylose contents (suppressed granule bound starch synthase, GBSS) showed very little APTS fluorescence and starch granules with low molecular weight amylopectin and/or high amylose contents showed high fluorescence. Growth ring structures were sharper in granules with normal or high amylose contents. High amylose granules showed a relatively even distribution in fluorescence while normal and low amylose granules had an intense fluorescence in the hilum indicating a high concentration of amylose in the centre of the granule. Antisense of the starch phosphorylating enzyme (GWD) resulted in low molecular weight amylopectin and small fissures in the granules. Starch granules with suppressed starch branching enzyme (SBE) had severe cracks and rough surfaces. Relationships between starch molecular structure, nano-scale crystalline arrangements and topographical-morphological features were estimated and discussed.

Fluorescent Dyes↗

PubMed2003

Fluorescence and electron microscopy probes for cellular and tissue uptake of poly(D,L-lactide-co-glycolide) nanoparticles.

Nanoparticles formulated from poly(D,L-lactide-co-glycolide) (PLGA) and poly(lactide) (PLA) are being extensively investigated for different therapeutic applications such as for sustained drug, vaccine, and gene delivery. For many of these applications, it is necessary to study the intracellular distribution as well as the tissue uptake of nanoparticles to optimize the efficacy of the encapsulated therapeutic agent. Fluorescence and electron microscopic techniques are usually used for the above purposes. Colloidal gold particles and fluorescent polystyrene, which are generally used as model particles for electron and fluorescence microscopy, respectively, may not be suitable alternatives to PLGA/PLA nanoparticles for these studies mainly because of the differences in their physical properties and also because they do not contain any therapeutic agent. The aim of the present study was to develop and characterize PLGA nanoparticle formulations that would be suitable for confocal/fluorescence and transmission electron microscopic (TEM) studies. Towards this objective, PLGA nanoparticles containing 6-coumarin as a fluorescent marker and osmium tetroxide as an electron microscopic marker with bovine serum albumin (BSA) as a model protein were formulated. Different physical properties of marker-loaded nanoparticles such as particle size, zeta potential, residual PVA content and protein-loading were compared with those of unloaded nanoparticles and were found to be not significantly different. Furthermore, marker-loaded nanoparticle formulations were non-toxic to the cells as unloaded nanoparticles. Nanoparticles loaded with 6-coumarin were found to be useful for studying intracellular nanoparticle uptake and distribution using confocal microscopy while osmium tetroxide-loaded nanoparticles were found to be useful for studying nanoparticle uptake and distribution in cells and tissue using TEM. It was concluded that 6-coumarin and osmium tetroxide could serve as useful fluorescence and electron microscopy probes, respectively, for incorporation into nanoparticles to study their cellular and tissue distribution.

Cells, Cultured↗

PubMed2003

Applying microscopy to the analysis of nuclear structure and function.

One of the ultimate goals of biological research is to understand mechanisms of cell function within living organisms. With this in mind, many sophisticated technologies that allow us to inspect macromolecular structure in exquisite detail have been developed. Although knowledge of structure derived from techniques such as X-ray crystallography and nuclear magnetic resonance is of vital importance, these approaches cannot reveal the remarkable complexity of molecular interactions that exists in vivo. With this in mind, this review focuses on the use of microscopy techniques to analyze cell structure and function. We describe the different basic microscopic methodologies and how the routine techniques are best applied to particular biological problems. We also emphasize the specific capabilities and uses of light and electron microscopy and highlight their individual advantages and disadvantages. For completion, we also comment on the alternative possibilities provided by a variety of advanced imaging technologies. We hope that this brief analysis of the undoubted power of microscopy techniques will be enough to stimulate a wider participation in this rapidly developing area of biological discovery.

Animals↗

PubMed2004

Contribution of microscopy to a better knowledge of the biology of Giardia lamblia.

Giardia lamblia is a flagellated protozoan of great medical and biological importance. It is the causative agent of giardiasis, one of the most prevalent diarrheal disease both in developed and third-world countries. Morphological studies have shown that G. lamblia does not present structures such as peroxisomes, mitochondria, and a well-elaborated Golgi complex. In this review, special emphasis is given to the contribution made by various microscopic techniques to a better knowledge of the biology of the protozoan. The application of video microscopy, immunofluorescence confocal laser scanning microscopy, and several techniques associated with transmission electron microscopy (thin section, enzyme cytochemistry, freeze-fracture, deep-etching, fracture-flip) to the study of the cell surface, peripheral vesicles, endoplasmic reticulum-Golgi complex system, and of the encystation vesicles found in trophozoites and during the process of trophozoite-cyst transformation are discussed.

Animals↗

PubMed2002

Application of laser scanning cytometry followed by epifluorescent and differential interference contrast microscopy for the detection and enumeration of Cryptosporidium and Giardia in raw and potable waters.

AIMS: The main goal of this study was to validate a new laser scanning cytometry method (ChemScanRDI) that couples immunofluorescence detection with differential interference contrast (DIC) confirmation, against manual microscopic enumeration of Giardia and Cryptosporidium (oo)cysts. This study also assessed the basic performance of the new Association Française de Normalisation (AFNOR) NF T 90-455 method for Giardia and Cryptosporidium (oo)cyst enumeration with respect to (oo)cyst yield, linearity, repeatability, influence of turbidity and detection limit in raw and potable waters. METHODS AND RESULTS: The new standard method relies on cartridge (Envirocheck) filtration, immunomagnetic separation purification, immunofluorescence staining and detection followed by DIC confirmation. The recovery was 30-50% for both parasites at seeding levels from 30 to 230 (oo)cysts. The method is linear from 0 to around 400 seeded (oo)cysts and the yield does not significantly vary for turbidity levels from 10 to 40 Formazin Nephelometric Units (FNU). The results were obtained using manual microscopic enumeration of the (oo)cysts. The ChemScanRDI yielded counts that were at least equivalent to those obtained using manual microscopy for both parasites in raw and potable water concentrates, for seeding levels of 10-300 or 10-100, respectively. The purification and labelling method proposed by the supplier of theChemScanRDI (Chemunex) reached very similar recoveries to the AFNOR protocol (70-86% in both cases). CONCLUSIONS: Laser scanning cytometry can be used as a more standardized alternative to manual enumeration as part of the new AFNOR standard method. SIGNIFICANCE AND IMPACT OF THE STUDY: By using laser scanning cytometry instead of manual microscopy, laboratories could circumvent the limitations of manual microscopy, namely: low sample throughput, operator subjectivity and operator fatigue. The study further supports the drive to incorporate laser scanning cytometry in the standard methods for Giardia and Cryptosporidium enumeration.

Animals↗