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PubMed2000

Direct oral microscopy and its value in diagnosing mucosal lesions: a pilot study.

OBJECTIVE: Clinical examination of the oral mucosa often leads to an uncertain diagnosis, and a supplementary biopsy with a histopathologic examination of the lesion is necessary to establish a definite diagnosis. However, the site for the biopsy is a subjective choice that sometimes raises doubts about its representativeness. So far, no simple and reliable method is available for selecting the most appropriate area for biopsy. STUDY DESIGN: In a prospective study, we performed direct oral microscopy (oral application of the colposcopy technique used in gynecology) in 35 patients with various clinical diagnoses, such as leukoplakia, oral lichenoid lesions, or suspected malignancy. First, the oral mucosa was examined with direct microscopy, and the most representative site, according to colposcopic criteria, was selected. Then, the mucosa was clinically inspected by an independent examiner. The best site for biopsy according to clinical criteria was noted, and any difference in biopsy sites was recorded. Biopsy specimens were taken from 2 of these sites. RESULTS: Twenty-nine patients (83%) showed changes in the vascular picture on microscopy, according to the colposcopy criteria. In 14 patients (40%), the biopsy sites identified by direct oral microscopy showed more advanced histologic signs than those selected by routine clinical examination (0.01 < P </=.05). Four patients (11%) had more advanced histologic signs in the biopsy samples, as identified during routine clinical examination. In 17 patients (49%), we found no differences between the biopsy specimens. CONCLUSION: Direct oral microscopy of mucosal lesions seems to offer advantages in selecting more representative sites for biopsy than routine clinical examination alone.

Adult↗
PubMed2004

Nanometer-localized multiple single-molecule fluorescence microscopy.

Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale "rulers" to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate single-molecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated.

DNA↗
PubMedDate not supplied

Electron microscopy in neuromuscular disorders.

Electron microscopy has a strategic position in the diagnosis of neuromuscular disorders. In muscular fibers, the main abnormalities include vacuoles, inclusion bodies, and myofibrillar disorganization with or without abnormal inclusion material. Vacuolar changes include lipidic and glycogenic storage vacuoles, rimmed vacuoles, and lysosomal and autophagic vacuoles. Accumulation of abnormal inclusion material is found in nemaline myopathy, actinopathies, and hyaline body myopathy. Myofibrillar disorganization involves cores, multiminicores, and myosin chain depletion. Myofibrillar myopathies associate a pathologic pattern of myofibrillar dissolution and ectopic protein expression. They can be divided into two groups: myofibrillar myopathies with multiple expression proteins and myofibrillar myopathies with desmin and alphaB-crystallin expression only. In these two conditions, electron microscopy shows accumulation of a granulofilamentous material immunoreactive for desmin. At least three genes are implicated: desmin, alphaB-crystallin, and myotilin. Lastly, electron microscopy serves to identify changes, pathogenic or not, which are not shown up by light microscopy. Moreover, electron microscopy gives insight on pathophysiological mechanisms and can guide molecular genetics analysis.

Humans↗
PubMedDate not supplied

Electron microscopy of wet tissues: a case study in renal pathology.

In this report we introduce wet-tissue scanning electron microscopy, a novel technique for direct imaging of wet tissue samples using backscattered electrons. Samples placed in sealed capsules are imaged through a resilient, electron-transparent membrane. The contrast of the imaged samples may be enhanced by chemical staining. The samples several millimeters thick and imaged without sectioning, makes this technique suitable for rapid analysis of tissue specimens. We applied this technique to D-limonene-induced nephropathy where accumulation of hyaline protein droplets is induced in proximal and distal convoluted tubules of the kidney. Images obtained by scanning electron microscopy of hydrated kidney specimens exhibited superior resolution, contrast, and magnification compared with those obtained by conventional light microscopy of paraffin sections. The electron micrographs can be obtained within an hour of tissue removal, whereas preparation for light microscopy requires at least 1 day. These advantages of the wet scanning electron microscopy technique indicate its potential utility in a wide range of applications in histopathology and toxicology.

Animals↗
PubMed2006

Accessible microscopy workstation for students and scientists with mobility impairments.

An integrated accessible microscopy workstation was designed and developed to allow persons with mobility impairments to control all aspects of light microscopy with minimal human assistance. This system, named AccessScope, is capable of performing brightfield and fluorescence microscopy, image analysis, and tissue morphometry requisite for undergraduate science courses to graduate-level research. An accessible microscope is necessary for students and scientists with mobility impairments to be able to use a microscope independently to better understand microscopical imaging concepts and cell biology. This knowledge is not always apparent by simply viewing a catalog of histological images. The ability to operate a microscope independently eliminates the need to hire an assistant or rely on a classmate and permits one to take practical laboratory examinations by oneself. Independent microscope handling is also crucial for graduate students and scientists with disabilities to perform scientific research. By making a personal computer as the user interface for controlling AccessScope functions, different upper limb mobility impairments could be accommodated by using various computer input devices and assistive technology software. Participants with a range of upper limb mobility impairments evaluated the prototype microscopy workstation. They were able to control all microscopy functions including loading different slides without assistance.

Adult↗
PubMed1975

New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy.

New immunochemical reagents consisting of antibodies bound to small latex spheres were used as visual markers for the detection and localization of cell surface antigens by scanning electron microscopy. Cross-linked latex spheres of various sizes from 300 to 3,4000 A in diameter were synthesized by aqueous emulsion copolymerization of methacrylate derivatives containing hydroxyl and carboxyl functional groups. Proteins and other molecules containing primary amino groups were covalently bonded to the acrylic spheres under a variety of mild conditions by the aqueous carbodiimide, cyanogen bromide, and glutaraldehyde methods. For use in the indirect immunochemical-labeling technique, goat antibodies directed against rabbit immunoglobulins were bonded to the spheres. These immunolatex reagents were shown to bind only to cells (red blood and lymphocytes) which had previously been sensitized with rabbit antibodies against cell surface antigens. Mouse spleen lymphocytes with exposed immunoglobulins on their surface (B cells) were labeled with these spheres and distinguished from unlabeled or T lymphocytes by scanning electron microscopy. The distribution of Ig receptors on lymphocytes was also studied using the spheres as visual markers. When lymphocytes were fixed with glutaraldehyde and subsequently labeled with the immunolatex reagents, a random distribution was observed by scanning electron microscopy; a patchy distribution was observed when unfixed lymphocytes were used. These results are consistent with studies using ferritin-labeled antibodies (S. De Petris and M. Raff. 1973. Nature [Lond.]. 241:257.) and support the view that Ig receptors on lymphocytes undergo translational diffusion. In addition to serving as visual markers for scanning electron microscopy, these latex spheres tagged with fluorescent or radioactive molecules have applications as highly sensitive markers for fluorescent microscopy and as reagents for quantitative studies of cell surface antigens and other receptors.

Animals↗
PubMed2004

Smooth-muscle regeneration after electrosurgical endopyelotomy in a porcine model as confirmed by electron microscopy.

BACKGROUND AND PURPOSE: Endopyelotomy is the preferred treatment for ureteropelvic junction (UPJ) obstruction because of its short operating time, limited morbidity, fast recovery, and reasonable efficacy. We used tissue and immunohistochemistry staining and electron microscopy to look at the muscle regeneration following an endopyelotomy incision in a porcine model. MATERIALS AND METHODS: Bilateral electrosurgical endopyelotomy was performed in six domestic pigs with placement of 7F 20-cm Percuflex double-J stents for up to 4 weeks, and urinary tracts were harvested at 3 or 5 months. Specimen evaluation included tissue staining with hematoxylin-eosin, Masson's trichrome, and Verhoeff's iodine and Van Gieson solution; histochemical staining for smooth-muscle actin, desmin and myosin staining, and electron microscopy. Each specimen was assigned a "healing" score of 0 (normal) 1 (slight changes), 2 (mild changes), or 3 (severe changes). The fibrosis score was based on six factors: muscle layer fibrosis, lamina propria fibrosis, amount of granulation tissue present, new deposits of collagen, fibrosis in the periureteral fat, and presence of myofibroblasts. The muscles were characterized with immunohistochemistry and electron microscopy. RESULTS: At both 3 and 5 months, the urothelium was healed, and the lamina propria was healed with focal loss. By 3 months, smooth-muscle bundles bridged the defect, and by 5 months, the whole defect was covered. Smooth muscle cells were evident by electron microscopy by 3 months, and actin and myosin could be detected by immunohistochemistry. Desmin-positive cells accounted for 50% of the population at 3 months and 40% at 5 months. The regenerated smooth-muscle bundles were oriented in different directions and intermingled with fibrous tissue. They could be distinguished easily from normal ureter under the microscope. CONCLUSION: Verifiable, functional smooth-muscle bundles bridge the endopyelotomy defect by 3 months, as confirmed by immunohistochemistry staining and electron microscopy.

Animals↗
PubMed1997

Confocal microscopy as a tool for the investigation of the anterior part of the eye.

In recent years, confocal microscopy has become a powerful tool for examining microscopic structures in the living eye. The decisive advantage of this technique is that it permits the investigation of optical sections of relatively thick (> 10 microns) specimens. Because confocal microscopy suppresses the out-of-focus blur, sharp three-dimensional images with excellent resolution can be obtained. Confocal microscopy is therefore able to provide more information than the classic methods--i.e., specular microscopy and slit-lamp biomicroscopy. This paper reviews recent applications of confocal microscopy in three fields of ophthalmology: the observation of the anatomy of the anterior parts of the eye, the investigation of these structures after local administration of drugs and, finally, the use of this technique for the diagnosis of infectious ocular diseases.

Animals↗
PubMed1982

Desmoplastic malignant melanoma: a study by conventional and electron microscopy.

A 61-year-old white man presented himself with a mass that had recurred on the chin. Conventional microscopy of sections from this mass showed atypical spindle cells surrounded by abundant collagen and resembled fibrosarcoma. Stains for melanin revealed pigment in the cytoplasms of the atypical spindle cells. Study of sections by electron microscopy demonstrated round, oval, and spindle-shaped cells having desmosomes and containing abundant melanosomes in varying stages of maturation. On the basis of both conventional microscopy and electron microscopy, the diagnosis was desmoplastic malignant melanoma. An unusual finding by electron microscopy was the presence of tubuloreticular intracytoplasmic inclusions in the neoplastic melanocytes.

Chin↗
PubMed1998

In vivo confocal microscopy of Fuchs' endothelial dystrophy.

PURPOSE: The purpose of this study is to analyze in vivo confocal microscopic findings of corneas with Fuchs' endothelial dystrophy. METHODS: Central corneas of 17 eyes of 11 patients aged 41-86 years were examined using in vivo scanning slit confocal microscopy after being diagnosed with Fuchs' endothelial dystrophy. The cellular structure of the corneas was analyzed morphologically and quantitatively and compared to control results from 22 healthy corneas. RESULTS: Bullae were detected in the basal epithelial layer of one eye. Eight of 17 eyes (47%) exhibited an abnormal Bowman's layer: diffuse bright reflection and absence of nerves. Eleven eyes (65%) exhibited abnormal anterior stroma: lacunae and diffuse increased light reflection due to edema. In 12 eyes (71%), lacunae or dark bands 5-20 microm wide against increased background reflection were noted in the posterior stroma. Descemet's membrane was thickened in all eyes. Dark bands were detected in six eyes (35%). Guttae (137-1,231/mm2) 20-40 microm in diameter were found in every endothelial cell layer. The mean endothelial cell count was 1,202 +/- 850 (cells/mm2 +/- SD; range, 0-2,735). There was a positive correlation between endothelial cell counts obtained by specular microscopy and those obtained by confocal microscopy (r = 0.95). CONCLUSION: In vivo confocal microscopic findings of Fuchs' endothelial dystrophy are described for the first time in a series of cases. Pathological changes in Fuchs' dystrophy were detected in all corneal layers, more frequently in the posterior layers. Endothelial cell counts obtained with confocal microscopy were statistically similar to those obtained with standard specular microscopy.

Adult↗
PubMed1997

Role of electron microscopy in interstitial lung disease.

Although electron microscopy no longer enjoys the important role in the diagnosis of interstitial lung diseases that it had in the 1960s and 1970s, it remains an important adjunct in the differential diagnosis of certain pulmonary diseases. Examples include various manifestations of systemic lupus erythematous pneumonitis, in which the presence of tubuloreticular structures and electron-dense deposits are useful for diagnosis; immotile cilia disorders, in which qualitative and now quantitative studies of the cilia of respiratory epithelial cells can help to establish the diagnosis; infections by viruses and other subcellular microorganisms as shown by the role played by electron microscopy in the initial diagnosis of the Hantavirus pulmonary syndrome; pneumoconioses, in which, in conjunction with elemental analysis probes, scanning electron microscopy is of critical importance in establishing the presence of offending foreign compounds in lung tissue or fluids; pulmonary fibrilloses, such as amyloidosis, light chain disease, and fibrillary glomerulonephritis, affecting the lung; and cases of alveolar proteinosis or Langerhans cell granulomatosis diagnosed from fluids such as bronchoalveolar lavages or small tissue samples. As important, electron microscopy remains of enormous usefulness in the study of early structural events leading to the pathogenesis of diseases. For example, recent uses of the technique have focused on the alveolar-capillary wall damage induced by alveolitis in hypersensitivity pneumonitis and sarcoidosis. In summary, electron microscopy remains a useful method in the study and diagnosis of some interstitial lung diseases, but because of its expense it is incumbent on the clinician to use good judgment in the selection of cases and diseases for study by this method.

Diagnosis, Differential↗
PubMed2005

Amiodarone keratopathy: an in vivo confocal microscopy study.

PURPOSE: To evaluate the characteristics and evolution of corneal morphologic changes induced by systemic amiodarone treatment by using in vivo slit scanning confocal microscopy in an attempt to understand the pathogenesis of corneal and other systemic side effects of this drug. METHODS: Forty-nine eyes of 25 consecutive subjects receiving amiodarone therapy (group A) and 26 eyes of 13 age- and sex-matched healthy control subjects (group B) were enrolled in the study. RESULTS: In group A, the mean dosage of amiodarone was 224.0 +/- 71.3 mg, and the mean duration of treatment was 21.3 +/- 20.9 months. In eight (8 of 49,16.3) eyes of four patients, no deposition was observed, neither by slitlamp nor by confocal microscopy. The deposition could be detected as early as 2 months after the onset of treatment in 41 (41 of 49, 83.7%) eyes, by slitlamp and confocal microscopy. Deposition was significantly correlated with the duration of treatment and, therefore, the cumulative dose of the drug ingested. Deposits were observed as bright, hyper-reflective spots that were localized intracellularly and were present at the level of basal lamina of all 41 (100%) corneas in which deposition could be observed at confocal microscopy. Deposits were also observed in the superficial epithelium, anterior stroma, mid stroma, and subepithelial nerves in eyes with grade 2 to 4 keratopathy. Additionally, morphologic abnormalities were observed in anterior stromal keratocytes, subepithelial and stromal nerves, and endothelium. The mean anterior and posterior keratocyte densities were statistically significantly higher than those in group B. CONCLUSIONS: In addition to showing drug deposition does not seem to occur before 2 months of treatment and does not seem to be correlated with tear function. Although the clinical significance of morphologic changes induced by the drug is known and although deposition could not be detected before it was evident with slitlamp biomicroscopy, with some improvement in instrumentation, the authors believe that confocal microscopy will also prove to be useful in early diagnosis and in understanding the pathophysiology of amiodarone keratopathy. This may help grow insight to the mechanism of other, sometimes lethal, systemic side effects of this drug.

Adult↗
PubMed2006

In vivo confocal microscopy findings in keratoconus.

PURPOSE: : To evaluate corneal structure in eyes with keratoconus by using in vivo confocal microscopy. METHODS: : In a prospective institutional study, 48 eyes of 24 consecutive patients with keratoconus were examined by in vivo confocal microscopy. Forty-four eyes of 22 healthy subjects served as the control. The main outcome measures were qualitative and quantitative changes encountered at confocal microscopy examination. RESULTS: : In the keratoconus group, in vivo confocal microscopy findings included elongated, exfoliating superficial epithelial cells; brightly reflective material deposition within the basal epithelial cells; prominent, thickened subbasal nerves; structural changes in subbasal nerve fibers; pronounced reflectivity and irregular arrangement of stromal keratocytes; structurally abnormal anterior stromal keratocyte nuclei; folds in the anterior, mid, and posterior stroma; folds in Descemet's membrane; pleomorphism and enlargement of endothelial cells; and endothelial guttata. No such abnormal findings were present in the control group. There were significant differences between the two groups in terms of the mean basal epithelial, anterior and posterior stromal keratocyte densities, the mean basal epithelial cell area, and the mean endothelial cell hexagonality percentages. CONCLUSIONS: : Quantitative and qualitative structural changes were observed in all corneal layers in eyes with keratoconus by using slit-scanning confocal microscopy. The changes at all levels were more prominent in eyes with severe keratoconus. This noninvasive in vivo technique may prove to be useful in the diagnosis and follow-up of keratoconus and in the understanding of its pathophysiology.

Adolescent↗
PubMed2006

Evaluation of intrastromal lipid deposits after intacs implantation using in vivo confocal microscopy.

PURPOSE: To assess the structure and location of intrastromal lipid deposits after implantation of Intacs by using in vivo confocal microscopy. METHODS: Seven eyes of six patients were examined by in vivo confocal microscopy 5 years (n = 6) or 2 months (n = 1) after uncomplicated implantation of Intacs for the correction of mild myopia. Selected images from all corneal layers were qualitatively evaluated for structural changes, with special attention paid to areas surrounding the Intacs implants. RESULTS: In the peripheral cornea of eyes examined 5 years after surgery, epithelial and endothelial cell layers appeared normal. Tandem scanning confocal microscopy showed stromal haze surrounding the implants in all eyes examined, but no keratocyte activation was seen. Reflective amorphous or crystalline structures consistent with lipid deposition were detected in all eyes with long-term implantation of Intacs. Deposits were localized to the inner and outer edges of Intacs segments and to the region anterior to the implant. Confocal microscopy did not show any deposits in the eye examined 2 months after surgery, although the region anterior to the implant appeared hazy and edematous. Areas central to the implant appeared normal in all eyes. CONCLUSIONS: The mechanical and physiologic stresses introduced by the implantation of Intacs lead to the accumulation of lipid deposits in the extracellular matrix. By using in vivo confocal microscopy, the location and structure of these deposits can be determined.

Adult↗
PubMed2006

Clinical and in vivo confocal microscopy findings in patients receiving tamoxifen citrate.

PURPOSE: : To describe the deposition rate of tamoxifen in the cornea and observe its impact on the cornea with confocal microscopy. METHODS: : Forty-four eyes of 22 women receiving tamoxifen at a dosage of 20 mg/day for at least 6 months for the adjuvant treatment of breast cancer and 30 eyes of 15 healthy age-matched women were examined for corneal drug deposition by slitlamp after pupil dilation. Ultrasound pachymetry, specular microscopy, in vivo confocal microscopy, and Schirmer tear test were also performed in all patients. RESULTS: : The mean duration of tamoxifen intake was 21.6 +/- 7.9 months (range, 13-44 months), and the mean cumulative dose was 12.9 +/- 4.7 g (range, 7.8-26.4 g). Drug depositions in the inferior paracentral cornea were identified in 32 (72%) eyes at pupil-dilated slitlamp examination. There were no significant differences between eyes with keratopathy, those without keratopathy, and control eyes in regard to the mean Schirmer test scores, mean central corneal thickness, mean endothelial cell count, mean basal epithelium cell density, mean anterior and posterior stromal keratocyte density, and mean endothelial cell density (P > 0.05). No pathologic alteration of structure was observed with in vivo confocal microscopy at any corneal level in patients receiving tamoxifen. CONCLUSIONS: : Low-dose tamoxifen use was associated with corneal depositions in 72% of patients. Slitlamp examination performed with pupil dilation was helpful in detection of subtle tamoxifen-related deposits. Amelioration of in vivo confocal microscopy systems may be helpful in accurate imaging of the paracentral and peripheral cornea.

Adult↗
PubMed2005

In vivo confocal microscopy of posterior polymorphous dystrophy.

PURPOSE: This study was designed to delineate the morphologic features of posterior polymorphous dystrophy (PPD) using in vivo confocal microscopy. METHODS: Six patients with clinically diagnosed PPD were examined by slit-lamp biomicroscopy, Orbscan II slit-scanning elevation topography, and in vivo confocal microscopy. RESULTS: Endothelial cell densities ranged from 613 to 3,405 cells/mm and endothelial polymegathism was noted in all cases, whereas endothelial pleomorphism was not a prominent feature. Three cases exhibited bright endothelial nuclei. A variety of abnormal curvilinear and vesicular abnormalities were imaged by in vivo confocal microscopy, with lesions ranging between 6 and 159 microm in diameter. Abnormal endothelial cells were visible within some of these lesions. Six cases showed hyperreflectivity at the level of Descemet's membrane around the lesions. Deep stromal keratocytes appeared to aggregate around, or were compressed by, the endothelial lesions in one case. CONCLUSIONS: We report the largest case series of PPD imaged by in vivo confocal microscopy. The ability of in vivo confocal microscopy to assess the living cornea over time enables monitoring of disease progression and thus the potential to identify and correlate development of, or changes in, microstructural features. As more data become available, these analyses may enable the formulation of prognostic and diagnostic criteria.

Adult↗
PubMed2005

Imaging the microstructural abnormalities of meesmann corneal dystrophy by in vivo confocal microscopy.

PURPOSE: To delineate the microstructural features of Meesmann corneal dystrophy using in vivo confocal microscopy. METHOD: Three subjects with clinically diagnosed Meesmann corneal dystrophy were examined by slit-lamp biomicroscopy and slit-scanning in vivo confocal microscopy. RESULTS: On slit-lamp biomicroscopy, all subjects demonstrated large bilateral multiple epithelial cystic lesions extending to the midperiphery. On in vivo confocal microscopy, these lesions appeared as hyporeflective areas in the basal epithelial layer. The majority were circular, oval or teardrop shaped and ranged between 48 mum and 145 mum in diameter. Large elongated intraepithelial clefts were also seen. Reflective spots were visible within most of the lesions and these may represent the fibrillogranular material (termed peculiar substance) and tonofilament bundles observed in electron microscopy studies. An additional finding was the fragmented appearance of the subbasal nerve plexus. CONCLUSION: We present the first case series of Meesmann corneal dystrophy imaged by in vivo confocal microscopy and describe the associated microstructural features. Delineation of these features facilitates the use of the confocal microscope to aid diagnosis and management of corneal dystrophies.

Corneal Dystrophies, Hereditary↗
PubMed2006

Diagnosis of epithelial ingrowth after penetrating keratoplasty with confocal microscopy.

PURPOSE: To report confocal microscopy use in the clinical diagnosis of epithelial ingrowth after penetrating keratoplasty (PKP). METHODS: A 36-year-old female patient with keratoconus developed a well-delimited posterior hazy membrane covering the inferior two thirds of the cornea 3 months after an uneventful PKP. A posterior corneal line was present resembling an endothelial graft rejection line, but with no keratic precipitates or corneal edema. Ocular hypertension was not observed. Confocal microscopy was performed to elucidate the diagnosis. RESULTS: Confocal microscopy showed epithelium and stroma with normal findings. Two distinct cellular types were presented at the endothelium layer. Enlarged endothelial cells were observed in the superior part of the cornea up to the leading edge of the hazy membrane. In the middle and inferior part of the graft, the cells were larger, with polygonal shape and easily recognizable hyperreflective nuclei, suggestive of epithelial cells. With these confocal microscopy findings, the patient was promptly submitted to another PKP. Histologic analysis confirmed the diagnosis of epithelial ingrowth. CONCLUSION: Confocal microscopy imaging technique seems to be a useful tool in the early diagnosis of epithelial ingrowth after PKP.

Adult↗