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PubMed1998

Clinical confocal microscopy.

Because it provides much higher magnification and better optical sectioning than a slit-lamp biomicroscope, confocal microscopy is ideally suited for clinical imaging of the cornea. One important clinical application of confocal microscopy has been the early detection and diagnosis of a number of infectious conditions, including infection with Acanthamoeba and microsporidium species, fungal keratitis, and contact lens-associated bacterial keratitis. Confocal microscopy has also been used for temporal evaluation of corneal wound healing following refractive surgery and penetrating keratoplasty. With the development of the new technique of quantitative confocal microscopy through-focusing, confocal microscopy can be used to measure epithelial, stromal, and corneal thickness accurately and reproducibly in human patients. Furthermore, conofocal microscopy through-focusing can be used to determine the initial photoablation depth, changes in epithelial, stromal, and corneal thickness, and subepithelial haze following photorefractive keratectomy.

Cornea↗
PubMed2005

Features of the iridocorneal endothelial syndrome on confocal microscopy.

PURPOSE: To present a subtle case of iridocorneal endothelial (ICE) syndrome and discuss the utility of in vivo confocal microscopy in the evaluation of this disorder. Previous reports of the confocal microscopic features of ICE syndrome are reviewed. METHODS: A 32-year-old man presented with decreased vision and halos. Slit-lamp biomicroscopy and in vivo confocal microscopy were used to evaluate the etiology of his complaints. Clinical photographs and confocal micrographs were analyzed. RESULTS: In vivo confocal microscopy revealed marked asymmetry between the right and left corneal endothelial layers with pleomorphic epithelioid cells on the right, some with hyperreflective nuclei. A transition between cells with uniform appearance and dark nuclei and a highly irregular cellular arrangement with hyperreflective nuclei was present. CONCLUSIONS: This report supports the clinical utility of in vivo confocal microscopy in the evaluation of ICE syndrome. It demonstrates that the histopathologic features of ICE syndrome on scanning electron microscopy may be shown clinically using high-resolution confocal microscopy.

Adult↗
PubMed2005

Confocal microscopy used as the definitive, early diagnostic method in Chandler syndrome.

PURPOSE: To report the early, rapid diagnosis of the Chandler variant of the iridocorneal endothelial (ICE) syndrome using confocal light microscopy. METHODS: A 62-year-old man with a long history of unilateral glaucoma reported progressively blurred vision in his right eye. Examination of both eyes included visual acuity, slit-lamp examination, pneumotonometry, visual field, gonioscopy, and confocal microscopy. RESULTS: On examination, visual acuity was 20/80 and 20/20 and the IOPs were 26 and 12. The anterior segment OD revealed 1+ inferior and axial corneal edema, while the OS was normal to biomicroscopy and posterior segment. Chandler syndrome or Fuchs endothelial dystrophy was suspected. In the affected eye, confocal light microscopy clearly showed an "epithelium-like" transformation of the corneal endothelium with irregularly shaped cells and hyperreflective nuclei, establishing the diagnosis of Chandler syndrome. CONCLUSIONS: In the presence of corneal edema or haze, corneal endothelium can be clearly visualized by confocal microscopy. "Epithelium-like" endothelial cells with highly reflective nuclei characteristic of Chandler syndrome were easily identified by confocal light microscopy to establish the diagnosis, despite the presence of corneal edema. Thus, confocal microscopy is a sensitive tool for the rapid, early diagnosis of ICE syndrome and may help distinguish among its variants.

Corneal Diseases↗
PubMed2005

Central corneal thickness measurements using Orbscan II scanning slit topography, noncontact specular microscopy, and ultrasonic pachymetry in eyes with keratoconus.

PURPOSE: To compare corneal thickness measurements using Orbscan II scanning slit topography, Topcon SP-2000P noncontact specular microscopy, and ultrasonic pachymetry in eyes with keratoconus. METHODS: Central corneal thickness was measured in 22 eyes with keratoconus. Eyes with apparent corneal opacity were excluded. Scanning slit topography, noncontact specular microscopy, and ultrasonic pachymetry were used in this sequence. The acoustic equivalent correlation factor (0.92) was used for Orbscan readings. RESULTS: Three devices gave significantly different corneal thickness readings (P < 0.001, repeated-measure analysis of variance). Measurements with Orbscan scanning slit topography (449.5 +/- 43.2 [SD] mum) were significantly smaller than those of ultrasonic pachymetry (485.0 +/- 29.3 microm; P < 0.001, Tukey multiple comparison) and SP-2000P noncontact specular microscopy (476.7 +/- 28.3 microm; P = 0.002). There were significant linear correlations between ultrasonic pachymetry and scanning slit topography (Pearson correlation coefficient r = 0.741, P < 0.001), between scanning slit topography and noncontact specular microscopy (r = 0.880, P < 0.001), and between noncontact specular microscopy and ultrasonic pachymetry (r = 0.811, P < 0.001). CONCLUSION: In eyes with keratoconus, Orbscan II scanning slit topography system gave significantly smaller corneal thickness readings than the other 2 devices. Measurements taken by noncontact specular microscopy and ultrasonic pachymetry were comparable. Three devices showed significant linear correlations with one another.

Adult↗
PubMed2005

Reflectance-mode confocal microscopy for the in vivo characterization of pagetoid melanocytosis in melanomas and nevi.

Pagetoid infiltration of the epidermis by melanocytes is a relevant criterion for the histologic diagnosis of melanoma, although sporadically observable in benign lesions. Since in vivo reflectance-mode confocal microscopy enables the visualization of superficial layers at cellular-level resolution, the different aspects and the diagnostic significance of epidermal alterations and pagetoid cell infiltration were investigated on 84 benign and malignant melanocytic lesions by confocal microscopy and compared with histopathology. The observation of a disarranged pattern in superficial layers appeared characteristic for malignant lesions. In vivo identification of pagetoid cells, clearly present in the majority of melanomas and in a few benign lesions, seemed useful for melanoma diagnosis. An excellent concordance between confocal microscopy and histopathology was achieved. Moreover, identification of some characteristic features by confocal microscopy, such as large and numerous closely arranged cells extended to the stratum corneum, was strongly correlated with malignancy. In conclusion, confocal microscopy enabled a very good identification of melanocytes spreading upward in a pagetoid fashion in melanocytic lesions. Thus, when pagetoid melanocytosis is observable by means of confocal microscopy, melanoma diagnosis should be considered, whereas it cannot be excluded in the absence of pagetoid cells, lacking in at least 10% of malignant lesions.

Humans↗
PubMed2004

5% imiquimod cream and reflectance-mode confocal microscopy as adjunct modalities to Mohs micrographic surgery for treatment of basal cell carcinoma.

BACKGROUND: Imiquimod is an immune response modifier that up-regulates cytokines and has been shown in clinical studies to reduce or clear basal cell carcinoma tumors when applied topically. OBJECTIVE: The objectives were to evaluate the efficacy of 5% imiquimod cream in treating basal cell carcinoma preceding excision by Mohs micrographic surgery and to determine if reflectance-mode confocal microscopy is useful to establish the need for surgical intervention after imiquimod treatment. METHODS: Subjects applied study cream to one biopsy-confirmed basal cell carcinoma tumor 5 x/week for 2, 4, or 6 weeks in this vehicle-controlled, double-blind study. Confocal microscopy was used for the 6-week treatment group to examine the target tumor area at each interval visit and immediately before Mohs micrographic surgery. After the Mohs micrographic surgery excision, the tissue was evaluated histologically, and the excision area was measured. Confocal microscopy readings were correlated to the histologic diagnosis. RESULTS: Tumors cleared or the target tumor area was reduced in subjects in the 4- and 6-week dosing regimens. Confocal microscopy assessments correlated well with the histologic diagnosis. CONCLUSION: Imiquimod improved excision results relative to vehicle when used for treating basal cell carcinoma before Mohs micrographic surgery. Confocal microscopy assessments correlated well with tumor response to therapy, suggesting that confocal microscopy may help determine the need for surgery.

Administration, Cutaneous↗
PubMed1992

The use of post-embedding immunoelectron microscopy in the diagnosis of glomerular diseases. Comparison of immunoelectron microscopic and immunofluorescence studies.

Fifty renal biopsies were studied by immunoelectron microscopy after embedding in a partly hydrophilic polyacrylic resin (LR White). Immunofluorescence studies were carried out on frozen sections of parallel tissue samples. Polyacrylic embedding gave good preservation of the renal ultrastructure and precise localization of immunoglobulin and C3c antibodies within glomerular electron-dense deposits. Non-specific staining of plasma proteins within vascular lumina could easily be detected. There was good correlation between immunoelectron and immunofluorescence microscopy. Immunoelectron microscopy is a very sensitive method, which can detect small amounts of antigen. More cases were, however, positive by immunofluorescence than by immunoelectron microscopy. This discrepancy may be explained by difference in sample size, and by difference in resolution of morphological details (electron microscopy versus fluorescence microscopy).

Acrylic Resins↗
PubMed1998

Comparison of PCR and microscopy for detection of Cryptosporidium parvum in human fecal specimens: clinical trial.

PCR technology offers alternatives to conventional diagnosis of Cryptosporidium for both clinical and environmental samples. We compared microscopic examination by a conventional acid-fast staining procedure with a recently developed PCR test that can not only detect Cryptosporidium but is also able to differentiate between what appear to be host-adapted genotypes of the parasite. Examinations were performed on 511 stool specimens referred for screening on the basis of diarrhea. PCR detected a total of 36 positives out of the 511 samples, while routine microscopy detected 29 positives. Additional positives detected by PCR were eventually confirmed to be positive by microscopy. A total of five samples that were positive by routine microscopy at Western Diagnostic Pathology but negative by PCR and by microscopy in our laboratory were treated as false positives. Microscopy therefore exhibited 83.7% sensitivity and 98.9% specificity compared to PCR. PCR was more sensitive and easier to interpret but required more hands-on time to perform and was more expensive than microscopy. PCR, however, was very adaptable to batch analysis, reducing the costs considerably. Bulk buying of reagents and modifications to the procedure would decrease the cost of the PCR test even more. An important advantage of the PCR test, its ability to directly differentiate between different Cryptosporidium genotypes, will assist in determining the source of cryptosporidial outbreaks. Sensitivity, specificity, ability to genotype, ease of use, and adaptability to batch testing make PCR a useful tool for future diagnosis and studies on the molecular epidemiology of Cryptosporidium infections.

Animals↗
PubMed2005

Utility of universal sample processing methodology, combining smear microscopy, culture, and PCR, for diagnosis of pulmonary tuberculosis.

The universal sample processing (USP) multipurpose methodology was developed for the diagnosis of tuberculosis (TB) and other mycobacterial diseases by using smear microscopy, culture, and PCR (S. Chakravorty and J. S. Tyagi, J. Clin. Microbiol. 43:2697-2702, 2005). Its performance was evaluated in a blinded study of 571 sputa and compared with that of the direct and N-acetyl L-cysteine (NALC)-NaOH methods of smear microscopy and culture. With culture used as the gold standard, USP smear microscopy demonstrated a sensitivity and specificity of 98.2% and 91.4%, respectively, compared to 68.6% and 92.6%, respectively, for the direct method. For a subset of 325 specimens, the USP method recorded a 97.1% sensitivity and 83.2% specificity compared to the NALC-NaOH method, which had a sensitivity and specificity of 80.0% and 89.7%, respectively, with culture used as the gold standard. Thus, the USP method exhibited a highly significant enhancement in sensitivity (P < 0.0001) compared to the direct and NALC-NaOH methods of smear microscopy. The USP culture sensitivity was 50.1% and was not significantly different from that of conventional methods (53.6%). The sensitivity and specificity of IS6110 PCR were 99.1% and 71.2%, respectively, with culture used as the gold standard, and increased to 99.7% and 78.8%, respectively, when compared with USP smear microscopy. Thus, the USP methodology was highly efficacious in diagnosing TB by smear microscopy, culture, and PCR in a clinical setting.

Acetylcysteine↗
PubMed1999

A rationalised virological electron microscope specimen testing policy. PHLS North West Viral Gastroenteritis and Electron Microscopy Subcommittee.

The aim of this project was to produce guidance for a rationalised virological electron microscopy specimen testing policy for PHLS North West, to facilitate centralisation of a groupwide diagnostic electron microscopy service on a single site. Careful specimen selection to limit numbers and the groupwide use of commercially available enzyme immunoassays has allowed PHLS North West to reduce the number of specimens prepared for electron microscopy. The rationalised virological electron microscopy specimen testing policy has enabled a diagnostic electron microscopy service to be provided from a single site with a manageable workload. Implementation of this specimen testing policy by PHLS North West has been successful and may be applicable to other laboratories (or groups of laboratories) to maximise the use of expensive electron microscopy facilities.

Aged↗
PubMed2001

Electron microscopy of myocardial tissue. A nine year review.

AIM: To review and reassess the role of this department's experience with routine electron microscopy of myocardial tissues. METHODS: A nine year series of myocardial samples that underwent electron microscopy analysis were audited. Fifty nine samples were derived from 46 male and 13 female subjects with an age range of 15-90 years (mean, 50.6). Forty two samples were endomyocardial specimens, with 13 being derived from explanted hearts, and four from necropsies. Two cases were from transplanted hearts. These were all reviewed in a blinded fashion, by all three authors separately, in terms of the myocardium at the ultrastructural level. Subsequently, the interpretations/diagnoses were cross compared with the light microscopy and clinical data results. [figure: see text] RESULTS: Four cases of amyloid were identified; in addition, one case of granulomatous inflammation and one case of basophilic degeneration were seen, although all these had been evident on light microscopy. One case of possible mitochondrial myopathy was found. A total of 18 cases revealed changes of a presumed non-specific type including glycogen, lipid, and mitochondrial accumulations. Varying types of degeneration involving myofibres were seen together with variations in interstitial fibrosis and occasional cytoplasmic inclusions. CONCLUSION: Overall, although interesting, the electron microscopy of myocardial tissue added little to the understanding of the patient's disease, with only one case showing changes not found at light microscopy or with other investigations. Further study might shed light on the "non-specific" ultrastructural findings encountered.

Adolescent↗
PubMed1986

Asbestos fibres in bronchoalveolar lavage fluid from asbestos workers: examination by electron microscopy.

The uncoated and coated fibre load in bronchoalveolar lavage (BAL) fluid was assessed using light microscopy, scanning electron microscopy, and x ray microanalysis in 15 subjects with previous, unprotected exposure to asbestos, including three with clinical and radiological evidence of asbestosis, and in 13 urban dwelling control subjects with no known occupational exposure to asbestos. The mean ferruginous body count per ml BAL fluid in asbestos exposed subjects as determined by light microscopy was 52 (range 0-333). No ferruginous bodies were detected in control subjects. The mean fibre count per ml BAL fluid in asbestos exposed subjects as determined by electron microscopy was 793 (133-3700), significantly greater than 239 (44-544) in controls (p less than 0.05). Electron microscopic counts correlated with duration of previous exposure to asbestos (r = 0.47, p less than 0.05) and with percentage neutrophil counts (r = 0.53, p less than 0.025). There was no relation between electron microscopic fibre counts and light microscopic ferruginous body counts. In 11 asbestos exposed cases x ray microanalysis confirmed the presence of asbestos and in six the asbestos fibre type was clearly identified. Of five subjects showing no asbestos bodies by light microscopy, all showed fibres by electron microscopy, and in three cases the presence of asbestos was confirmed by microanalysis. Among control subjects, fibres were either large organic fibres or smaller particles which microanalysis showed were not asbestos. In only one control case were a few fibres identified which were confirmed as asbestos fibres on microanalysis. Electron microscopic examination of BAL fluid may confirm past exposure to asbestos and probably gives a crude quantitative estimate of asbestos load.

Adult↗
PubMed1988

The Bacteroides glycocalyx as visualized by differential interference contrast microscopy.

The glycocalyx of eight strains representing six species of Bacteroides was examined by differential interference contrast microscopy. Wet mounts in India ink were prepared from bacteria cultured in broth and on an agar medium; the wet mounts were observed by phase-contrast microscopy and differential interference contrast microscopy. With differential interference contrast microscopy, all bacteria demonstrated a glycocalyx, which included capsules surrounding single cells and microcolonies, strands of glycocalyx connecting cells and microcolonies, detached slime, and solid masses of glycocalyx in which innumerable bacteria were enmeshed. Bacteria showed comparable amounts of glycocalyx by visual observation with differential interference contrast microscopy whether grown on plates or in broth. Serial transfers of cultures did not diminish the amount of glycocalyx. Differential interference contrast microscopy proved to be a superior method to phase contrast for examining wet preparations of Bacteroides.

Bacteroides↗
PubMedDate not supplied

Roughness of excimer laser ablated corneas with and without smoothing measured with atomic force microscopy.

PURPOSE: To analyze the surface roughness of porcine corneas after excimer laser ablation with and without the smoothing procedure by means of atomic force microscopy. METHODS: Excimer laser photorefractive keratectomy (PRK) was performed on eight porcine corneas. Immediately following the procedure, smoothing was performed on four corneas using a viscous solution of 0.25% sodium hyaluronate. The corneas were examined in balanced salt solution after fixation in 2.5% glutaraldehyde solution using atomic force microscopy. Quantitative analysis of the ablated stromal surface topography was performed using the section analysis module of the atomic force microscopy software. Repeated measurements were made over small areas (< or =50 microm2) near the center of each ablation, with a vertical resolution of <1 nm. RESULTS: Images of the ablated stromal surface showed undulations and granule-like features on the ablated surface of the specimens. The specimens on which the smoothing procedure was performed (root-mean-square [RMS] rough: 0.152 +/- 0.014 microm) were more regular (P < .001) than those on which PRK alone was performed (RMS rough: 0.229 +/- 0.018 microm). CONCLUSIONS: Atomic force microscopy analysis requires a simpler preparation of the specimens with respect to that necessary for scanning electron microscopy; for this reason, atomic force microscopy techniques are more reliable for the study of biological surfaces and prove to be a feasible method to establish the differences when comparing different laser techniques. Our investigations highlight that although the laser cut of scanning-spot excimer laser systems is precise in removing even the smallest amounts of tissue, the smoothing technique may still be useful to reduce post-ablation roughness.

Animals↗
PubMed1999

A comparison of fluorescence microscopy with the Ziehl-Neelsen technique in the examination of sputum for acid-fast bacilli.

SETTING: National Tuberculosis Reference Laboratory in Dakar, Senegal. OBJECTIVES: Comparison of results with fluorescence and bright-field microscopy for acid-fast bacilli. METHODOLOGY: Two smears from 2,630 consecutive sputum specimens between January 1996 and June 1998 were prepared for blinded examination of one smear each by the Ziehl-Neelsen technique and fluorescence microscopy at 1,000x magnification. The time required to declare a slide as negative was determined for both techniques in a sample of 68 slides. RESULTS: Concordancewas 96.9% and 92.3% for diagnostic and follow-up examinations, respectively. The yield was similar with both techniques for specimens with at least 10 bacilli per 100 fields, but higher with fluorescence microscopy in those with fewer than 10 bacilli per 100 fields. The mean time required by fluorescence microscopy before declaring a slide as negative with the same magnification was 3 minutes 34 seconds, compared to 7 minutes 44 seconds with the Ziehl-Neelsen technique. CONCLUSIONS: The results obtained with one technique are highly reproducible by the other. Fluorescence microscopy appears to be more likely to detect bacilli in paucibacillary cases than bright-field microscopy, and it more than halves the required examination time.

Humans↗
PubMed2002

Electron and immuno-electron microscopy of abdominal fat identifies and characterizes amyloid fibrils in suspected cardiac amyloidosis.

We evaluated the role of electron microscopy and immuno-electron microscopy studies on abdominal fat fine-needle biopsy samples in diagnosis and characterization of cardiac amyloidosis. The series consists of 15 patients with echocardiographic evidence of "restrictive cardiomyopathy" suspected to be due to amyloidosis. Patients underwent: clinical examination, electrocardiography, 2-D and Doppler echocardiography, immunofixation of serum and urine for detection of monoclonal immunoglobulins, and abdominalfat biopsies that were investigated with polarized light (Congo red), electron and immuno-electron microscopy using specific antibodies to kappa and lambda light chains, apolipoprotein A1, serum amyloid A (SAA), and transthyretin (TTR). Ultrastructural study of abdominal fat samples identified amyloid deposits in 15/15 cases. Immuno-electron microscopy specifically stained amyloid fibrils with antibodies anti-lambda (n = 8), -kappa (n = 2), -apolipoprotein A1 (n = 2) and -TTR (n = 3). Immuno-electron microscopy revealed TTR immuno-labelling in 2 patients with accidental monoclonal components, and a A reaction in I patient without monoclonal components. TTR and apolipoprotein A1 positive cases carried missense mutations in the corresponding genes. Our results demonstrate that amyloid deposits are present in the abdominalfat of patients suspected to have cardiac amyloidosis and that immuno-electron microscopy was able to characterize the amyloid protein in all cases.

Abdomen↗
PubMed2002

Evaluation of direct light microscopy for rapid detection of microorganisms in blood cultures.

OBJECTIVE: The aim of this study was to evaluate the use of direct light microscopy (x 40 objective) of unstained uncentrifuged blood-broth mixtures for rapid detection of microorganisms in blood cultures. METHODS: Two thousand two hundred and ninety-four blood cultures were investigated in this study. Blood cultures were processed in the Clinical Microbiology Laboratory, Queen Alia General Hospital, Amman, Jordan from January to December 2001. We compared the results of direct light microscopy and subcultures in 3 stages: macroscopically positive blood culture bottles, macroscopically negative blood culture bottles after 9-17 hours, and macroscopically negative blood culture bottles after 7 days of incubation. RESULTS: The total positive blood cultures was 434 (18.9%). Three hundred and fifty three (81.3%) were positive by macroscopic examination and direct light microscopy and grew viable organisms, 34 were macroscopically negative blood cultures that grew viable organisms after 9-17 hours of incubation in which 32 (94.1%) were positive by direct light microscopy and 47 macroscopically negative blood cultures that grew viable organisms after 7 days of incubation in which 45 (95.7%) were positive by direct light microscopy. CONCLUSION: The direct light microscopy methodology proved to be simple, rapid, cost effective, accurate and sensitive technique for the early detection of bacteremia.

Adult↗
PubMed2004

Dr. McCrone's teaching methods in forensic microscopy, their nature, history, and durability.

The microscopy teaching activities of Walter C. McCrone started long before the McCrone Research Institute (McRI) was incorporated as a not-for-profit research institute in Chicago. McCrone obtained his first microscopy training at Cornell University, with Emile Monnin Chamot, and was shortly thereafter appointed a full instructor in chemical microscopy before obtaining his Ph.D. (in 1941). After leaving Cornell, he had classes at the Armour Research Foundation (now Illinois Institute of Technology Research Institute--IITRI) from 1942-1956 and founded McRI in 1960. The course and student totals from McCrone's educational activities are impressive. As of January, 1, 2002, the cumulative for McRI (1942-2002) is 2,130 courses for 22,557 students. There has been an average of 600 students in an average of 60 classes for the last several years. Nearly all of the courses contain one week of intensive hands-on microscopy training with usually only one instructor for the entire duration of the class, making it a unique teaching experience for both student and Instructor. Thousands of students have successfully completed at least one of McCrone's specialized forensic microscopy (trace evidence) courses and the number will steadily increase as a result of McRI's continued efforts to interest forensic investigators in microscopy.

Forensic Medicine↗