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PubMed1996

Assessment of nerve ultrastructure by fibre-optic confocal microscopy.

Fibre-optic technology combined with confocality produces a microscope capable of optical thin sectioning. In this original study, tibial nerves have been stained in a rat model with a vital dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide, and analysed by fibre-optic confocal microscopy to produce detailed images of nerve ultrastructure. Schwann cells, nodes of Ranvier and longitudinal myelinated sheaths enclosing axons were clearly visible. Single axons appeared as brightly staining longitudinal structures. This allowed easy tracing of multiple signal axons within the nerve tissue. An accurate measurement of internodal lengths was easily accomplished. This technique is comparable to current histological techniques, but does not require biopsy, thin sectioning or tissue fixing. This study offers a standard for further in vivo microscopy, including the possibility of monitoring the progression of nerve regeneration following microsurgical neurorraphy.

Animals↗
PubMedDate not supplied

Cosmetic assessment of the human hair by confocal microscopy.

The optical sectioning property of the confocal microscope offers a breakthrough from the classic observation of the hair in a scanning electron microscope (SEM). Confocal microscopy requires minimal sampling preparation, and the hair can be observed in its natural environment with less damage than by other microscopic methods such as SEM. While used in the reflection mode, the true morphology of the cuticle and the various exogenous deposits at the surface can be identified and quantified. This relatively noninvasive, nondestructive technique is routinely used by us to monitor the efficiency of cleansing shampoos, to assess the homogeneity of layering polymers, and to evaluate the changes they induce in the optical properties of the hair surface in terms of opacity, transparency, and brilliancy. A second important field of investigation uses the fluorescence channel which reveals the internal structure of the hair. Fluorescent probes (rhodamine and its derivatives) demonstrate the routes of penetration and outline the geometry of cortical cells and of the medulla according to their lipophilic or hydrophilic properties. A volume rendering of a hair cylinder provides a better understanding of the interrelationships between cuticle cells, cortical cells, and the medullar channel. This recent technology is becoming an invaluable tool for the cosmetic assessment of the hair.

Hair↗
PubMedDate not supplied

Skin optics revisited by in vivo confocal microscopy: melanin and sun exposure.

A new confocal prototype dedicated to the exploration of in vivo human skin has been constructed around a laser confocal module (Oz Noran, Inc.) and a skin contact device, assuring perfect stability of skin images. The power of the Argon/Krypton laser source has been limited to 2mW to secure safety, and the laser provides three visible wavelengths: 488, 568, and 647 nm. Optical sections were digitized at video rate, providing easy and rapid measurements of the thickness of epidermal layers and time-resolved information. Unexpected details of the epidermis were recorded with the blue laser line. Melanin provided strong reflection of the basal keratinocytes instead of the absorption expected. The 3D reconstruction of the melanin cap in basal keratinocytes confirmed the behavior of melanosomes acting as myriads of nanomirrors that reflected light. Confocal images of the posterior aspect of the forearm were recorded before sun exposure and then for one month after exposure. There was a 25% increase in the thickness of the stratum corneum. Bright inclusions into the dark nucleus of numerous spinous cells were interpreted as local condensation of chromatin. Numerous bright intercellular filaments were attributed to melanosomes filling up dendrites of melanocytes. A striking observation concerned the lack of melanosome caps in basal keratinocytes. In vivo confocal microscopy affords new insight to the role of melanin and its gradual migration after sun exposure.

Adult↗
PubMed2005

In vivo confocal microscopy of megalocornea with central mosaic dystrophy.

In vivo confocal microscopy was performed on the central cornea of both eyes of a patient with megalocornea and central mosaic dystrophy. In the stroma, starting just below Bowman's membrane, polygonal, moderately reflective areas of opacification separated by diagonal hyporeflective striations were observed. The opaque areas appeared smaller in the anterior stromal layers and seemed to get larger towards the posterior stroma. The epithelium, Bowman's membrane and endothelium appeared normal. In vivo confocal microscopy is helpful in evaluating the morphological characteristics of corneal dystrophies, degenerations or developmental abnormalities of the cornea, and may prove to be helpful in understanding their pathophysiology.

Cell Count↗
PubMed1995

Stereoscopic and biplanar microphotography of Golgi-impregnated neurons: a correlative study using conventional and real-time, direct-image confocal microscopies.

A correlative study of neuronal reconstruction methods was made using both conventional (non-confocal) and real-time confocal microscopies. Simple and sophisticated (totally automated) methods are described for making both biplanar microphotographs using conventional transmitted light, and stereoscopic microphotographs using real-time confocal microscopy of Golgi-impregnated neurons. Confocal microscopy discriminates against out-of-focus layers to produce optical sections which can be summed on photographic film to obtain neuronal reconstructions. Biplanar images are obtained by fusion, using a stereo-viewer, of two adjacent optical sections obtained with a conventional transmitted light microscope. Stereoscopic and biplanar microphotographs of 3-day-old chick neurons are presented.

Animals↗
PubMed2004

In vivo confocal microscopy of epithelial inclusions from aberrant wound healing after astigmatic keratotomy.

OBJECTIVE: To report confocal microscopic findings in vivo of delayed prominent epithelial inclusions at a gaped incision groove after astigmatic keratotomy (AK). METHODS: Astigmatic keratotomy using paired arcuate incisions was performed on the right eye of a 59-year-old man who had a preoperative refraction of +2.50 DS, -7.00 DC x 80. The procedure and initial postoperative course were uneventful, and his refraction was OD +1.00 DS, -2.50 DC x 60 at 16 months. However, at 17 months postsurgery, tiny pearl-like lesions appeared along one of the incision grooves. In vivo confocal microscopy was performed to investigate these lesions. RESULTS: Under confocal microscopy, clusters of epithelial inclusions inside the gaped incision groove corresponded to the pearl-like lesions observed clinically. A confluent layer of flat, regular and polygonal epithelial cells covered the wall of the groove. Activated keratocytes were observed adjacently. The number of keratocytes around the groove, however, did not appear to increase in comparison to normal corneal wound healing and scar formation. No foreign body, infective, or inflammatory signs were observed. CONCLUSIONS: Aberrant wound healing was identified in post-AK incisions, similar to post-radial keratotomy cases. Confocal microscopy is a useful tool to study the wound healing of AK incisions and to rule out foreign bodies or infective elements as illustrated by this case. To the best of our knowledge, this is the first reported in vivo confocal study of AK wound healing in humans.

Astigmatism↗
PubMed2006

Quantitative analysis of corneal microstructure in keratoconus utilising in vivo confocal microscopy.

PURPOSE: To establish and quantify the in vivo confocal microscopic features of moderate to advanced keratoconus. METHODS: Nineteen keratoconus subjects were catergorised using Orbscan-derived corneal apex power and pachymetry as exhibiting moderate (n=7) and advanced (n=12) keratoconus. Control subjects included 23 noncontact lens wearers (Group A) and 15 contact lens wearers (Group B). All subjects underwent Confoscan slit scanning in vivo confocal microscopy. RESULTS: Compared with Group A (4912+/-434 cells/mm(2)), basal epithelial density was significantly lower in both moderate (4592+/-414 cells/mm(2), P<0.05) and advanced keratoconus (4530+/-596 cells/mm(2), P=0.01). In comparison to Group A (761+/-118 cells/mm(2)), anterior stroma keratocyte density was significantly greater in both moderate keratoconus (883+/-111 cells/mm(2), P=0.001) and advanced keratoconus (952+/-122 cells/mm(2), P<0.001). Compared to Group A (504+/-80 cells/mm(2)) posterior stroma keratocyte density was also significantly greater in advanced keratoconus (599+/-97 cells/mm(2), P<0.001) and posterior stromal keratocyte density appeared to increase with increasing severity of keratoconus (P<0.05). However, comparing control Groups A and B, contact lens wear per se, was associated with significantly reduced (P=0.000) keratocyte density in the anterior stroma (609+/-66 cells/mm(2)) and demonstrated a trend (P=0.056) in the posterior stroma (470+/-63 cells/mm(2)). Keratoconic corneas (429+/-72 microm) were significantly thinner than control Groups A (508+/-77 mm) and B (495+/-80 microm). The presence of keratoconus did not affect the endothelial cell density (P=0.54). CONCLUSION: In vivo confocal microscopy can provide insight into the microstructural changes that occur in keratoconus.

Adult↗
PubMed2005

Confocal microscopy of posterior polymorphous endothelial dystrophy.

PURPOSE: To report the in vivo confocal microscopic findings of posterior polymorphous endothelial dystrophy (PPED). METHODS: Four patients with PPED from 2 unrelated families and 2 asymptomatic children of an index patient were included in this observational case series. The eyes of the 6 subjects were examined by confocal light microscopy. RESULTS: Confocal microscopy demonstrated craters, streaks, and cracks over the corneal endothelium surface. Pleomorphism and polymegathism were present in eyes with PPED. Guttae and clusters of abnormal endothelial cells were also identified in corneas of these PPED patients. These findings were absent in eyes without clinical manifestations of PPED. CONCLUSIONS: In vivo confocal microscopy is potentially useful for excluding suspected cases of subclinical PPED. Abnormalities in the Descemet membrane and endothelium were observed.

Adolescent↗
PubMed2003

In vivo confocal microscopy in the patients with cornea farinata.

PURPOSE: To report in vivo corneal confocal microscopic findings of patients with cornea farinata. PATIENTS AND METHODS: Two unrelated patients, a 47-year-old man and a 77-year-old woman, with cornea farinata were studied. Examination with a confocal microscope was performed in addition to routine slit-lamp biomicroscopy. RESULTS: In both cases, slit-lamp biomicroscopy showed numerous small, faint opacities in the deep stroma in both eyes. Using confocal microscopy, highly reflective small particles were observed in the cytoplasm of keratocytes in the deep stroma adjacent to the corneal endothelial layer. No abnormalities could be detected in the epithelial layer, in the mid-stromal layer, at the level of Descemet's membrane, and in the endothelial layer. CONCLUSIONS: In vivo corneal confocal microscopy is useful for observing stromal abnormalities in cornea farinata. Further investigation of posterior stromal opacities using confocal microscopy may be useful to understand and differentiate various corneal conditions involving primarily deep stromal layers.

Aged↗
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↗
PubMed1999

Practical confocal microscopy and the evaluation of system performance.

The laser scanning confocal microscope has enormous potential in many fields of biology. Currently there is a subjective nature in the assessment of a confocal microscope's performance by primarily evaluating the system with a specific test slide provided by the user's laboratory. To achieve better performance from the equipment, it is necessary to run a series of tests to ensure that the optical machine is functioning properly. We have devised these methods on the Leica TCS-SP and TCS-4D systems. Tests measuring field illumination, lens clarity, laser power output, dichroic functioning, spectral alignment, axial resolution, laser power stability, machine performance, and system noise were derived to test the Leica laser scanning confocal microscopy system. These tests should be applicable to other manufacturers' systems as well. The relationship between photomultiplier tube (PMT) voltage, laser power, and averaging using a 10-microm-diameter test bead has shown that the noise (coefficient of variation of bead intensity, CV) in an image increases as the PMT increases. Therefore increasing the PMT setting results in increased noise. For ideal image quality, it appears that it is better to decrease the PMT setting and increase laser power, as noise generated by high PMT settings will reduce the image quality far more than the bleaching caused by higher laser power. Averaging can be used to improve the image at high PMT values, provided the sample is not bleached by repeated passes of the laser.

Artifacts↗
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↗
PubMed1998

pHi and pHo at different depths in perfused myocardium measured by confocal fluorescence microscopy.

Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pHi) or extracellular pH (pHo) in isolated, arterially perfused rabbit papillary muscles. Single-excitation, dual-emission fluorescent images of the endocardial surface and underlying myocardium to a depth of 300 micron were simultaneously recorded from perfused cylindrical muscles suspended in a controlled atmosphere oriented oblique to the focal plane. Contraction was inhibited by the addition of butanedione monoxime. In separate muscles, pHo was measured during continuous perfusion of SNARF-1 free acid. pHi measurements were made after the muscle was loaded with SNARF-1/AM and the extracellular space was cleared of residual fluorophore. Initial experiments demonstrated the uniformity of ratiometric measurements as a function of pH, image depth, and fluorophore concentration, thereby establishing the potential feasibility of this method for quantitative intramural pH measurements. In subsequent experiments, the method was validated in isolated, arterially perfused rabbit papillary muscle during normal arterial perfusion and as pHi and pHo were altered by applying CO2 externally, exchanging HEPES and bicarbonate buffers, and changing pHi with NH4Cl washout. We conclude that in situ confocal fluorescent microscopy can measure pHi and pHo changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pHi regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pHi.

Acidosis↗
PubMed2005

In vivo confocal microscopy study of blebs after filtering surgery.

OBJECTIVE: To analyze bleb structure after filtering surgery at the cellular level using a new generation in vivo confocal microscope. DESIGN: Observational case series. PARTICIPANTS: We retrospectively evaluated 17 filtering blebs of 13 patients after trabeculectomy. METHODS: Ophthalmologic examinations included slit-lamp examination, applanation tonometry, and in vivo confocal microscopy (Heidelberg Retina Tomograph II, Rostock Cornea Module). Eyes were classified into 3 groups: (1) functioning blebs (6 eyes), (2) nonfunctioning blebs (6 eyes), and (3) functioning blebs after application of mitomycin C (5 eyes). Cellular patterns, morphologic appearance, and functional aspects of functioning and nonfunctioning blebs were compared in a masked manner. MAIN OUTCOME MEASURES: In vivo confocal microscopy images were analyzed for number of intraepithelial microcysts, density of subepithelial connective tissue, presence of blood vessels, or encapsulation. RESULTS: All functioning blebs had numerous intraepithelial optically-empty microcysts, whereas all nonfunctioning blebs had none or few. Subepithelial connective tissue was widely spaced in all functioning blebs, whereas the tissue was dense in 83.3% of nonfunctioning blebs. Functioning blebs with mitomycin C had numerous microcysts and loosely arranged subepithelial connective tissue as compared with nonfunctioning blebs. CONCLUSIONS: In vivo confocal microscopy study of blebs is an original method that agrees well with ex vivo histologic examination. The number of microcysts and the density of the subepithelial connective tissue observed with in vivo confocal microscopy are correlated with bleb function. By providing details of the structures of filtering blebs at the cellular level, in vivo confocal microscopy constitutes a new promising way to understand wound healing mechanisms after filtering surgery.

Adult↗
PubMed1996

Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new, efficient upconverting fluorophore.

Three-dimensional confocal imaging of polymer samples was achieved by the use of two-photon excited fluorescence in both positive and negative contrast modes. The fluorophore was a new and highly efficient two-photon induced upconverter, resulting in improved signal strength at low pumping power. Because of the relatively long wavelength of the excitation source (798 nm from a mode-locked Ti:Sapphire laser), this technique shows a larger penetration depth into the samples than provided by conventional single-photon fluorescence confocal microscopy. Single-photon and two-photon images of the same area of each sample show significant differences. The results suggest the possibility of using two-photon confocal microscopy, in conjunction with highly efficient fluorophores, as a tool to study the surface, interface, and fracture in material science applications.

Fluorescent Dyes↗
PubMed1992

Immunofluorescent analysis of creatine kinase in cultured astrocytes by conventional and confocal microscopy: a nuclear localization.

The subcellular localization of creatine kinase (CK) was examined in primary cultures of astrocytes with immunofluorescent labeling methods and detection by both standard fluorescence microscopy and confocal laser-scanning microscopy. With conventional microscopy, the pattern of CK staining was uniform throughout the cell cytoplasm and appeared to stain the nuclear region intensely. Staining of CK in the nuclear region co-localized with the DNA-specific Hoechst nuclear stain. CK produced a diffuse cytoplasmic staining pattern that was different from the staining pattern produced by the cytoskeletal proteins glial fibrillary acidic protein and tubulin, both of which showed a filamentous cytoskeletal network that excluded the nucleus. To examine the structural details of CK in the nuclear region, serial optical sections were taken through the cell monolayer with a confocal microscope. The cells were immunostained for CK, and the CK-staining pattern was compared with the staining pattern produced by propidium iodide, which is specific for DNA in RNase-treated samples and stains total nucleic acid in untreated samples. CK staining was present within the nucleus in each section taken through the monolayer. The nucleolus did not stain for CK. The pattern of CK staining in the nucleus (and cytoplasm) was distinctly different from the staining pattern of either DNA or total nucleic acid. Nuclear CK appeared to have a granular, particulate pattern, which is suggestive of a nucleoplasmic distribution.

Animals↗
PubMed2004

Evaluation of the corneal effects of topical ophthalmic fluoroquinolones using in vivo confocal microscopy.

PURPOSE: To compare the effects of several fluoroquinolone antibiotics on the corneal epithelium and stroma using in vivo confocal microscopy. METHODS: Five antibiotic solutions were evaluated: 1) 0.3% ofloxacin (Oflox) solution with 0.005% benzalkonium chloride (BAC); 2) 0.3% gatifloxacin (Gati) solution with 0.005% BAC; 3) 0.3% ciprofloxacin (Cipro) solution with 0.006% BAC; 4) 0.5% levofloxacin (Levo) with 0.005% BAC; and 5) 0.5% moxifloxacin (Moxi) solution with no BAC. Preservative-free artificial tears (Tears) were used as a control. New Zealand white rabbits were used for this study (six per solution group). Ten days prior to exposure to any solution, central corneal epithelial thickness and stromal thickness were measured using in vivo confocal microscopy through focusing. Images of the superficial epithelium were also acquired. Both eyes of each rabbit then received one drop of the assigned solution six times the first day and then four times per day for 6 days. On day 7, in vivo confocal microscopy was repeated. RESULTS: A significant decrease in epithelial thickness was induced by 7 days of exposure to Levo, Gati, Oflox, and Cipro (P < 0.05, two-way repeated-measures ANOVA, Tukey test). Tears and Moxi, which do not contain BAC, did not induce significant changes in epithelial thickness. No significant changes in stromal thickness were detected (P = 0.266), and no keratocyte activation was observed for any of the solutions evaluated. CONCLUSION: We have previously used confocal microscopy to establish a correlation between epithelial thinning (due to superficial cell loss) and slight ocular irritation. The results of this study suggest that Moxi induces less damage to the corneal epithelium than other antibiotic solutions, perhaps because it does not contain BAC.

Animals↗
PubMed2000

In vivo confocal microscopy of patients with corneal recurrent erosion syndrome or epithelial basement membrane dystrophy.

OBJECTIVE: To characterize morphologic changes in corneas of patients with recurrent erosion syndrome or epithelial basement membrane dystrophy using in vivo confocal microscopy. DESIGN: Observational case series PARTICIPANTS: Fourteen eyes of eight patients with diagnosed epithelial basement membrane dystrophy and 13 eyes of seven patients with recurrent erosion syndrome were examined. METHODS: Slit-lamp examination and in vivo confocal microscopy. The pathologic findings are presented as digitized images obtained from video tape recorded during the confocal microscopy. MAIN OUTCOME MEASURES: The morphology of corneal surface epithelial cells, basal epithelial cells, subbasal nerve plexus, Bowman's layer, stromal keratocytes, and endothelium was analyzed. RESULTS: The surface epithelium was intact in all but two eyes. One cornea (a basement membrane disorder with clinically visible dots) had multinucleate surface epithelial cells, and one eye with recurrent corneal erosions showed a freely floating surface epithelium sheet in the tear fluid. Patients in both groups showed islets of highly reflective cells with presumed intracellular deposits surrounded by normal cells in the basal epithelial cell layer. The basal epithelial cell area also showed other pathologic changes, including drop-shaped configurations, streaks, or ridges. Folding of the Bowman's layer was also observed in both groups. Anterior keratocytes showed signs of activation (highly reflective nuclei with visible processes) in some of the patients regardless of the clinical diagnosis, and in recurrent erosions even increased deposition of abnormal extracellular matrix in the anterior stroma was suspected. Posterior corneal keratocytes and endothelium appeared normal when examined. The subbasal nerve plexus showed various pathologic changes, such as short or strangely shaped nerve fiber bundles, decreased numbers of long nerve fiber bundles, only faintly visible long nerve fiber bundles (instead of the normally observed long parallel running interconnected bundles), or increased amounts of Langerhans cells, but only one patient (with recurrent erosion syndrome) lacked the subbasal nerve plexus. CONCLUSIONS: In vivo confocal microscopy of corneas with recurrent erosions or epithelial basement membrane dystrophy showed deposits in basal epithelial cells, subbasal microfolds and streaks, damaged subbasal nerves, or altered morphology of the anterior stroma. Confocal microscopy cannot replace biomicroscopy in making a specific diagnosis, but it sometimes helps the diagnosis in corneas that appear normal under a biomicroscope.

Adult↗