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PubMed1997

Confocal microscopy visualization of antifolate uptake by the reduced folate carrier in human leukaemic cells.

Confocal microscopy was used to visualize the intracellular uptake of the fluorescent methotrexate analogue, fluorescein-MTX (F-MTX), in human leukaemic cell lines and leukaemic blasts. Cytosolic labelling of wild-type K562 human erythroleukaemia cells was detected during 3-60 min incubations with F-MTX (1 microM) and was completely inhibited by co-exposure to either methotrexate or the thymidylate synthase inhibitor, ZD1694. There was no significant intracellular F-MTX accumulation over this period in a K562 subline (K500E) with a documented defect (approximately 10% of wild type) in membrane transport by the reduced folate carrier (RFC). F-MTX uptake was re-established in K500E cells transfected with a cDNA to human RFC, establishing a role for RFC in the cellular uptake of this compound. High levels of intracellular labelling were detected in all cell lines after prolonged (24 h) F-MTX incubations, however F-MTX accumulation at this time was not inhibited by ZD1694. F-MTX uptake by RFC was also detected in leukaemic blasts from children with acute lymphoblastic leukaemia and could be blocked with ZD1694. In leukaemic blasts with a documented defect in MTX uptake, F-MTX accumulation was abolished in almost all the cells. These results display the power of confocal microscopy for directly visualizing RFC-mediated anti-folate uptake. Over short intervals, F-MTX uptake is mediated by RFC, however, RFC-independent processes predominate during long drug exposures. Direct assay by confocal microscopy may be better suited than other indirect methods (i.e. flow cytometry) for detecting low levels of RFC transport in leukaemic blasts from patients undergoing chemotherapy with methotrexate.

Biological Transport↗
PubMed2001

Image restoration for confocal microscopy: improving the limits of deconvolution, with application to the visualization of the mammalian hearing organ.

Deconvolution algorithms have proven very effective in conventional (wide-field) fluorescence microscopy. Their application to confocal microscopy is hampered, in biological experiments, by the presence of important levels of noise in the images and by the lack of a precise knowledge of the point spread function (PSF) of the system. We investigate the application of wavelet-based processing tools to deal with these problems, in particular wavelet denoising methods, which turn out to be very effective in application to three-dimensional confocal images. When used in combination with more classical deconvolution algorithms, these methods provide a robust and efficient restoration scheme allowing one to deal with difficult imaging conditions. To make our approach applicable in practical situations, we measured the PSF of a Biorad-MRC1024 confocal microscope under a large set of imaging conditions, including in situ acquisitions. As a specific biological application, we present several examples of restorations of three-dimensional confocal images acquired inside an intact preparation of the hearing organ. We also provide a quantitative assessment of the gain in quality achieved by wavelet-aided restorations over classical deconvolution schemes, based on a set of numerical experiments that we performed with test images.

Algorithms↗
PubMed2005

Mapping of the normal human corneal sub-Basal nerve plexus by in vivo laser scanning confocal microscopy.

PURPOSE: To produce a two-dimensional reconstruction map of the living human sub-basal corneal nerve plexus using in vivo confocal microscopy. METHODS: Laser scanning in vivo confocal microscopy was performed on three normal eyes of three healthy human subjects. Subjects were asked to fixate on targets arranged in a grid to enable examination of the cornea in a wide range of positions. Using the section mode, a mean of 573 +/- 176 images of the sub-basal plexus were obtained for each subject. The data were arranged and images were mapped for each subject into confluent montages. RESULTS: Mean dimensions of the corneal areas mapped were 4.95 +/- 0.53 mm horizontally and 5.14 +/- 0.53 mm vertically. In all subjects, the sub-basal nerve plexus appeared to radiate toward a whorl-like complex centered 1 to 2 mm inferior to the corneal apex. Outside this area, the nerve fiber bundles in the remainder of the cornea were arranged in a radiating pattern. Mean nerve density was significantly higher in the inferocentral whorl region (25,249 +/- 616 microm/mm2) compared with the central cornea (21,668 +/- 1411 microm/mm2) (Mann-Whitney U test; P = 0.05). CONCLUSIONS: This is the first study to elucidate the overall distribution of sub-basal nerves in the healthy, live central to mid-peripheral human cornea by laser scanning in vivo confocal microscopy. The whorl pattern of the sub-basal nerves is similar to that seen in the epithelium in corneal verticillata and may lend support to the theory that epithelial cells and nerves migrate centripetally in tandem.

Adult↗
PubMedDate not supplied

In vivo observation of the human tear film by tandem scanning confocal microscopy.

A new method for observing normal and pathologic states of the human tear film using tandem scanning confocal microscopy is presented. The confocal microscope is configured with a horizontal light path, a 10 x dry objective, and an image-intensified camera for collecting images at a magnification of approximately 150x. The advantages of confocal microscopy can be used to collect reflected images of the human tear film with improved detail and resolution.

Humans↗
PubMed2002

Three-dimensional confocal microscopy of the living human eye.

Three-dimensional confocal microscopy of the living eye is a major development in instrumentation for biomicroscopy of the eye. This noninvasive optical technology has its roots in the application of optics to reflected light imaging of the eye. These instrument developments began with Leeuwenhoek's use of his single lens microscope to investigate the structure of the eye. There followed a series of connected instruments: the ophthalmoscope, the slit lamp, the specular microscope, and the clinical confocal microscope. In vivo confocal microscopy produces high contrast, reflected light images or optical sections through the depth of living ocular tissue. Stacks of registered optical sections can be transformed by computer visualization techniques into three-dimensional volume images of ocular tissues: cornea, ocular lens, retina, and optic nerve. The clinical confocal microscope has resulted in new diagnostic techniques and new cellular descriptions of ocular disorders and pathology.

Animals↗
PubMed2003

In vivo confocal microscopy of corneal insect foreign body.

PURPOSE: To report a case of insect corneal foreign body with in vivo confocal microscopy performed. METHODS: Clinical presentation, clinical pictures, and treatment are discussed. Confocal microscopy of the injured cornea was performed in areas showing foreign bodies with and without infiltrations seen under the slit-lamp. CONCLUSION: Confocal microscopy is useful in detection of subtle inflammatory reaction, monitoring the progress of the injury and decision of treatment plans.

Administration, Topical↗
PubMed2004

Corneal thickness measurement by confocal microscopy, ultrasound, and scanning slit methods.

PURPOSE: To measure corneal thickness by using a calibrated confocal microscope and to compare this measurement to thickness determined by ultrasonic and noncontact scanning slit pachymetry. DESIGN: Comparison of corneal thickness measured by using four instruments in normal subjects. METHODS: Thickness measured by a clinical confocal microscope (Tandem Scanning) was calibrated from measurements of polymethylmethacrylate contact lenses with known thickness. Corneal thickness was measured in one eye of 24 normal subjects by using this instrument, two ultrasonic pachymeters (DHG-1000 and Sonogage), and a noncontact optical scanning slit pachymeter (Orbscan II). RESULTS: Mean corneal thickness measured by confocal microscopy was 516 +/- 30 microm (+/-SD). This was less than the mean thickness measured by both ultrasonic pachymeters, 554 +/- 28 microm by the DGH, and 555 +/- 28 microm by the Sonogage (P <.001). Thickness measured by the Orbscan II pachymeter was 540 +/- 35 microm (P <.001, compared with either confocal or ultrasound) after applying an "acoustic factor" of 0.92, a default correction of the software. CONCLUSION: Corneal thickness measured by calibrated confocal microscopy is approximately 39 microm (7.0%) less than thickness measured by two commonly used ultrasonic pachymeters and approximately 24 microm (4.4%) less than thickness measured by the corrected Orbscan II pachymeter. These differences are important for planning and measuring the effects of refractive and other surgical procedures. The precision of confocal microscopy is limited by corneal motion in an anterior-posterior direction. The difference between instruments suggests that verification of clinical ultrasonic pachymeters should be revisited.

Adult↗
PubMed1999

Confocal microscopy in lattice corneal dystrophy.

BACKGROUND: The purpose of the study was to assess the appearance of lattice corneal dystrophy by means of white-light confocal microscopy. METHODS: Two consecutive patients with lattice corneal dystrophy were prospectively examined. In vivo white-light tandem-scanning confocal microscopy was performed in the right eye of the first patient. Her left eye had undergone penetrating keratoplasty 4 years earlier. Histologic findings of the corneal button were compared with confocal microscopic findings of the right eye. The other patient was monocular and confocal microscopy was performed only in the non-seeing eye. RESULTS: In both patients, linear and branching structures with changing reflectivity and poorly demarcated margins were visualized in the stroma. The linear structures measured approximately 40-80 microm in width. CONCLUSION: Lattice corneal dystrophy presents characteristic linear images on confocal microscopy and should not be misdiagnosed as fungal hyphae in cases of corneal infection.

Aged↗
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↗
PubMed2006

Mapping the corneal sub-basal nerve plexus in keratoconus by in vivo laser scanning confocal microscopy.

PURPOSE: To produce a two-dimensional reconstruction map of the living corneal sub-basal nerve plexus in keratoconus with in vivo confocal microscopy. METHODS: Four eyes of four subjects with keratoconus were examined by slit lamp biomicroscopy, Orbscan II slit-scanning elevation topography (Bausch & Lomb Surgical, Rochester, NY), and laser scanning in vivo confocal microscopy with the Heidelberg Retina Tomograph II, Rostock Corneal Module (Heidelberg Engineering, Heidelberg, Germany). Subjects were asked to fixate on targets arranged in a grid to enable in vivo confocal microscopy of the cornea in a wide range of positions. RESULTS: A mean of 402 +/- 57 images were obtained for each cornea, to create confluent montages. The mean dimensions of the corneal areas mapped were 6.60 +/- 0.70 mm horizontally and 5.91 +/- 0.72 mm vertically. All corneas exhibited abnormal sub-basal nerve architecture compared with patterns previously observed in normal corneas. At the apex of the cone, a tortuous network of nerve fiber bundles was noted, many of which formed closed loops. At the topographic base of the cone, nerve fiber bundles appeared to follow the contour of the base, with many of the bundles running concentrically in this region. Central sub-basal nerve density was significantly lower in keratoconus corneas (10,478 +/- 2,188 microm/mm2) compared with normal corneas (21,668 +/- 1,411 microm/mm2; Mann-Whitney; P < 0.01). CONCLUSIONS: This is the first study to elucidate the overall distribution of sub-basal nerves in the living central to midperipheral human cornea in keratoconus, using laser scanning in vivo confocal microscopy.

Adult↗
PubMed2006

In vivo confocal microscopy of filtering blebs after trabeculectomy.

OBJECTIVES: To analyze filtering blebs after trabeculectomy by means of in vivo confocal microscopy and to correlate the images with clinical bleb appearance and function. METHOD: In vivo confocal microscopy using the Heidelberg Retina Tomograph/Rostock Cornea Module (Heidelberg Engineering, Heidelberg, Germany) was performed in 53 filtering blebs in 45 patients 6 days to 30 years postoperatively. RESULTS: In vivo confocal microscopic findings significantly correlated with good bleb function included the number of epithelial microcysts (P = .03), a large total stromal cyst area (P = .009), the absence of encapsulated stromal cysts (P = .002), minimal vascularization (P = .05), and the absence of tortuous conjunctival vessels (P = .01). In contrast, a hyperreflective condensed bleb stroma was significantly associated with bleb failure (P<.001). Bleb stroma mainly consisting of a rarified collagenlike network was significantly linked to trabeculectomy performed with mitomycin C (P = .001). Epithelial and stromal inflammation were observed at a median of 1 and 4 months after surgery, respectively. CONCLUSIONS: In vivo confocal microscopy using the Heidelberg Retina Tomograph/Rostock Cornea Module permits diagnostic imaging of filtering blebs and differentiation between good and insufficient bleb function. Moreover, the postoperative inflammatory reaction can be monitored directly for adapted postoperative anti-inflammatory treatment.

Adult↗
PubMed2006

Confocal microscopy of corneal flap microfolds after LASIK.

PURPOSE: To describe the morphological characteristics of microfolds that appear at the corneal flap after LASIK, as seen under confocal microscopy. METHODS: Twenty-one eyes that had undergone LASIK were examined, all within 3 weeks to 1 month after surgery. A central scan of the total corneal thickness was obtained by using confocal microscopy in vivo. Confocal images were captured and digitized. The longitudinal orientation (vertical, horizontal, and oblique) and morphological characteristics of the microfolds were described and recorded. RESULTS: Six eyes had folds at the central corneal flap, visible as linear distortions in the confocal images: one fold had a vertical orientation, two were horizontal, and three were oblique. The folds were visible from the epithelial basal cell layer to the stromal portion of the flap and were deeper than Bowman's layer. CONCLUSIONS: Confocal microscopy allowed visualization of microfolds after LASIK. With the appropriate software, it is possible to analyze the morphological characteristics of these folds. Flap microfolds after LASIK are deeper than Bowman's layer.

Cornea↗
PubMed1995

Quantitative recording of vitality patterns in living multicellular spheroids by confocal microscopy.

Fluorescent dyes were used in conjunction with confocal microscopy to record the vitality status of cells in multicellular glioma spheroids. Multicellular spheroids are in vitro models for micrometastases or intravascular microregions of large tumors. With progressing growth three distinct concentric annular shells develop. A rim of proliferating cells in the periphery is followed towards the center by layers of quiescent cells and at a defined spheroid diameter cell death occurs in the central core. Fluorescein diacetate (FDA) and Calcein/AM were used as vital stains and Lucifer Yellow/VS (LYVS) was used as a marker for dead cells. For loading multicellular spheroids with the esterase substrate dyes we used a two step cold incubation technique to avoid dye accumulation in the most peripheral cell layers. Homogenously stained tissue allowed to describe the fluorescence attenuation in depth as a monoexponential decay. An attenuation coefficient C was calculated from calibration experiments to be 12.5 x 10(-3) in vital stained tissue and 17.9 x 10(-3) in lethal stained tissue. Using the respective attenuation coefficient the raw data were corrected for light absorption and scattering in depth. In radial recordings of the vitality status of multicellular glioma spheroids using CLSM-technique we showed that spheroids up to a diameter of 250 microns were homogenously stained with Calcein/AM and FDA. Spheroids larger than 250 microns consist of vital stained cells and unstained cells. They do not show dead cell staining until they reach a diameter of about 400 microns. The thickness of the rim of vital stained cells decreased with increasing diameter of the spheroids to 64 +/- 7 microns in spheroids of a diameter of 550 +/- 25 microns. Thereafter the thickness of the Calcein/AM or FDA stained rim augmented again, reaching 93 +/- 9 microns in spheroids of 700 microns in diameter. The first signs of dead cell staining in the central core occurred at a diameter of 400 +/- 25 microns. The radius of the core increased in an exponential way. The cell layer which was stained neither by vital nor by lethal dyes showed a thickness of 150 microns in spheroids of 550 +/- 25 microns in diameter. Our staining technique and the radial recording of mean field fluorescence signals in living multicellular spheroids will be a valuable tool for experimental cancer research providing a non invasive quantification of cell vitality in living multicellular spheroids.

Cell Survival↗
PubMed2006

Reflectance confocal microscopy of pigmented basal cell carcinoma.

BACKGROUND: Reflectance confocal microscopy (RCM) is a high-resolution imaging tool for in vivo noninvasive evaluation of skin lesions. OBJECTIVE: We sought to describe the relevant RCM features for pigmented basal cell carcinoma (BCC). METHODS: Pigmented skin lesions with a differential diagnosis of pigmented BCC were imaged using dermoscopy and RCM, followed by excision for histologic analysis. RESULTS: RCM demonstrated aggregations of tightly packed cells with palisading, forming cordlike structures and nodules with irregular borders and variable brightness; these represented nests of pigmented basaloid tumor cells on histopathology, and blue-gray ovoid areas on dermoscopy. These tumor nests were associated with bright dendritic structures, identified histologically as either melanocytes or Langerhans cells, together with numerous bright oval to stellate-shaped structures with indistinct borders representing melanophages, and with highly refractile granules of melanin. LIMITATIONS: The pigmented BCCs imaged in this study were predominantly nodular; a different set or additional criteria may be necessary for detection of infiltrative and metatypical BCCs. CONCLUSION: RCM may permit in vivo diagnosis of pigmented BCC.

Aged↗
PubMed1999

Delayed mustard gas keratopathy: clinical findings and confocal microscopy.

PURPOSE: To describe the clinical manifestations and confocal microscopic findings in a patient with delayed mustard gas keratopathy. METHOD: Case report. A 32-year-old veteran who had participated in the Iran Iraq conflict was exposed to mustard gas in 1988. Ocular abnormalities in 1996 and 1998 and corneal confocal microscopic findings in 1998 are presented. RESULTS: In 1996, slit-lamp examination disclosed bilateral limbal changes with tortuous blood vessels and full-thickness corneal alterations. In 1998, the right eye had porcelain-white episcleral changes and adjacent peripheral ulcerative keratopathy. Confocal microscopy demonstrated irregular-appearing epithelial and basal epithelial cells. The anterior stroma was remarkable for spindle-like keratocytes, diffuse fibrillar inhomogeneities and the presence of highly reflective material. CONCLUSIONS: Mustard gas keratopathy is a uncommon cause of ocular damage, but it may lead to delayed ocular damage.

Adult↗
PubMed2005

In vivo findings of the bulbar/palpebral conjunctiva and presumed meibomian glands by laser scanning confocal microscopy.

OBJECTIVE: To report in vivo laser scanning confocal microscopic findings of the normal human conjunctiva and presumed meibomian glands and to investigate the potential application of this confocal microscope (HRT II Rostock Cornea Module) as a diagnostic device for normal and abnormal ocular surface structures. METHOD: Four healthy volunteers (51-, 49-, 31-, and 30-year-old men) participated in this study. The temporal bulbar conjunctiva, approximately 5 mm away from the limbus, and the upper tarsus conjunctiva of each patient were examined in vivo by a laser confocal microscope. Also, presumed meibomian glands beneath the tarsal conjunctiva were examined. RESULTS: In all subjects, the in vivo laser confocal microscope obtained similar images. In the superficial epithelial cell layer of the bulbar conjunctiva, numerous bright nuclei were observed. We also observed many Langerhans cells with characteristic dendritic morphology and goblet cells with relatively homogeneous brightness in the bulbar conjunctiva. Most interestingly, the palpebral conjunctival epithelium with invaginations or crypts was observed. In addition, weblike structures, presumably meibomian glands, were observed beneath the palpebral conjunctival epithelium. CONCLUSIONS: These results indicate that in vivo laser confocal microscopy can be used not only to visualize the bulbar conjunctiva but the upper tarsus conjunctiva and possibly subconjunctival meibomian glands. Further investigations of conjunctival and meibomian gland pathologies may further elucidate the potential of this device in clinical ophthalmology.

Adult↗
PubMedDate not supplied

Confocal microscopy in early diagnosis of Acanthamoeba keratitis.

PURPOSE: This study correlated confocal microscopic images obtained using the Nidek ConfoScan 2.0 System in corneas with clinical suspicion of Acanthamoeba keratitis, with diagnosis confirmed by either cytological and/or histological analysis. METHODS: Fifteen eyes of 14 patients with a clinical diagnosis of Acanthamoeba keratitis underwent confocal microscopy evaluation. RESULTS: Fifteen eyes of 14 patients (one bilateral case) showed Acanthamoeba keratitis alterations that ranged from massive infestation to cicatricial opacity in the stroma. Ten patients (71%) were females. Mean age was 26 years (range 19 to 37 yr). All patients were contact lens wearers. CONCLUSION: Confocal microscopy was a useful, noninvasive technique in the diagnosis and treatment of Acanthamoeba keratitis, especially in those cases in which corneal scraping, cytological analysis, and culture are negative. It also eliminated the necessity of tissue biopsy, considered an invasive procedure.

Acanthamoeba Keratitis↗
PubMed2006

Corneal and conjunctival manifestations in Fabry disease: in vivo confocal microscopy study.

PURPOSE: To describe the microscopic corneal and conjunctival findings in patients with Fabry disease (FD) related keratopathy by using in vivo confocal microscopy. DESIGN: Prospective observational case series. METHODS: Twelve eyes of six patients affected by Fabry disease, belonging to two different families, underwent in vivo confocal microscopic examination. Corneal and conjunctival morphology were assessed by means of a scanning slit corneal confocal white-light microscopy and confocal laser-scanning microscope. RESULTS: Confocal microscopy examination evidenced two different types of corneal epithelial changes. The three hemizygous patients presented bright hyper-reflective intracellular inclusions located within the basal epithelial cells, while the three heterozygous patients showed fine diffusion of reflective substance at the level of superficial, basal epithelial cells and basal membrane, in all eyes. The complex basal-Bowman's membrane appeared irregular, distorted, and nonhomogeneous in all subjects. Stromal increased reflectivity attributable to haze and epithelial ingrowth with bright intracellular inclusions was noticed in one hemizygous patient. In all patients, conjunctival epithelial involvement represented by bright roundish intracellular inclusions was evidenced, appearing more pronounced in tarsal than in bulbar conjunctiva. CONCLUSIONS: Although FD-related cornea verticillata attributable to glycosphingolipids accumulation is considered to be primarily a corneal disease, in vivo confocal microscopy demonstrated structural alterations throughout the entire ocular surface epithelia. It is still unclear whether the different type of corneal epithelial lesions observed for hemizygous and heterozygous patients is related to different physiopathological mechanisms. Confocal microscopy may assist ophthalmologists in the diagnosis of FD-related ocular surface and corneal manifestations.

Adult↗