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Results for “Confocal microscopy”

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At least 433 records · Page 24Linked to original sources
PubMed2003

Macro-microscopic fluorescence of human bladder cancer using hypericin fluorescence cystoscopy and laser confocal microscopy.

The early detection of carcinoma is very essential for the diagnosis and prognosis of a bladder cancer patient. In this study we have investigated the use of hypericin as a fluorescent tumour marker and laser confocal microscopy as a diagnostic tool to aid the diagnosis of such cancers. Both cellular and clinical studies have been conducted. In the cellular studies, we have compared two bladder cell lines for the uptake and sub-cellular localization of hypericin. It was found that there was a rapid uptake and clearance of hypericin and significant localization in mitochondria and lysosomes. The study also revealed that there was a time-dependent increase in fluorescence intensity in bladder cells. The optimum localization was found to be 2-4 h post drug incubation. In the clinical study, consisting of 30 patients, both white light and fluorescence cystoscopy were performed after hypericin instillation. Biopsies taken from fluorescing regions were imaged using the confocal microscope. The order of fluorescence was detected to be as follows: normal < inflammation < grade 1 < grade 2 < CIS < grade 3. It was also found that the fluorescence intensity increased with the stage of the disease thereby enabling the determination of the degree of invasiveness of cancer. This enables the use of hypericin as a prognostic marker and laser confocal microscopy as a tool to aid in diagnosis of bladder cancer.

Anthracenes↗
PubMed2000

Confocal microscopy of a patient with irregular astigmatism after LASIK reoperations and relaxation incisions.

PURPOSE: Laser-assisted in situ keratomileusis (LASIK) is widely used for correcting refractive errors. If the predicted refractive result is not achieved after the first operation, a re-operation can be performed by ablating more stromal tissue after reopening the flap. The goal of this study was to analyze, by using in vivo confocal microscopy, the morphologic changes associated with repeated LASIKs. METHODS: Clinical examination, computed corneal topography, and real-time in vivo confocal microscopy were performed on both eyes of a 50-year-old patient with induced irregular astigmatism leading to decreased best-corrected vision in the left eye after LASIK. The left cornea had been operated on 5 times (LASIK with two reoperations followed by two relaxing incisions), and the right cornea twice (LASIK with one reoperation). RESULTS: Microfolds at the level of the Bowman's layer and highly reflective particles at the flap interface were observed in both corneas. The subbasal nerve plexus was severed in the left eye. In addition, we identified epithelial material in the flap margin and inside one of the two relaxing incisions placed inferotemporally. CONCLUSION: Repeated LASIKs may stretch the flap and result in microfolding at the Bowman's layer. This and deposition of particles in the flap interface may increase with the number of reoperations, challenging the healing response. Microfolding and occurrence of foreign material in the interface may add to the irregular astigmatism and poor visual outcome after LASIK. Clinical in vivo confocal microscopy offers new possibilities for the assessment of ultrastructural changes after corneal refractive surgery.

Astigmatism↗
PubMed2004

Use of in vivo confocal microscopy in malignant melanoma: an aid in diagnosis and assessment of surgical and nonsurgical therapeutic approaches.

BACKGROUND: Melanomas with poorly defined borders, lack of pigmentation, lentiginous extension, and location in cosmetically sensitive regions represent diagnostic and therapeutic challenges. Repeated surgical reexcisions are frequently required to achieve tumor-free margins. The use of reflectance mode confocal microscopy as an noninvasive method has shown to be a promising tool for diagnosing pigmented lesions in vivo. OBSERVATIONS: We report 3 clinical cases of melanoma: amelanotic melanoma (case 1), locally recurrent melanoma (case 2), and lentigo maligna melanoma (case 3). In case 1, in vivo confocal microscopy was instrumental in making the diagnosis and in monitoring the response to imiquimod therapy for in situ residual disease. It was also used to successfully delineate preoperative surgical margins in cases 2 and 3. CONCLUSION: As new methods for treating melanoma emerge and become more available, confocal microscopy can play a significant role by improving sensitivity in diagnosis, by increasing rates of successful initial excision, and by serving as a noninvasive means of monitoring therapy.

Aminoquinolines↗
PubMed2002

Measurement of nuclear factor-kappa B translocation on lipopolysaccharide-activated human dendritic cells by confocal microscopy and flow cytometry.

BACKGROUND: Nuclear factor kappa B (NF-kappaB) is a ubiquitously expressed transcription factor that regulates cytokine and immunoglobulin (Ig) gene expression. In most cell types, the inactive p50/p65 NF-kappaB heterodimer is located in the cytoplasm, complexed to its IkappaB inhibitory unit. Stimulation of cells by various reagents such as bacterial endotoxin or cytokines leads to a dissociation of NF-kappaB from IkappaB and a rapid translocation of free NF-kappaB to the nucleus. The aim of this article is to define optimal conditions for the measurement of NF-kappaB translocation by both confocal microscopy and flow cytometry. METHODS: Four commercial anti-NF-kappaB antibodies were evaluated by confocal microscopy, after using two methods of fixation and permeabilization of the cells. These antibodies were examined further by flow cytometry on purified nuclei. RESULTS: Paraformaldehyde-methanol treatment of dendritic cells is a good combination to visualize NF-kappaB translocation by confocal microscopy. Three of the four antibodies tested gave good results on nonactivated and on lipopolysaccharide (LPS)-activated dendritic cells. The measurement of NF-kappaB translocation by flow cytometry on purified nuclei is a quick and sensitive method. Only one of the four evaluated antibodies showed a significant difference between nonactivated and activated cells. CONCLUSIONS; Microscopy and flow cytometry are quick and reproducible methods to measure NF-kappaB translocation and can be adapted to identify new molecules that activate dendritic cells.

Animals↗
PubMed2001

Morphology of corneal nerves using confocal microscopy.

PURPOSE: The aim of the current study was to evaluate the distribution and morphology of corneal nerves as seen by means of white light confocal microscopy. METHODS: This study analyzed images of corneal nerves that were obtained using the Tomey Confoscan slit scanning confocal microscope (40x/0.75 objective lens). The images were classified according to their location within the cornea. The objective and subjective evaluation of the images involved measuring, grading, or judging a number of parameters from both individual pictures and from each single nerve fiber within any image. RESULTS: The in vivo observations made in this work are in agreement with those of previous histologic studies. The general scheme of corneal innervation is described as originating from thick and straight stromal nerve trunks that extend lateral and anteriorly and give rise to plexiform arrangements of progressively thinner nerve fibers at several levels within the stroma. From there, nerve fibers perforate Bowman's layer and eventually form a dense neural plexus just beneath the basal epithelial cell layer, which is characterized by tortuous and thin beaded nerve fibers interconnected by numerous nerve elements; nerve fibers from this plexus are known to be responsible for the innervation of the epithelium. CONCLUSION: This study provides convincing evidence of the suitability of confocal microscopy to image corneal nerves, the only drawback being the limited resolution in terms of the differentiation of the ultrastructure of nerve bundles.

Adult↗
PubMed2006

Quantitative analysis of immunofluorescent punctate staining of synaptically localized proteins using confocal microscopy and stereology.

We established a protocol for the immunofluorescent detection of glutamate receptor subunits at synaptic sites using laser scanning confocal microscopy and stereological procedures. An in vitro model of eyeblink classical conditioning from turtles was used for this study. Triple-labeling of the presynaptic marker synaptophysin, the NR1 subunit of NMDA receptors, and the GluR4 subunit of AMPA receptors was performed on pseudoconditioned (control) and conditioned in vitro brain stem preparations in which punctate staining for each individual protein, as well as for the colocalization of GluR4 and NR1 with synaptophysin, was analyzed. For every tissue section analyzed, images of two consecutive optical planes were taken using confocal microscopy. Protein puncta were counted in one optical section (sample section) if they were not present in the optical section immediately above the sample section (look-up section). We found a significant increase in the colocalization of GluR4-containing AMPA receptors with synaptophysin after conditioning compared with the control group. Colocalization of NR1 subunits with synaptophysin was unchanged after conditioning. The described protocol, therefore, can be used for the quantitative analysis of changes in synaptic localization of different types of proteins. The protocol is designed to provide a more accurate and uniform approach in studying receptor trafficking during various forms of synaptic plasticity.

Animals↗
PubMed2006

In vivo confocal microscopy in fungal keratitis.

BACKGROUND: Fungal keratitis is a major blinding eye disease found throughout the world, particularly in developing countries. Given the recent increase in Fusarium keratitis infections in contact lens wearers owing to contact lens solutions, a warning was recently issued by the Food and Drug Administration, making it a public health concern in developed countries. OBJECTIVE: To show the advantages of in vivo confocal microscopy imaging using the Heidelberg Retina Tomograph II-Rostock Cornea Module (HRTII-RCM) in the early diagnosis of fungal keratitis. METHODS: HRTII-RCM confocal microscopy was performed on five patients presenting with fungal keratitis and on three donor corneas contaminated with Fusarium solani, Aspergillus fumigatus and Candida albicans. RESULTS: Direct microscopic evaluation of corneal smears and culture revealed the presence of F solani in four cases and C albicans in one case. HRTII-RCM examination of the infected patients and contaminated donor corneas revealed numerous high-contrast elements resembling Fusarium, Aspergillus hyphae or Candida pseudofilaments in the anterior stroma. CONCLUSION: HRTII-RCM in vivo confocal microscopy is a new, non-invasive and rapid technique for the early diagnosis of fungal keratitis, showing high-resolution images resembling fungal structures in the early phase of the disease.

Adult↗
PubMed2000

Confirmation of onychomycosis by in vivo confocal microscopy.

Onychomycosis is common in adults and constitutes 20% of all nail disease. Widely used methods to confirm a clinical diagnosis of onychomycosis often yield negative results (ie, potassium hydroxide [KOH] preparation) or are slow (ie, dermatophyte cultures). We report a case of onychomycosis in which we used in vivo confocal microscopy to diagnose the disease; we also correlated our findings with results from routine KOH preparations. On the basis of our findings, we hypothesize that in vivo confocal microscopy may be faster and more accurate than the conventional microscope used in KOH preparations in the diagnosis of onychomycosis.

Adult↗
PubMed2005

Non-invasive visualization of melanin and melanocytes by reflectance-mode confocal microscopy.

In vivo visualization of epidermal melanin was performed by reflectance-mode confocal microscopy (RCM). Firstly, we examined the distribution of epidermal melanin in pigmented animals and compared with that of the human skin. Melanocytes in the skin of pigmented animals were found to accumulate a large amount of melanin that can be easily visualized because of its brightness. Their RCM images correlated well with the Fontana-Masson-stained sections for melanin. In contrast, in the human skin, typical dendritic melanocytes were hardly observed even in pigmented lesions, although supranuclear melanin caps were easily visible. These results suggested that human melanocytes rapidly transfer the produced melanin to keratinocytes and do not accumulate it. Secondly, to elucidate the production of melanin by human melanocytes, we evaluated the changes of melanin after a single ultraviolet (UV) exposure. The melanin-accumulating melanocytes were clearly visualized during the skin pigmentation process. The RCM images showed the brightness because of melanin gradually increased from day 4, then dendrite-elongated melanocytes appearing from day 8, and finally melanin caps formed from day 29. In conclusion, RCM successfully evidenced the difference in melanin distribution between the pigmented animals and humans, and the UV-induced pigmentation process in vivo as well.

Animals↗
PubMed1997

[Observation of the free calcium of lens epithelial cell using laser scanning confocal microscopy].

OBJECTIVE: Using a new fluorescent Ca++ indicator (fluo-3) and laser scanning confocal microscopy (LSCM) to monitor the intracellular Ca++ of rat lens epithelial cell (LEC). METHODS: The LEC was loaded with fluo-3, then the distribution of free Ca++ in the LEC was observed by using LSCM monitoring the distribution of fluorescence that is given due to the chelation between fluo-3 and Ca++. At last, ionophore (A23187) and heavy-metal ions (Mn++) were applied to calibrate fluo-3 intensities to obtain [Ca++]i value. RESULTS: The free calcium in LEC is mainly located in the nucleus. Free calcium concentration of rat LEC is 259.79 +/- 49.24 nmol/L. CONCLUSION: Fluo-3 can be loaded into LEC by incubation with acetoxymethyl (AM) ester of the dye, and LSCM can be used to observe the cellular free calcium directly by surveying the fluorescence. This method will pave a way technically for the further study of intracellular free Ca++ of the LEC.

Animals↗
PubMed2006

Confocal microscopy in multiple myeloma associated crystalline keratopathy: case report.

We report on the confocal microscopical findings of both corneas of a patient presenting a multiple myeloma associated crystalline keratopathy and the response to treatment. Blurred vision was the first sign of progression of the multiple myeloma. Confocal microscopy images show needle-like structures in the epithelium and stroma of the cornea which regressed after treatment of the multiple myeloma.

Corneal Opacity↗
PubMed2003

Corneal wound healing after photorefractive keratectomy: a 3-year confocal microscopy study.

PURPOSE: To perform a sequential quantitative analysis of corneal wound healing after photorefractive keratectomy (PRK) by using confocal microscopy in vivo. METHODS: In a prospective, nonrandomized, comparative trial performed in an institutional setting, 24 eyes of 14 patients received PRK to correct refractive errors between -1.25 and -5.75 D. Central corneas were examined preoperatively and at 1 day, 5 days, and 1, 3, 6, 12, 24, and 36 months after PRK by using confocal microscopy. A masked observer randomly examined 3 to 6 confocal scans per eye per visit to determine epithelial and stromal thickness, keratocyte density in 5 anterior-posterior stromal layers, corneal nerve density in the subbasal region and the stroma, and corneal light backscattering (corneal haze). RESULTS: Epithelial thickness increased 21% (P < .001) by 12 months after PRK and thereafter remained unchanged to 36 months after PRK. There was no change in stromal thickness between 1 and 36 months after PRK (P = .35). The dense keratocyte population in the preoperative anterior 10% of the stroma (32,380 +/- 5,848 cells/mm3) that was partially or completely removed during photoablation was not reconstituted at 36 months in the anterior 10% of the post-PRK stroma (17,720 +/- 4,308 cells/mm3, P < .001). Subbasal nerve fiber bundle density was decreased 60% at 12 months after PRK (P < .001) before returning to densities at 24 and 36 months after PRK that were not significantly different from preoperative values (P = 1.0). Activated keratocytes and corneal haze peaked at 3 months after PRK. CONCLUSIONS: Wounding of the cornea by PRK alters the normal structure, cellularity, and innervation of the cornea for up to 36 months.

Adult↗
PubMed1993

Visualization of synaptic structure and function with confocal microscopy: calcium fluctuations and oscillations.

This article summarizes the basic principles of confocal microscopy and how they can be employed to visualize synaptic structure and function. Optical 'sectioning' of living cells allows the examination of a large number of biological processes at different subcellular localities. Different fluorescent markers enable the study of processes in the extracellular, intracellular and membrane domains of the nerve cell. The excellent spatial resolution of confocal microscopy permits to study the changes in intracellular calcium concentration in single synaptic boutons, without a substantial interference from supporting cells. Intracellular calcium concentration shows coordinated fluctuations in space and periodic oscillations. Periodic oscillations can serve as time keeping devices in nerve terminals. Oscillations were previously observed also in the process of transmitter release. We speculate therefore that these calcium oscillations may be of significance, if the quantal transmitter release is governed by a sequence of calcium dependent steps, which have a different affinity for calcium.

Animals↗
PubMed1994

Probing individual molecules with confocal fluorescence microscopy.

Confocal fluorescence microscopy coupled with a diffraction-limited laser beam and a high-efficiency detection system has been used to study the diffusive movement and emission process of individual fluorescent molecules in the liquid phase at room temperature. The high detection sensitivity achieved at fast data acquisition speeds (greater than 1 kilohertz) allows real-time observation of single-molecule fluorescence without statistical analysis. The results show fluorescence-cycle saturation at the single-molecule level and multiple recrossings of a single molecule into and out of the probe volume as well as the triplet state.

Fluorescein↗
PubMed2003

Detection of rabbit corneal epithelium over amniotic membrane patch by white-light and laser confocal microscopy.

PURPOSE: To report confocal microscopic observation of rabbit corneal epithelium through amniotic membrane patch (AMP). DESIGN: Experimental study. METHOD: Six rabbit eyes were patched by amniotic membrane. Then, white-light and laser confocal microscopic observation was performed. RESULTS: Images of human amniotic epithelium, amniotic basement membrane, and amniotic stroma were obtained using both devices, followed by detection of rabbit corneal epithelium. CONCLUSION: This study indicates that observation of corneal epithelium through AMP is possible in rabbit eyes using both these methods. This implies that both devices might be used clinically to observe the epithelial healing process under an AMP to better determine when it should be removed.

Amnion↗
PubMed2006

Evaluation of confocal microscopy system performance.

The confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. When tests are made to evaluate the performance of a CLSM, the usual subjective assessment is accomplished by using a histological test slide to create a "pretty picture." Without the use of functional tests, many of the machines could be working at suboptimal performance levels, delivering suboptimum performance and possibly misleading data. To replace the subjectivity in evaluating a confocal microscope, tests were derived or perfected that measure field illumination, lens clarity, laser power, laser stability, dichroic functionality, spectral registration, axial resolution, scanning stability, photomultiplier tube quality, overall machine stability, and system noise. These tests will help serve as a guide for other investigators to ensure that their machines are working correctly to provide data that are accurate with the necessary resolution, sensitivity, and precision. Utilization of this proposed testing approach will help eliminate the subjective nature of assessing the CLSM and allow different machines to be compared. These tests are essential if one is to make intensity measurements.

Animals↗
PubMed2005

Configuration of the connective tissue in the posterior atlanto-occipital interspace: a sheet plastination and confocal microscopy study.

STUDY DESIGN: The connective tissue structures in the posterior atlanto-occipital region were investigated using E12 sheet plastinations and confocal microscopy. OBJECTIVES: To define the relationship between rectus capitis posterior minor (RCPm), posterior atlanto-occipital (PAO) membrane, nuchal ligament, and the spinal dura in the PAO interspace. SUMMARY OF BACKGROUND DATA: It has been speculated that connections between the dura and muscles and/or ligaments in the PAO interspace may transmit forces from the cervical spine joint complexes to the pain-sensitive dura, generating cervicogenic headaches. Anatomic structures involved in these connections include the RCPm, PAO membrane, and nuchal ligament. However, there is little information about the nature of these connections and the relationships between these anatomic structures. METHODS: The study used a combined approach, consisting of the gross anatomic dissection of nine cadavers and the E12 sheet plastination method for thirteen adult human cadavers, five of which were further examined using confocal microscopy. RESULTS: The study demonstrates that (1) the tendinous fibers from the medial and deep part of the RCPm muscle are continuous antero-inferiorly with the spinal dura; (2) the PAO membrane is part of the RCPm fascia and tendon and the perivascular sheathes; (3) antero-inferiorly the PAO membrane fuses with the spinal dura rather than the atlas; and (4) the nuchal ligament does not exist in the PAO interspace. CONCLUSIONS: The connective tissue structures that connect the spinal dura to the RCPm muscle in the PAO interspace are the RCPm fascia and tendinous fibers and perivascular sheathes.

Aged↗
PubMed1995

Using laser scanning confocal microscopy as a guide for electron microscopic study: a simple method for correlation of light and electron microscopy.

Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large or have fine branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of light microscopic appearance and electron microscopic detail is very time-consuming. We report here a simple method combining laser scanning confocal microscopy and electron microscopy for study of the synaptic relationships of the neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Neurons were labeled intracellularly with neurobiotin or biocytin, two widely used stains. The tissue was then sectioned on a vibratome and processed with both streptavidin-nanogold (for electron microscopic study) and streptavidin-Cy3 (for confocal microscopic study) and embedded in epon/araldite. Interesting areas of the labeled neuron were imaged in the epon/araldite blocks with laser scanning confocal microscopy and then thin-sectioned at the indicated depth for electron microscopic study. This method provides an easy, reliable way to correlate three-dimensional light microscopic information with electron microscopic detail, and can be very useful in studies of synaptic connections.

Animals↗