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Confocal microscopy of false-negative breast aspirates.

Even when using a novel optimized FNA-21 for breast fine-needle aspiration biopsy, our series of 163 breast lesions with consecutive histology, including 110 carcinomas, contained eight cancer cases with a negative cytologic diagnosis. Two consisted exclusively of thick, undiagnosable epithelial fragments and were devoid of detached malignant cells on which the proper diagnosis could have been made. Optical sectioning of the original smears by the confocal microscope allowed us to reclaim from these epithelial fragments the diagnostic abnormal chromatin patterns, even in the air-dried smears. In the thin optical sections the cancerous tubular or cribriform growing pattern was visualized. The high spatial resolution of the confocal microscope can be further exploited with the application visualization system (AVS). The AVS three-dimensional reconstructions allow us to see the surface of these malignant cells in a detail approaching that of scanning electron microscopy. Furthermore, the internal aspects of the malignant nuclei can be visualized with these techniques.

Biopsy, Needle↗

Use of flow cytometry and confocal microscopy techniques to investigate early CdCl(2)-induced nephrotoxicity in vitro.

CdCl(2) is a well-known toxic compound for the kidney in vivo and in vitro. We report here part of the results of an ECVAM (European Centre for the Validation of Alternative Methods) contract study, aimed at establishing and assessing several flow cytometric and confocal microscopic endpoints for use in an in vitro nephrotoxicity model. Three renal tubule cell lines, OK (opossum, proximal tubule origin), LLC-PK1 (pig, proximal tubule origin) and MDCK (dog, distal tubule origin) were exposed for 1, 5 and 24 h to 25 microM and 100 microM CdCl(2). The results obtained for mitochondrial membrane potential showed a decrease in all the cell lines after 5 h of treatment with both CdCl(2) concentrations. In some cases, this decrease was detected by flow cytometry after a 1-h exposure. On the contrary, intracellular Ca(2+) increased in a time-dependent and concentration-dependent fashion. This increase was especially high in the MDCK cell line after a 24-h exposure to 100 microM CdCl(2). However, cell viability was not affected by 25 microM CdCl(2). Our results demonstrate early changes in mitochondrial membrane potential and cytoplasmic Ca(2+) levels in renal tubular epithelial cell lines treated with CdCl(2).

Animal Testing Alternatives↗

[Evaluation of diffuse lamellar keratitis after LASIK with confocal microscopy].

OBJECTIVE: To investigate the clinical pathologic characteristics and the pathogenesis of diffuse lamellar keratitis (DLK) after laser in situ keratomileusis (LASIK). METHODS: On 30 patients with 39 eyes being diagnosed with DLK (grade I-grade IV) after LASIK, slit-lamp examinations were performed 1, 3, 5, 7 and 30 days postoperatively, and scanning slit confocal microscope in vivo were performed 3, 7 and 30 days postoperatively. RESULTS: Typical characteristics of grade I and grade II DLK by confocal microscope examination showed up at 3 days postoperatively. We found that the epithelium, the posterior stroma and the endothelium were normal. In the lamella in front of the incision, many round or oval-shaped cells with diameter of approximately 12 approximately 20 microm were detected. These cells had eccentric, highly reflective nuclei and less reflective intracellular structures. They were most likely to be mononuclear cells. These cells distributed diffusely or arranged in line shape. In the lamella, clusters and lines of small, highly reflective, irregular shaped cells 8 to 10 microm in diameter were seen. They were similar to granulocytes or lymphocytes. Seven days postoperatively, these cells almost disappeared. Grade III DLK appeared from 3 to 5 days postoperatively, with more dense infiltration and more highly reflective irregular shape materials in the lamella. Grade IV DLK appeared 5 to 7 days postoperatively. Anterior stroma structure became unclear, with highly reflective and folded corneal flap. Numerous highly reflective scarring formed late. CONCLUSIONS: DLK after LASIK is a inflammatory response in corneal lamella, its typical pathologic characteristic is inflammatory cells infiltration in the lamella. Several factors work together to cause DLK through endogenous and exogenous mechanisms.

Cornea↗

A preferential pole for exocytosis in cultured chromaffin cells revealed by confocal microscopy.

Histological studies suggest that adrenal medulla chromaffin cells in situ are polarised, but functional evidence is lacking. We present here the first demonstration for polarisation of exocytosis in isolated, spherical, bovine chromaffin cells. Cells were stimulated with 70 mM K(+) to cause a marked enhancement of catecholamine release, monitored amperometrically. FM1-43 and dopamine beta-hydroxylase antibodies provided fluorescence confocal pictures that were 2-3-fold more intense in the bottom of the cells, as compared to equatorial or apex planes. This suggests that the solid phase to which the cell attaches serves as a 'trophic' signal for the polarisation of its secretory apparatus.

Animals↗

Sources of scattering in cervical tissue: determination of the scattering coefficient by confocal microscopy.

Most models of light propagation through tissue assume that the scattering properties of various tissue layers are the same. We present evidence that the scattering coefficient of cervical epithelium varies by a factor of 3 within the epithelium owing to variations in nuclear density and to the presence of keratin. We estimated the scattering coefficient from regions of normal and precancerous cervical epithelium by fitting reflectance measurements from confocal images to an exponential function of depth based on Beer's law of attenuation. The results suggest that the normal cervix is characterized by highly variable scattering in the superficial epithelium, low scattering in the intermediate epithelium, and high scattering in the basal and stromal regions. In high-grade dysplasia, high scattering from high-density nuclei is observed throughout the entire epithelium.

Algorithms↗

Confocal microscopy using variable-focal-length microlenses and an optical fiber bundle.

The use of variable-focal-length (VFL) microlenses can provide a way to axially scan the foci across a sample by electronic control. We demonstrate an approach to coupling VFL microlenses individually to a fiber bundle as a way to create a high-throughput aperture array with a controllable aperture pattern. It would potentially be applied in real-time confocal imaging in vivo for biological specimens. The VFL microlenses that we used consist of a liquid-crystal film sandwiched between a pair of conductive substrates for which one has a hole-patterned electrode. One obtains the variation of the focal length by changing the applied voltage. The fiber bundle has been characterized by coupling with both coherent and incoherent light sources. We further demonstrate the use of a VFL microlens array in combination with the fiber bundle to build up a confocal system. The axial response of the confocal system has been measured without mechanical movement of the sample or the objective, and the FWHM is estimated to be approximately 16 microm, with asymmetric sidelobes.

Equipment Design↗

Refractive index of tissue measured with confocal microscopy.

Refractive index of tissue is an essential parameter in many bio-optical experiments, yet little data can be found in literature. Several methods have been proposed to measure refractive index in tissue samples, but all have specific limitations, such as low accuracy, the need for large amounts of tissue, or the complexity of the measurement setup. We propose a new method using a standard confocal microscope and requiring only small tissue samples. A thin slice of tissue is put next to a layer of immersion fluid of exactly the same thickness. The actual thickness of the fluid layer is directly measured with the microscope, as there is no refractive index mismatch. A difference between index of refraction of the tissue and of the immersion medium causes an axial scaling factor. The optical thickness of the specimen is thus measured with the microscope, and as its actual thickness equals the known thickness of the fluid layer, the axial scaling factor is readily determined. From this factor, we calculate the refractive index of the tissue. We use a diffraction model to take the point spread function (PSF) of the microscope into account, so we can determine the index of refraction to a very high accuracy. We demonstrate the method on bovine muscle tissue and find a value of n=1.382+/-0.004, at 592 nm.

Algorithms↗

Localization of ribosomal genes in human spermatogonia by fluorescent in situ hybridization and confocal microscopy.

The distribution of ribosomal RNA genes in nucleoli is still a matter of controversy. We have investigated the nucleolus of human type A spermatogonia, which displays a single, large fibrillar center. Silver-staining was used to localize the fibrillar center by light microscopy. Fluorescent in situ hybridization was performed on the same cell, using a 5.8-kb probe specific for a transcribed region of the ribosomal genes. The fluorescent area exactly corresponded to the silver-stained area. The three-dimensional distribution of rDNA was studied in horizontal and orthogonal sections using a confocal laser scanning microscope. The presence of the fluorescent signal throughout the whole silver-stained structure demonstrated that the fibrillar center, and at least a part of the dense fibrillar component, contained most of the rDNA.

Cell Nucleolus↗

IgG-induced Ca2+ oscillations in differentiated U937 cells; a study using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes.

We have investigated, at the single cell level, intracellular Ca2+ ([Ca2+]i) modulations triggered by the high affinity receptor for IgG, Fc gamma RI, in the monocytic cell line, U937. Cells were co-loaded with the Ca(2+)-sensitive dyes, Fluo-3 and Fura-Red, by incubation with their acetoxymethyl (AM) esters and confocal ratio imaging was used to monitor the [Ca2+]i changes induced by antibody cross-linking of IgG-loaded Fc gamma RI. A single Ca2+ spike was observed in 81% of untreated cells whereas dibutyryl cAMP-induced differentiation into a more macrophage cell type resulted in a sub-population of cells (44%) responding to receptor cross-linking with calcium oscillations. This change in calcium signalling may explain the difference in functional responses triggered by Fc gamma RI in monocytes and macrophages. Analysis of the Fluo-3 and Fura-Red fluorescence, after AM-ester loading, showed that both dyes have similar photobleach rates and intracellular localization allowing compensation for shifts in focal plane, dye photobleaching and non-uniformity of dye loading. In addition, because the binding kinetics of both dyes are equivalent, accurate temporal information can be gained about [Ca2+] changes. There are, however, two major problems with this dual indicator technique. Firstly, loading from AM esters results in considerable variation between cells in the intracellular concentration ratio of the two dyes, making calibration difficult. Secondly, the fluorescence ratio, Fluo-3/Fura-Red, behaves non-linearly at Ca2+ concentrations less than approximately 500 nM and comparison with Fura-2-loaded single cell photometry studies suggests there is considerable amplitude distortion of the signal when the ratios are displayed on a linear scale. These problems may considerably limit the application of Fluo-3/Fura-Red ratiometric measurements.

Adenosine Triphosphate↗

[Evaluation of confocal microscopy in the analysis of the external trabecular membrane during deep nonpenetrating sclerectomy].

PURPOSE: Deep non penetrating sclerectomy (DNPS) is a new filtering surgical procedure whose aim is a selective ablation of the external portion of the trabecular meshwork (TM) which is involved in the aqueous outflow resistance, i.e. the inner wall of Schlemm's canal (SC) and the external trabecular layers, especially the cribriform TM. We evaluated, with a confocal microscope, the structural characteristics of this part of the TM in glaucomatous patients. METHODS: Thirty-six external trabecular membranes (ETM) were obtained from 33 consecutive glaucomatous patients (mean age: 56.5+/-14.5 years) and from four post-mortem normal donors (60.5 +/-7.7 years), which underwent DNPS according to the same surgical procedure. Under conjunctival and scleral flaps, the roof of the SC was opened and removed. A deeper dissection led to the removal of the inner wall of the SC and the adjacent ETM, i.e the TEM, which allowed a satisfactory aqueous flow through the remaining internal TM layers. After fixation with acetone or triton X100 and immunostaining with anti-fibronectin or vimentin antibodies, the samples were analyzed with a confocal microscope (Nikon EZ 2000). RESULTS: The mean thickness of the ETM was 34.4+/-7.3 microm in glaucomatous eyes, not significantly different from the controls (39.0+/-10.7 microm). The main characteristic of the glaucomatous ETM membrane is a paucicellularity as compared to the controls (respectively 21.6+/-12.1 cells/area and 156.1+/-28.8 cells/area). The confocal microscope analysis shows that the ETM involves two different portions of the trabecular meshwork. The architectural characteristics of the outermost portion of the ETM with its star-shaped cells arranged in a homogenous extra-cellular matrix, suggest that it is the trabecular cribriform meshwork. The inner portion of the ETM shows cells arranged regularly in a fibrillar extra-cellular matrix as described in the corneoscleral trabecular meshwork. CONCLUSION: These results confirm the previous reported histopathological changes of the glaucomatous TM. The thickness of the removed ETM and its structural characteristics show that the ablation of the TM layers necessary to obtain a satisfactory aqueous filtration, not only involves the cribriform layers but also one of the inner parts of the TM, i.e. the corneoscleral TM. This result suggests that aqueous humor resistance not only involves the cribriform trabecular meshwork but also a part of the corneoscleral trabecular meshwork.

Aged↗

Patterns of morphological variation within myelin internodes of normal peripheral nerve: quantitative analysis by confocal microscopy.

Knowledge of variations in the morphology of normal myelinated peripheral nerve fibres is fundamental to subsequent interpretation of neuropathology. It would be advantageous for structural analysis of normal variations to be based on entire myelin internodes, but acquisition of such data via the classic approach of nerve fibre teasing has been hindered by limitations in optical resolution and specimen preparation. This study addressed these limitations through a new confocal imaging method which permits detailed visualisation of individual myelinated fibres in intact peripheral nerve trunks, and quantitated previously unrecognised patterns of morphological variation within normal internodes. The study focused particularly on Schmidt-Lanterman incisures, the narrow cytoplasmic channels which traverse normal compact myelin and provide foci for disruption of the compact sheath in a number of peripheral neuropathies. Analysis was based on confocal fluorescence images of multiple sequential internodes, traced within posterior tibial nerve trunks of adult male Sprague-Dawley rats. The strength of relationships between internodal size variables (length, fibre diameter, myelin sheath thickness) and total number of incisures per internode were documented. Each internode was divided into 4 regions of equivalent length (regions 1-4), and variations in the distribution of incisures and Schwann cell nuclear location were evaluated. Regional variations were consistent, irrespective of differences in fibre diameter, myelin sheath thickness, and internodal length. Expressed in terms of proximodistal orientation, there was a unimodal distribution of incisures within internodes of this fibre population (diameter range 5-9 microns), with region 3 containing the highest number of incisures and region 4 the lowest (P < 0.05). The Schwann cell nucleus was located more frequently in region 3 than in region 2 (P < 0.01). Contrary to previous reports, an incisure was found in close association with the nucleus in at least 50% of internodes. Documentation of frequent incisure-nuclear association and consistent patterns of variation within internodes extends knowledge of the microanatomy of normal peripheral nerve, and may provide insight into the functional role of incisures. Demonstration of such patterns in normal nerve may contribute to the understanding of pathological change, for example progression of ovoid formation from midinternodal regions during wallerian degeneration.

Animals↗

Technology insight: Laser-scanning confocal microscopy and endocytoscopy for cellular observation of the gastrointestinal tract.

Recent advances in endoscopic imaging technology have enabled the visualization of early-stage cancer and its precursors in the gastrointestinal tract. Chromoendoscopy, magnifying endoscopy, endoscopic optical coherent tomography, spectroscopy, and various combinations of these technologies, are all important for the recognition of small and unclear lesions. To observe cancer cells in vivo, two types of ultra-high magnifying endoscope--'laser-scanning confocal endoscopy series' and 'contact endoscopy series'--that have a maximum of more than 1,000x magnifying power have been developed. These endoscopes can generate high-quality images of both living cancer cells and normal cells in the gastrointestinal tract, with a quality comparable to that possible with conventional cytology. These novel imaging technologies may make in vivo histological diagnosis by virtual histology possible.

Endoscopy, Gastrointestinal↗

Chronic ethanol modulates delta and mu-opioid receptor expression in rat CNS: immunohistochemical analysis with quantitiative confocal microscopy.

Ethanol consumption affects levels of endogenous opioids as well as opioid receptors in both animals and humans. We studied the expression of delta (delta) and mu (mu) opioid receptors (ORs) in brain sections of adult male Sprague-Dawley rats after 2 weeks of consuming ethanol in a liquid diet, with comparisons to sections from pair-fed control animals. Immunohistochemical staining for the ORs, using selective antibodies, and quantitation of confocal images, revealed increased expression of delta-ORs in hippocampal CA1 of the chronic ethanol-treated rats. In contrast, mu-ORs decreased in their expression after ethanol treatment in multiple brain areas, including cortex, hippocampus, midbrain colliculi, striatum and nucleus accumbens. The alterations in immunoreactive OR expression may be related to reduced functional coupling of the ORs to G-proteins, as found in prior studies in several brain regions, using the same chronic ethanol diet protocol. Changes in OR expression and functional coupling in the CNS may be factors in ongoing ethanol consumption and tolerance.

Alcoholism↗

Limitations on optical sectioning in live-cell confocal microscopy.

In three-dimensional (3-D) live-cell microscopy, it has been common to treat cells as having a constant refractive index (RI). Although the variations in RI associated with the nucleus and other organelles were recognized from phase- and differential interference contrast (DIC) images, it was assumed that they were small and would not affect 3-D fluorescence images obtained using widefield/deconvolution, confocal of multiphoton imaging. This paper makes clear that this confidence was misplaced. Confocal images made using backscattered light (BSL) to image the flat, glass/water interfaces above and below living microscope specimens should reveal these structures as flat and featureless. That the image of the interface on the far side of the cells is neither flat nor featureless indicates that the "optical section" surface can be profoundly distorted by the RI irregularities associated with the presence of nuclei and other subcellar structures. This observation calls into question the reliability of images made using any of the current methods for performing 3-D light microscopy of living cells.

Cheek↗

Detection of doubly stained fluorescent specimens using confocal microscopy.

Studies of doubly stained specimens were performed with a confocal scanning microscope. The instrument used provides the possibility of making separate detections of the fluorescent dyes. The optimal choice of excitation wavelengths and optical filters are discussed. The fluorphores that were used are Lucifer Yellow, Texas Red, fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate.

Animals↗

Intranuclear co-location of newly replicated DNA and PCNA by simultaneous immunofluorescent labelling and confocal microscopy in MCF-7 cells.

The intranuclear distribution of newly replicated DNA and of the proliferating cell nuclear antigen (PCNA) was mapped by confocal laser scanning microscopy after simultaneous immunofluorescent labelling of incorporated bromodeoxyuridine (BrdUrd) and PCNA. A mild hydrolysis with HCl followed by an enzymic digestion of DNA was used to produce single-stranded DNA required for BrdUrd immunorevelation, since this procedure preserves PCNA antigenicity. Optical sections obtained with a laser scanning microscope clearly showed a similar distribution of PCNA and BrdUrd within the nuclei, thus confirming previous observations on parallel labelled synchronized cultures. The intranuclear distribution of PCNA and BrdUrd varies concomitantly during the S phase of MCF-7 cells.

Autoantigens↗

Segmentation and counting of FISH signals in confocal microscopy images.

In this paper we have presented a semi-automatic method for segmenting and counting the Fluorescence In Situ Hybridization (FISH) signals per cell nucleus in a 3D tissue image. The number of FISH signals indicate the gain (trisomy) or loss (monosomy) of certain base-sequences in deoxyribonucleic acid (DNA). The quantitative evaluation of the loss or gain in DNA is necessary in qualitative diagnostic molecular pathology. Multispectral volumetric images are obtained using the Confocal Microscope. Each consists of a red channel depicting the 3D morphology of the tissue and green channel containing the FISH signals. The red channel tissue image is segmented first to determine the membership of the FISH signal to a particular nuclei. Various segmentation methods starting from simple local thresholding to volume growing and active volumes are used for segmentation. A brief comparative study of the visual signal count by pathologist and our automatic count is also presented.

Cell Nucleus↗

Quality assessment of confocal microscopy slide-based systems: instability.

BACKGROUND: All slide-based fluorescence cytometry detections systems basically include an excitation light source, intermediate optics, and a detection device (CCD or PMT). Occasionally, this equipment becomes unstable, generating unreliable and inferior data. METHODS: A number of tests have been devised to evaluate equipment performance and instability. The following four instability tests are described: galvanometer scanning, stage drift, correct wavelength spectral detection, and long-term laser power. RESULTS: Quality assurance tests revealed that a confocal microscope can become unstable in the following parameters, yielding inaccurate data: laser power, PMTs functionality, spectrophotometer accuracy, galvanometer scanning and laser stability, and stage drift. Long-term laser power stability has been observed to vary greatly. CONCLUSIONS: Confocal systems can become unstable in the following parameters: long-term laser power, galvanometer scanning, spectrophotometer accuracy, and stage stability. Instability in any of these parameters will affect image quality. Laser power fluctuations result from either a defective Acousto-optic tunable filter or improper heat dissipation. Spectrophotometer instability will generate unreliable spectra data, extra light reflections, and poor image quality. Galvanometer scanning instability yields poor image quality while microscope stage drift results in a sample going out of the plane of focus. With minor modifications, these tests may be applicable to other slide-based systems.

Animals↗