Confocal fluorescence microscopy and three‐dimensional reconstruction

Wright, Shirley J.; Schatten, Gerald

doi:10.1002/jemt.1060180103

Cited by 31 publications

(17 citation statements)

References 39 publications

(66 reference statements)

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“…By combining a number of optical sections, regis tered at different depths, a relatively fast three-dimensional reconstruction o f the specimen can be made [Carlsson et al, 1989;Carlsson. 1991;Wright and Schatten. 1991], Digital image processing can be used to determine surface features, area and volume analysis of given structures, and views of the total structure from any angle in three dimensions [Carlsson, 1991].…”

Section: Discussionmentioning

confidence: 99%

Measurement of Enamel Demineralization using Microradiography and Confocal Microscopy

Fontana¹,

Li²,

Dunipace³

et al. 1996

Caries Res

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Substantial amounts of tooth minerals are lost during dental caries formation. Transversal microradiography, a well-accepted method used to quantify mineral loss, is a time-consuming technique which requires a thin enamel section (100 µm) and involves the use of x-rays. In an attempt to solve these difficulties, a procedure has been developed in which a human tooth specimen with demineralized enamel is cut in half (HT), stained with a fluorescent dye (rhodamine B) and analyzed using a laser scanning confocal microscope. A series of three studies was conducted to correlate measurements of enamel demineralization obtained from enamel thin (100 µm) sections (TS) using transversal microradiography with three parameters (area of the lesion; total and average dye fluorescence intensities) measured on the same TS or on a thicker section (HT) of the same specimen by laser scanning confocal microscopy. Results showed that a 0.1 mM rhodamine B solution provided the most adequate imaging conditions for confocal microscopy. Pearson’s correlation coefficients, calculated between microradiography and confocal microscopy data obtained using a 0.1 mM rhodamine B solution, were: ΔZ vs. HT lesion area = 0.95; ΔZ vs. HT total fluorescence = 0.80; ΔZ vs. HT average fluorescence = 0.74; ΔZ vs. TS lesion area = 0.95; ΔZ vs. TS total fluorescence = 0.74; ΔZ vs. TS average fluorescence = 0.55. All these correlations coefficients were statistically significant (p < 0.01). It is concluded that in enamel demineralization studies statistically significant correlations exist between parameters measured using transversal microradiography and parameters quantified using confocal microscopy.

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Section: Discussionmentioning

confidence: 99%

Measurement of Enamel Demineralization using Microradiography and Confocal Microscopy

Fontana¹,

Li²,

Dunipace³

et al. 1996

Caries Res

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“…Teased fibres are subject, however, to preparation artefacts and interpretation is limited further by the inherent 0300-4864/94 9 1994 Chapman and Hall disruption of neighbouring, potentially significant, cellular and extra-cellular relationships (Murray, 1992). Alternatively, serial sectioning and subsequent 3-dimensional reconstruction can be used to investigate the course and structure of individual fibres (Fraher, 1978a,b;Kidd & Heath, 1988a,b;Tuisku & Hildebrand, 1992) but this approach is technically demanding and rarely undertaken (Wright & Schatten, 1991;Murray, 1992).…”

Section: Introductionmentioning

confidence: 97%

Imaging myelinated nerve fibres by confocal fluorescence microscopy: individual fibres in whole nerve trunks traced through multiple consecutive internodes

et al. 1994

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Current methods of morphological analysis do not permit detailed imaging of individual myelinated fibres over substantial lengths without disruption of neighbouring, potentially significant, cellular and extracellular relationships. We report a new method which overcomes this limitation by combining aldehyde-induced fluorescence with confocal microscopy. Myelin fluorescence was intense relative to that from other tissue components, enabling individual myelinated nerve fibres to be traced for distances of many millimeters in whole PNS nerve trunks. Image obtained with a Bio-Rad MRC-600 confocal laser scanning microscope clearly displayed features of PNS and CNS myelinated fibres including nodes of Ranvier; fibre diameter; sheath thickness and contour; branch points at nodes; as well as (in the PNS) Schmidt-Lanterman incisures and the position of Schwann cell nuclei. Direct comparisons using the same specimens (whole nerve trunks; also teased fibres) showed confocal imaging to be markedly superior to conventional fluorescence microscopy in terms of contrast, apparent resolution and resistance to photobleaching. Development of the fluorophore was examined systemically in sciatic nerves of young adult rats. In separate experiments, animals were perfused systemically using (1) 5% glutaraldehyde; (2) Karnovsky's solution; (3) 4% paraformaldehyde; buffered with either 0.1 M sodium phosphate or sodium cacodylate (pH 7.4). The concentration of glutaraldehyde in the fixative solution was the principal determinant of fluorescence intensity. Confocal imaging was achieved immediately following perfusion with 5% glutaraldehyde or Karnovsky's. Fluorescence intensity increased markedly during overnight storage in these fixatives and continued to increase during subsequent storage in buffer alone. The fluorophore was stable and resistant to fading during storage (15 months at least), enabling data collection over extended periods. To demonstrate application of the method in neuropathology, individual fibres in transected sciatic nerve trunks were traced through multiple successive internodes: Classical features of Wallerian degeneration (axonal swelling and debris; ovoid formation and incisure changes; variation among fibres in the extent of degeneration) were displayed. The method is compatible with subsequent ultrastructural examination and will complement existing methods of investigation of myelinated fibre anatomy and pathology, particularly where preservation of 3-dimensional relationships or elucidation of spatial gradients are required.

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“…Since this microscopy combined with computed image analysis was introduced, its applications have expanded at an astonishing rate. CLSM has enabled variable regions of routinely processed light microscopic specimens to be observed not only at the microscopic but also at the subcellular level [18][19][20], because the optical sectioning minimizes flaring and optimizes the fluorescence signals, and images taken by CLSM can be digitized and made available for manipulation and computer-assisted 3D reconstruction [10,13,20,30,40,43,44,53,55].…”

Section: Confocal Laser Scanning Microscopy (Clsm) Configurationmentioning

confidence: 99%

Confocal Laser Scanning Microscopic Imaging of Subcellular Organelles, mRNA, Protein Products, and the Microvessel Environment.

Itoh

Matsuno

Yamamoto³

et al. 2001

Acta Histochem. Cytochem

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The confocal laser scanning microscope (CLSM) was employed to visualize highly contrasted images of histochemically stained subcellular organelles. In early studies, only fluorescent signals were detectable by CLSM, however, recent innovations have enabled the visualization of non-fluorescent probes such as horseradish peroxidase (HRP)-diaminobenzidine (DAB). The non-fluorescent signals are permanently preserved and allow repeated or retrospective examinations. We applied CLSM to specimens prepared for light microscopy, and visualized subcellular organelles and pituitary hormone mRNA. The images were comparable to those obtained by electron microscopy (EM). We also applied this technique to the observation of tumor angiogenesis and the microvessel environment of hormone-secreting cells. Both the visualization of subcellular organelles, mRNA and protein products, and 3D images of the microvessel environment of hormone-secreting cells are discussed in this review.

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Confocal fluorescence microscopy and three‐dimensional reconstruction

Cited by 31 publications

References 39 publications

Measurement of Enamel Demineralization using Microradiography and Confocal Microscopy

Measurement of Enamel Demineralization using Microradiography and Confocal Microscopy

Imaging myelinated nerve fibres by confocal fluorescence microscopy: individual fibres in whole nerve trunks traced through multiple consecutive internodes

Confocal Laser Scanning Microscopic Imaging of Subcellular Organelles, mRNA, Protein Products, and the Microvessel Environment.

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