We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after ∼140 min) into the right jugular vein, and the fluorescence signal (at 650–670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30–40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 ± 0.26, mean ± SE, n = 10), indicating a “priming,” or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 ± 0.07, n = 6; P < 0.01). Cy2- or Cy3-labeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand β2-glycoprotein.

Video rate confocal laser scanning microscopy at the highest spatial and temporal resolution of backscattered light (BSL) imaging allowed for regular observation of fast intracellular motion (FIM) first revealed in living neoplastic cells. However, the absence of an objective evaluation has hampered further study of the mechanisms and biological significance of FIM. Particularly, a quantification of apparent differences in velocities that would complement and improve the current demonstration of FIM by color coding using the combination of red-green-blue (RGB) images had been missing. Standard methods of tracking or pattern recognition could not be applied because of the fuzzy nature of images of FIM. A search for a suitable method led to correlation analysis. It was calibrated on Brownian motion and a known type of motion, such as cell marginal ruffling, compared with FIM. Results approved its explanatory potential. Therefore, several crucial incidences of FIM could be analyzed. Apart from an argument against viewing FIM as a manifestation of simple Brownian motion, the correlation analysis of FIM in the adjacent peripheries of a rat fibroblast and a K4 rat sarcoma cell confirmed the notion of higher and uneven distribution of velocity of FIM in a tumor cell so far shown in color-coded images only. This result and other yet unpublished observations indicate that the velocity and topology of FIM can also contribute to a biological distinction between neoplastic and normal cells. Regular application of the correlation analysis should further expand the study of FIM for its mechanisms and predictive value. Such an approach should be thoroughly examined for a contribution to the knowledge of cancer cells.

To investigate aging bone structure of humans—here, in the lumbar vertebral bodies—requires methodologies that have sufficiently high resolving power yet still have sufficient width and depth of field. No clinical imaging method can come close to meeting the first requirement, leading to the disadvantage of being limited to postmortem studies. Few microscopic methods meet the second and third requisites. The three-dimensional (3D) images of bone in this article were obtained using deep-field 3D optical imaging, X-ray imaging, and scanning electron microscopy (SEM) of macerated plane parallel slices. The study of bone as a 3D object provides a different perspective from conventional two-dimensional images, and enriches our understanding of how modeling and remodeling processes regulate bone structure and connectivity. The study of ultraflat block surfaces by quantitative back-scattered electron imaging permits acquisition of data on mineral distributions and densities within a very thin layer (a continuous and perfect very thin section) in the block face. With this information, bone can be viewed as a spectrum of tissue types varying in degree of mineralization.

The structure and relative degree of mineralisation of antler and pedicle bone of yearling red deer stags exposed either to low or high levels of environmental fluoride were determined by digital quantitative backscattered electron (BSE) imaging. Bone fluoride content (BFC) in antlers (845±86 mg F − /kg ash, arithmetic mean± S.E.M. ) and pedicles (1448±154 mg F − /kg ash) of deer from a highly fluoride polluted area in North Bohemia (Czech Republic) were significantly higher ( P < 0.001) than those of controls from uncontaminated regions in West Germany (antlers: 206±41, pedicles: 322±52 mg F − /kg ash). Mean (56.5±4.5%) and maximum (84.9±2.1%) mineralised bone area of the control antlers significantly ( P < 0.05 and P < 0.001, respectively) exceeded the corresponding values for the N. Bohemian deer (43.3±1.3 and 73.3±1.9%, respectively), while the pedicles from the 2 groups did not differ significantly. In the pooled antler samples (n = 18), negative correlations existed between BFC and mean (r s = −0.62, P < 0.01) as well as maximum (r s = −0.69, P < 0.01) mineralised bone area. Morphological imaging revealed a decreased width and an increased porosity of the antler cortex in the N. Bohemian specimens. Mean (148.5±1.7) and maximum (154.2±1.7) BSE‐signal intensities (= grey levels; range between a monobrominated (grey level 0) and a monoiodinated (grey level 255) dimethacrylate resin standard) of the antlers from the controls were significantly higher than those of the N. Bohemian deer (140.7±2.1 and 145.7±2.2, respectively; P < 0.05 for both comparisons). In the pooled antler samples, negative correlations between BFC and mean (r s = −0.51, P < 0.05) as well as maximum (r s = −0.52, P < 0.05) BSE‐signal intensities were observed. No significant differences in mineralisation density parameters were found for the 2 pedicle samples, and BFC and mineralisation density of the pooled pedicles were uncorrelated. Morphological imaging revealed bone mottling (denoting increased remodelling activity) and frequent occurrence of apparently increased osteocyte lacunae in some of the pedicles from the N. Bohemian deer. It is concluded that the reduced amount of mineralised bone in, and the lower mineralisation density of, the N. Bohemian antlers resulted from a fluoride induced disturbance of bone mineralisation. The rapid growth of antlers leads both to a high mineral demand and a high rate of fluoride uptake during antlerogenesis. This, and the limited lifespan of antlers, which does not allow for a compensation of a delay in the onset or progression of the mineralisation process, renders antler bone particularly susceptible to fluoride. Antlers are therefore considered a useful model for studying fluoride effects on bone formation. Furthermore, analysis of cast antlers enables a noninvasive monitoring of environmental pollution by fluorides.

Newer methods of scanning microscopy using both light and electrons are particularly relevant to the study of bone cells, bone matrix organization, matrix mineralization, bone modeling and remodeling, and the adaptation of cells and matrix to implants. Most of such studies are conducted on retrieved implants, at least after the death of the related tissue. Because the retention of the tissue-implant relationship in such preserved tissue is crucial for critical evaluation of the implant, methods based on the study of flat surfaces of embedded tissue blocks are very important. Using electrons, the backscattered electrons in a scanning electron microscope can be employed to evaluate mean atomic number (density) and cathodoluminescence can identify polymers and fluorescent labels. Using light, confocal microscopical techniques permit the examination of layers deep to the block face. Confocal reflected and fluorescence methods allow the study of cell behavior upon both transparent and opaque substrates in the laboratory. Examples of the above are presented and interpretation problems discussed. Current experiments are aimed at enabling the study of bone wound healing and bone adaptation to implanted materials in vivo, through the implantation of optical quality windows and/or newly conceived and designed microscopical objective lenses. (Implant Dent 1992;1:117–125)

The aim of this study was to look at the bone bonding potential of six formulations of a novel glass-ceramic system. Cylinders of the ceramics were implanted in rabbit tibiae for 4 and 7 weeks. Histological tests, both quantitative and qualitative, as well as push-out tests, were carried out during the bonding assessment. Bone growth was quite prolific, even at 4 weeks, as evidenced by growth up to and along the implant surfaces. The interfacial shear strengths compared well with other biomaterials in use as endosseous implants. Therefore it seems pertinent to pursue further long-term experimentation with this material.

Anorganic unerupted developing teeth and airdired erupted teeth of the platypus (Ornithorhynchus anatinus) were examined in a scanning electron microscope and in a tandem scanning reflected light microscope. Typically mammalian developing fronts of enamel and dentine were identified in the anorganic unerupted specimens. The developing teeth were particularly small and fragile and the enamel elusive and difficult to examine in the normal way for morphological detail. Prepared fractured surfaces of unerupted specimens revealed preferentially oriented crystallite groups in the enamel generally perpendicular to the developing front and a highly globular, mineralized pattern in the dentine with fine diameter, sparsely distributed dentinal tubules.

Annals of the New York Academy of SciencesVolume 483, Issue 1 p. 428-439 Applications of Tandem Scanning Reflected Light Microscopy and Three-Dimensional Imaginga ALAN BOYDE, ALAN BOYDE Department of Anatomy and Embryology, University College London, London WCIE 6BT, EnglandSearch for more papers by this author ALAN BOYDE, ALAN BOYDE Department of Anatomy and Embryology, University College London, London WCIE 6BT, EnglandSearch for more papers by this author First published: December 1986 https://doi.org/10.1111/j.1749-6632.1986.tb34553.xCitations: 30 a Supported by grants from MRC and SERC. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 Petran, M., M. Hadravsky, J. Benes & A. Boyde. 1986. In vivo microscopy using the tandem scanning microscope. This volume. 2 Egger, M. D. & M. Petran. 1967. New reflected-light microscope for viewing unstained brain and ganglion cells. Science 157: 305–307. 3 Petran, M., M. Hadravsky, M. D. Egger & R. Galambos. 1968. Tandem scanning reflected light microscope. J. Opt. Soc. Am. 58: 661–664. 4 Petran, M., M. Hadravsky & A. Boyde. 1985. 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We consider results from tissue culture studies and the comparative histology of mineralized tissues and other natural tissue interfaces which may have some relevance in understanding the abnormal biology of the immediate environment of an implant in bone. We discuss factors influencing setting, colonization, and migration on natural and artificial substrates by various cell types which may make or remove matrix near the implant. A knowledge of mechanisms of mineral and organic matrix destruction by osteoclasts and other cells must be important in addition to an understanding of the interaction of local and systemic hormones with bone cells. More studies of the role of the immune system in implant failure are urgently required.

Journal Article SCANNING ELECTRON MICROSCOPY OF THE BASAL SURFACE OF THE SEPARATED SUCTION BLISTER TOP Get access ALAN BOYDE, ALAN BOYDE Department of Anatomy, University College London, London, W.C.1, St John's Hospital for Disease of the Skin, Lisle Street, London, W.C.2, and Institute of Dermatology, Homerton Grove, London, E.9 Search for other works by this author on: Oxford Academic Google Scholar ROBIN D. G. PEACHEY, ROBIN D. G. PEACHEY Department of Anatomy, University College London, London, W.C.1, St John's Hospital for Disease of the Skin, Lisle Street, London, W.C.2, and Institute of Dermatology, Homerton Grove, London, E.9 Search for other works by this author on: Oxford Academic Google Scholar JOHN E. W. WHITE JOHN E. W. WHITE Department of Anatomy, University College London, London, W.C.1, St John's Hospital for Disease of the Skin, Lisle Street, London, W.C.2, and Institute of Dermatology, Homerton Grove, London, E.9 Search for other works by this author on: Oxford Academic Google Scholar British Journal of Dermatology, Volume 84, Issue 4, 1 April 1971, Pages 346–352, https://doi.org/10.1111/j.1365-2133.1971.tb14230.x Published: 01 April 1971 Article history Accepted: 11 December 1970 Published: 01 April 1971