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PubMed1996

Atomism, Lynceus, and the fate of seventeenth-century microscopy.

Recent scholarship, focusing on the rapid decline of microscopy after the late 1680's, has shown that the limitations of microscopy and the ambivalent meaning of its findings led to a wide-spread sense of frustration with the new instrument. The present article tries to connect this fall from favor with the microscope's equally surprising but hitherto little noticed late rise to prominence. The crucial point is that when the microscope, more than a decade after the telescope, finally managed to arouse the interest of natural philosophers, it did so as a corpuscularian tool, and as such it came to share the difficult fate of seventeenth-century corpuscularianism. The essay ends with the claim that the fall of microscopy was not only due to the failure of microscopy to corroborate corpuscularianism, but also to the changing definition of natural philosophy in the late seventeenth and early eighteenth centuries and to the separation of the domains of living matter (to which the microscope found itself confined) from a physica whose recent mathematical framework excluded the organic world.

Academies and Institutes↗
PubMed2005

Single-particle light microscopy of bacteriophages.

Bacteriophage capsids have a protein shell with a symmetrical, fullerene-like arrangement of subunits. In the case of double-stranded DNA bacteriophages, the capsid joins with accessory proteins to form a DNA packaging motor that packages a genome in a cavity of the capsid. The motor cleaves ATP to obtain the needed energy. Light microscopy of single bacteriophages and single bacteriophage DNA packaging intermediates is being developed for the following reasons: (1) A synchronization-independent, fractionation-independent procedure is needed for the in vitro analysis of bacteriophage DNA packaging motors. (2) A non-biological procedure is needed for identifying and characterizing new bacteriophages needed for studies of bacteriophage gene homologies. In a recent study, light microscopy-based nanometry is used to follow the in vitro packaging of DNA in real time. Fluorescence microscopy of stained DNA is similarly used. Towards a more thorough analysis by fluorescence microscopy, single bacteriophage capsids are visualized by the unenhanced fluorescence of covalently bound protein-specific dyes. Dimerization of capsids is observed in real time. The dimerizing capsids had been restricted to a thin planar zone so that single-particle tracking was performed before, during, and after dimerization. Photobleaching is not a major problem. Thermal motion-based procedures are used for distinguishing binding from accidental co-migration. The long-range objective is the simultaneous real time monitoring of multiple state variables during cycling of a single DNA packaging motor. The results of these basic studies are applicable to both nanotechnological drug delivery and biological therapy.

Bacteriophages↗
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↗
PubMed1995

Capillary microscopy is a diagnostic aid in patients with acral ischemia.

The diagnosis of peripheral ischemic diseases is a challenge to the vascular specialist, since discrimination of the different entities on clinical grounds is not always clear. Capillary microscopy is a noninvasive diagnostic technique that can visualize in particular the morphology and perfusion of the digital capillaries. The authors investigated all 522 patients who were referred to the vascular laboratory over a six-year period, in an attempt to obtain more certainty about the diagnosis of their acral ischemic complaints. Patients with clinically manifest Raynaud's phenomenon and control subjects were compared to obtain differentiation criteria. Thus, capillary microscopy scored a sensitivity of 67%, a specificity of 84%, and an accuracy of 81% to detect Raynaud's phenomenon. A disturbed capillary morphology was virtually pathognomonic in Raynaud's disease secondary to systemic disorders (specificity 100%); the accuracy was but 74%. In 69% of the patients with clinically atypical acral ischemia, capillary microscopy yielded a diagnosis. Digital blood pressure measurements, however, appeared of no use in the diagnosis of Raynaud's phenomenon. Hence, capillary microscopy appears to be a useful aid in the diagnosis of acral ischemic syndromes.

Adolescent↗
PubMed1997

Efficient immunocytochemical labeling of leukocyte microtubules with FluoroNanogold: an important tool for correlative microscopy.

We tested the immunoprobe FluoroNanogold (FNG) for its utility as an immunocytochemical labeling reagent. This immunoprobe consists of a 1.4-nm gold particle to which a specific Fab' fragment and a fluorochrome are conjugated. We employed the microtubules (MTs) of human phagocytic leukocytes as a model system for testing the usefulness of FNG as a secondary antibody for immunocytochemistry. We show that these fluorescently labeled ultrasmall immunogold particles are very efficient for labeling MTs in these cells. The signal from FNG can be detected directly by fluorescence microscopy or indirectly by other modes of optical microscopy and electron microscopy, after silver-enhancement of the gold. The spatial resolution of immunolabeled MTs obtained with FNG and silver enhancement was comparable to that of conventional immunofluorescence detection. Colloidal gold (5-nm and 10-nm in diameter), on the other hand, failed to label MTs in cells prepared in a similar manner. This difference in labeling was due in large part to greater penetration of 1.4-nm gold into aldehyde-fixed cells than either 5-nm or 10-nm gold particles. The fluorescent 1.4-nm immunoprobe was shown to be an important new tool for general use in correlative microscopy.

Animals↗
PubMed1983

Electron microscopy applied to fine-needle aspiration. A report of six cases from various sites.

The authors report the results obtained from the application of electron microscopy techniques to the cytology of fine-needle-aspirated samples of neoplastic lesions from various body sites. These results show that the tissue structure, which is usually lost during the squashing necessary for light microscopy cytology, is preserved when the samples are processed for ultrastructural analysis. Electron microscopy also allows a highly detailed study of the cell's inner structures. Thus, when this technique is applied, fine needle-aspirated samples can be regarded as actual microbiopsies. However, because of the high cost of ultrastructural techniques, we suggest that actual analysis be performed only in selected cases, whereas fixation and inclusion for electron microscopy could be done routinely.

Adult↗
PubMed1976

Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.

Animals↗
PubMed1992

Subcellular visualization of light microscopic specimens by laser scanning microscopy and computer analysis: a new application of image analysis.

To identify subcellular organelles or to observe their pathological changes in sections prepared for light microscopy, immuno- and/or enzyme histochemical staining for the marker substances or enzymes of those subcellular organelles are frequently employed. With conventional light microscopes (CLM), however, it is hardly possible to determine whether or not the target organelles are properly stained and to confirm their fine structure. In the present study, the laser scanning microscope (LSM) was employed to obtain highly contrasted images of histochemically stained subcellular organelles at the limit of resolution in light microscopy. To refine or characterize those images, images built up as electronic signals in LSM were further processed in the Image Analysis System (IAS) with pipeline. Thus, the approximate figures of subcellular organelles such as microtubules, endoplasmic reticula, secretory granules, and mitochondria were visualized in brightfield on sections prepared for light microscopy (paraffin, frozen sections and cultured living cells). The validity of the images obtained by LSM or LSM-IAS was confirmed by immunoelectron microscopy when possible. The LSM images of histochemically stained suborganelles of various cells were definitely improved (refined and/or strengthened) by processing them with IAS.

Endoplasmic Reticulum↗
PubMed1996

The osmium tetroxide-p-phenylenediamine procedure reveals the chromatid cores and kinetochores of meiotic chromosomes by light and electron microscopy.

We analyzed first-metaphase meiotic chromosomes of the grasshopper Chorthippus jucundus by two different methods, i.e., a silver impregnation technique and the osmium tetroxide-p-phenylenediamine (Os-PPD) procedure. The former was applied on squashed testes previously fixed in ethanol-acetic acid, whereas for Os-PPD the material was not subjected to any previous extraction treatment but was fixed in OsO4, treated with PPD, and embedded in Epon 812. Both techniques revealed chromatid cores and kinetochores regardless of the processing of the material (squashed or sectioned). Unstained Os-PPD sections were analyzed by light microscopy and transmission electron microscopy (TEM). The Os-PPD technique provided a high contrast of chromatid cores and kinetochores in relation to the chromatin, which revealed a low electron density. To determine the Os-PPD reaction mechanism, the PAS procedure, as well as scanning electron microscopy (SEM) backscattering and SEM X-ray microanalysis, was performed on sections. By use of the Os-PPD-PAS procedure, glycol groups formed by oxidation of osmium bound to aromatic substrates were detected in chromatid cores and kinetochores by brightfield and fluorescence microscopy. A high Z contrast was detected in these structures by backscattered electron imaging. SEM X-ray microanalysis showed osmium and phosphorus to be the main elements present on the chromatid cores. Taking into account the known reactivity of OsO4 and the present results, the possible participation of nucleic acids as well as proteins in the Os-PPD reaction mechanism and in the composition of chromatid cores and kinetochores is discussed.

Animals↗
PubMed2002

Histometric data obtained by in vivo confocal laser scanning microscopy in patients with systemic sclerosis.

BACKGROUND: It would be a benefit if time-saving, non-invasive methods could give hints for diagnosing systemic sclerosis. To investigate the skin of patients with systemic sclerosis using confocal laser scanning microscopy in vivo and to develop histometric parameters to describe characteristic cutaneous changes of systemic sclerosis observed by this new technique, we conducted an exploratory study. MATERIALS AND METHODS: Fifteen patients with systemic sclerosis treated with extracorporal photopheresis were compared with 15 healthy volunteers and 10 patients with other disorders also treated with extracorporal photopheresis. All subjects were investigated using confocal laser scanning microscopy in vivo. RESULTS: Micromorphologic characteristics of skin of patients with systemic sclerosis and measuring parameters for melanisation, epidermal hypotrophy, and fibrosis for dislocation of capillaries by collagen deposits in the papillary dermis were evaluated. An interesting finding was an increased thickness of the tissue in the dermal papillae superior to the first dermal papilla vessel. It was also possible to reproduce characteristic histologic features by confocal laser scanning microscopy in vivo. Histometric parameters for fibrosis and vascular features developed in this study showed significant differences in patients with systemic sclerosis compared to controls. CONCLUSIONS: Although the predominant histopathological features in systemic sclerosis are findings of the reticular dermis and the subcutis, and in histopathological investigation the epidermis seems to remain unaffected by the disease, we have demonstrate some characteristic differences in the epidermis and papillary dermis by confocal laser scanning microscopy in vivo. Some of them have not been described so far. However, to use this technique as a tool for diagnosis and/or staging of systemic sclerosis, further studies are needed investigating the sensitivity and specificity of the histometric parameters developed in this study.

Adult↗
PubMed2003

Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy.

A new vibrational imaging method based on coherent anti-Stokes Raman scattering (CARS) has been used for high-speed, selective imaging of neutral lipid droplets (LDs) in unstained live fibroblast cells. LDs have a high density of C-H bonds and show a high contrast in laser-scanning CARS images taken at 2,845 cm-1, the frequency for aliphatic C-H vibrations. The contrast from LDs was confirmed by comparing CARS and Oil Red O (ORO)-stained fluorescence images. The fluorescent labeling processes were examined with CARS microscopy. It was found that ORO staining of fixed cells caused aggregation of LDs, whereas fixing with formaldehyde or staining with Nile Red did not affect LDs. CARS microscopy was also used to monitor the 3T3-L1 cell differentiation process, revealing that there was an obvious clearance of LDs at the early stage of differentiation. After that, the cells started to differentiate and reaccumulate LDs in the cytoplasm in a largely unsynchronized manner. Differentiated cells formed small colonies surrounded by undifferentiated cells that were devoid of LDs. These observations demonstrate that CARS microscopy can follow dynamic changes in live cells with chemical selectivity and noninvasiveness. CARS microscopy, in tandem with other techniques, provides exciting possibilities for studying LD dynamics under physiological conditions without perturbation of cell functions.

3T3-L1 Cells↗
PubMed1995

Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy.

A Ca2+ wave at fertilization triggers cortical granule exocytosis in sea urchin eggs. New methods for visualizing exocytosis of individual cortical granules were developed using fluorescent probes and confocal microscopy. Electron microscopy previously provided evidence that cortical granule exocytosis results in the formation of long-lived depressions in the cell surface. Fluorescent dextran or ovalbumin in the sea water seemed to label these depressions and appeared by confocal microscopy as disks. FM 1-43, a water-soluble fluorescent dye which labels membranes in contact with the sea water, seemed to label the membrane of these depressions and appeared as rings. In double-labeling experiments, the disk and ring labeling by the two types of fluorescent dyes were coincident to within 0.5 second. The fluorescent labeling is coincident with the disappearance of cortical granules by transmitted light microscopy, demonstrating that the labeling corresponds to cortical granule exocytosis. Fluorescent labeling was simultaneous with an expansion of the space occupied by the cortical granule, and labeling by the fluorescent dextran was found to take 0.1-0.2 second. These results are consistent with, and reinforce the previous electron microscopic evidence for, long-lived depressions formed by exocytosis; in addition, the new methods provide new ways to investigate cortical granule exocytosis in living eggs. The fluorescence labeling methods were used with the Ca2+ indicator Ca Green-dextran to test if Ca2+ and cortical granule exocytosis are closely related spatially and temporally. In any given region of the cortex, Ca2+ increased relatively slowly.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Sensitivity of microscopy for the rapid diagnosis of gonorrhoea in men and women and the role of gonorrhoea serovars.

Auditing the sensitivity of microscopic diagnosis of gonorrhoea is recommended by the current guidelines. A retrospective study was performed of 596 cases of positive cultures for Neisseria gonorrhoeae in modified New York City culture (MNYC) media diagnosed from 1995 to 1999. The sensitivity of the cervical slides in women was 51% while in men who have sex with men (MSM) the sensitivity of urethral and rectal slides were 89% and 54% respectively. The sensitivity of urethral slides in heterosexual men was 84%. Neisseria serovar 1B02 among MSM and serovar 1B31 among women were mostly undiagnosed with microscopy. Serovars 1A05, 1A21, B08 among heterosexual men were exclusively associated with negative microscopy. Microscopy is important in the rapid detection and treatment of gonorrhoea. Infections with certain serovars are less likely to be detected by microscopy; making them more likely to spread within the community. Culture from different ano-genital sites is essential to maximize detection of gonorrhoea in all patients.

Adult↗
PubMed2006

Rectal gonorrhoea in men -- is microscopy still a useful tool?

Rectal gonorrhoea (GC) in men may cause anal discharge or proctitis, but these symptoms have been shown to correlate poorly with rectal infection. Culture of Neisseria gonorrhoeae from an exposed site offers a readily available, sensitive and cheap diagnostic test, and is currently the gold standard for diagnosis; however, these results can take a few days and therefore do not offer an instant diagnosis. Gram staining of rectal smears for N. gonorrhoeae has a low sensitivity but a high specificity when performed by experienced personnel. We audited whether rectal microscopy increased the number of patients diagnosed and treated for rectal GC at initial presentation at one inner London genitourinary clinic over a 12-month period. One hundred and thirty-six episodes of rectal GC were identified in 132 men. In all, 134/136 had rectal microscopy of whom, 47/134 (35%) were smear-positive for GC. Of the 136 cases, 90 received antibiotics for GC at their first presentation. Twenty-four of 90 (27%) would not have been treated until culture results were available, if rectal microscopy had not been performed. These results suggest that rectal microscopy remains an important tool and increases the proportion of men treated for GC at their first attendance.

Adult↗
PubMed2000

A new method of quantitative fecal fat microscopy and its correlation with chemically measured fecal fat output.

Fecal fat microscopy using the Sudan stain has suffered from a relative lack of specificity, and results are "qualitative." Therefore, we developed a quantitative fecal fat microscopic method with hopes of improving diagnostic accuracy. One hundred eighty patients with chronic diarrhea collected stools for 1 to 3 days, and fecal fat output was measured by a standard chemical method, and microscopy was performed by the old qualitative and new quantitative methods. There was a highly statistically significant linear correlation between quantitative fecal fat microscopy and chemically measured fecal fat output. The quantitative microscopic method had a sensitivity of 94% and a specificity of 95%; the traditional method had a sensitivity and specificity of 76% and 99%, respectively. Fecal fat Sudan microscopy performed by a dedicated approach to counting and size measurement of fat globules can yield a quantitative result that correlates well with chemically measured fecal fat output and has a high diagnostic accuracy.

Azo Compounds↗
PubMed2006

Two-photon excitation fluorescence microscopy with a high depth of field using an axicon.

In conventional two-photon excitation fluorescence microscopy, the numerical aperture of the objective determines the lateral resolution and the depth of field. In some situations, as with functional imaging of dynamic events distributed in live biological tissue, an improved temporal resolution is needed; as a consequence, it is imperative to use optics with a high depth of field to simultaneously image objects at different axial positions. With a conventional microscope objective, increasing the depth of field is achieved at the expense of lateral resolution. To overcome this limitation, we have incorporated an axicon in a two-photon excitation fluorescence microscopy system; measurements have shown that an axicon provides a depth of field in excess of a millimeter, while the lateral resolution is maintained at the micrometer scale. Thus axicon-based two-photon microscopy has been shown to yield a high-resolution projection image of a sample with a single 2D scan of the laser beam while maintaining the improved tissue penetration typical of two-photon microscopy.

Equipment Design↗
PubMed2006

Cell imaging and manipulation by nonlinear optical microscopy.

Advances in the technologies for labeling and imaging biological samples drive a constant progress in our capability of studying structures and their dynamics within cells and tissues. In the last decade, the development of numerous nonlinear optical microscopies has led to a new prospective both in basic research and in the potential development of very powerful noninvasive diagnostic tools. These techniques offer large advantages over conventional linear microscopy with regard to penetration depth, spatial resolution, three-dimensional optical sectioning, and lower photobleaching. Additionally, some of these techniques offer the opportunity for optically probing biological functions directly in living cells, as highlighted, for example, by the application of second harmonic generation to the optical measurement of electrical potential and activity in excitable cells. In parallel with imaging techniques, nonlinear microscopy has been developed into a new area for the selective disruption and manipulation of intracellular structures, providing an extremely useful tool of investigation in cell biology. In this review we present some basic features of nonlinear microscopy with regard both to imaging and manipulation, and show some examples to illustrate the advantages offered by these novel methodologies.

Animals↗
PubMed2001

The fibronexus in reactive and tumoral myofibroblasts: further characterisation by electron microscopy.

Forty two surgical specimens containing myofibroblasts were studied to clarify the criteria for identifying the fibronexus, an ultrastructural feature regarded as a marker for myofibroblastic differentiation. Granulation tissue, tumour stroma, fibro-proliferative lesions (nodular fasciitis, myofibromatosis, inflammatory myofibroblastic tumour) and malignancies (myofibrosarcoma and fibrosarcoma) were studied. Comparable results were found throughout these specimens, although fibronexus junctions were better developed in reactive compared with tumoral myofibroblasts. By electron microscopy, myofibroblasts were identified by abundant rough endoplasmic reticulum, peripheral smooth-muscle myofilaments with focal densities, and fibronexus junctions. The latter were recognised as the points of convergence on the myofibroblast surfaces of intracellular myofilaments and extracellular fibronectin fibrils. The fibronectin fibrils were often co-linear with myofilaments. Also, fibronectin fibrils were dark-staining, straight and rigid-looking, and had a longitudinal filamentous substructure. A striking feature was the tendency of fibronectin fibrils to project into the surrounding extracellular space, away from the myofibroblast surface: in these respects, they differed significantly from lamina ("basement membrane"). The presence of fibronectin fibrils correlated positively with fibronectin immunostaining by light and electron microscopy. Laminin and collagen IV showed variable and weak staining in the intercellular spaces in a minority of cases and never strongly stained myofibroblast surfaces. The data emphasise that the fibronexus has a number of distinctive features permitting identification, and constitute a reference-point for pathologists wishing to use electron microscopy to refine light microscopy diagnoses of putative myofibroblastic lesions. The role of the fibronexus in the definition of the myofibroblast is discussed.

Carcinoma, Squamous Cell↗