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PubMed2005

Fluorescence in situ hybridisation coupled to ultra small immunogold detection to identify prokaryotic cells using transmission and scanning electron microscopy.

We describe a method based on fluorescence in situ hybridisation (FISH) that allows the identification of individual cells by electron microscopy. We hybridised universal and specific fluorescein-labelled oligonucleotide probes to the ribosomal RNA of prokaryotic microorganisms in heterogeneous cell mixtures. We then used antibodies against fluorescein coupled to sub-nanometer gold particles to label the hybridised probes in the ribosome. After increasing the diameter of the metal particles by silver enhancement, the specific gold-silver signal was visualised by optical microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is the first time that SEM is applied to the detection of gold nanoparticles hybridised to an intracellular target, such as the ribosome. The possibility to couple phylogenetic identification by FISH to cell surface and ultrastructure observation at electron microscopy resolution has promising potential applications in microbial ecology.

Deinococcus↗

PubMed1991

Detection of B19 parvovirus in human fetal tissues by electron microscopy.

We present the electron microscopy observations on samples from 38 pregnancies that were investigated for B19 parvovirus infection. Thirty-four had resulted in fetal loss thought to be due to a virus infection and 22 of the 38 were positive for B19 parvovirus in one or more of the tissues. Twenty-one placentas and 75 fetal tissue samples were examined. Fresh samples were investigated by immune electron microscopy while formalin-fixed tissues were examined as thin sections and by negative staining of tissue extracts with direct electron microscopy. Electron microscopy was more sensitive on fresh than on fixed samples. The ultrastructural observations on thin sections of fixed tissues yielded new information locating B19 parvovirus particles in both nucleus and cytoplasm of infected fetal cells. The diagnostic results of the range of electron microscopy assays were compared with those of two hybridization methods. The fresh samples yielded comparable results from electron microscopy and hybridisation assays but on formalin-fixed materials hybridisation was more sensitive.

Erythema Infectiosum↗

PubMed1999

Assessing confocal microscopy systems for purchase.

Confocal laser scanning microscopy now has so many functions and applications that choosing a new multiuser confocal laser microscopy system can be a daunting task, particularly for a first-time buyer and new users of confocal microscopy. How does one determine which features are most appropriate for any particular laboratory, application, or imaging environment? Each confocal microscopy system has its own individual advantages and limitations, which ultimately defines its market niche. Here, we describe the features that differentiate the four confocal microscopy systems we assessed. The decisive factors in choosing a confocal microscopy system for our anatomical laboratory were user-friendly software for on-line acquisition and off-line processing; the working distances of objective lenses; applicability for a multiuser environment; interactions with company representatives; and turnaround times for questions, service, and accessories.

Computers↗

PubMed2005

The contribution of microscopy to the study of Alzheimer's disease, amyloid plaques and Abeta fibrillogenesis.

A broad survey is presented in this chapter, dealing with the impact that microscopy has made to the study of Alzheimer's disease, amyloid plaques and amyloid-beta fibrillogenesis. This includes classical light microscopy and the modem immunolabelling and confocal microscopies, together with the contribution of transmission electron microscopy and atomic force microscopy. Whilst usefully standing alone, the individual microscopies often contribute most effectively when they are integrated with cellular, biophysical and molecular approaches.

Alzheimer Disease↗

PubMed1999

Confocal laser scanning microscopy: A nondestructive subsurface histotomography of healthy human bone.

Microscopy of bony tissue usually requires special treatment for decalcification and processing of thin sections. Confocal laser scanning microscopy (CLSM) allows the nondestructive histotomography of organic hard tissue. The aim of this study was to visualize healthy human bone structures and to correlate identical areas in CLSM and conventional light microscopy. Each sample of healthy human lower jaw (n = 20) was divided into three parts: (1) fresh, untreated bony blocks studied by CLSM; (2) MMA-embedded thin sections (without decalcification), HE stained and studied by CLSM and conventional light microscopy (correlation of identical areas); (3) decalcificated, HE stained, histological sections studied by conventional light microscopy. In untreated bony blocks, microstructures such as osteocytes and lamellae were identified by CLSM. These structures could be correlated with conventional light microscopy. In CLSM, subcellular structures cannot yet be interpreted, whereas cytoplastic processes of osteocytes were seen with high contrast. With CLSM, nondestructive histology of cortical bone can be obtained. The risk of artifacts due to pretreatment is minimized, and subsurface visualization does not affect the interpretation.

Histological Techniques↗

PubMed1998

The use of intravital microscopy in surgical research. 26-years of experience analyzed by studies presented at the Surgical Forum of the Annual Congress of the German Society of Surgery.

Recent developments in intravital microscopy make this technique an attractive approach to studying microvascular, cellular, and molecular mechanisms of distinct surgical diseases. We investigated the value of this technique in surgical research laboratories by analyzing the studies presented during the past 26 years (1972-1997) at the Surgical Forum of the Annual Congress of the German Society of Surgery. From a total of 2279 papers 188 contributions (8.3%) presented data which derived from the analysis of the microcirculation using techniques, such as H2 and 133Xe clearance, autoradiography, thermodiffusion, laser Doppler fluxmetry, laser speckle, radioactive and fluorescent microspheres, polarographic oximetry, and intravital microscopy. There were 72 presentations (3.2% of all contributions) reporting the use of intravital microscopy, thus reflecting 38.3% of all microcirculatory analyses. Although these numbers may be considered quite small, analysis over time revealed a significant (P<0.05) increase in the number of microcirculatory studies (11.4%) and in particular of those using intravital microscopy (6.3%) in the 1990s when compared to the 1970s (5.3%; 0.1%) and 80th (7.1 %; 1.3%). In 1997, 27 of 165 contributions (16.4%) included microcirculatory analyses, and 18 of the 165 contributions (10.9%) reported results analyzed by intravital microscopy. Thus our analysis reflects an increasing interest of surgical researchers to study in vivo the microcirculation, and by doing so to use intravital microscopy for the elucidation of mechanisms of surgical disease.

Germany↗

PubMedDate not supplied

Application of confocal laser scanning microscopy to the observation of bone biopsy specimens.

Iliac bone biopsy specimens from five patients with endstage renal disease were observed by confocal laser scanning microscopy. Images of affected bone specimens were accurately focused, whereas images viewed with conventional light microscopy from thick ground sections were obscure. Especially at high magnifications, fine structures of bone cells, otherwise blurred, were clearly observed with confocal scanning microscopy. From thin-cut sections, images satisfactory for pathological diagnosis were viewed with light microscopy even at high magnification. However, the sections tended to shrink vertically compared with the cross-sectional images of the blocks observed directly by confocal laser scanning microscopy. A three-dimensional image of bone tissue was also constructed from serial optical sections. Confocal laser scanning microscopy is a useful technique for observing bone tissues, and may become essential for the evaluation of bone biopsy specimens.

Adult↗

PubMed2006

Analytical performance of the Iris iQ200 automated urine microscopy analyzer.

BACKGROUND: We evaluated the Iris iQ200 Automated Urine Microscopy Analyzer to find out if the instrument performed better than traditional visual bright field microscopy in detecting basic urine particles, as assessed against reference phase contrast microscopy. METHODS: The HUSLAB quality system was followed in planning and completing the evaluation process. The iQ200 instrument results from 167 mid-stream, uncentrifuged urine specimens were compared to those obtained with phase contrast reference microscopy, and to those with routine bright field microscopy. Linearity, carry-over and precision were tested according to well-established protocols. RESULTS: The iQ200 counted erythrocytes (RBC) at r=0.894 (R(2)=0.799) with Automated Particle Recognition (APR) software alone and at r=0.948 (R(2)=0.898) after re-classification. The performance for leukocytes (WBC) was r=0.885 with APR and r=0.978 after re-classification. The correlations of counting after user re-classification were r=0.927 for squamous epithelial cells (SQEP), r=0.856 for casts, and r=0.706 for non-squamous epithelial cells. The iQ200 showed good linearity and precision and no carry-over was detected. CONCLUSIONS: The Iris iQ200 was capable to count reliably RBC, WBC, and SQEP cells and to identify a fraction of bacteria and renal elements. Counting results equalled or exceeded that of routine bright field microscopy or earlier flow cytometric technology. The instrument eliminates manual sample preparation but requires a well-trained technologist for re-grouping of findings.

Automation↗

PubMed2004

The use of scanning ion conductance microscopy to image A6 cells.

BACKGROUND: Continuous high spatial resolution observations of living A6 cells would greatly aid the elucidation of the relationship between structure and function and facilitate the study of major physiological processes such as the mechanism of action of aldosterone. Unfortunately, observing the micro-structural and functional changes in the membrane of living cells is still a formidable challenge for a microscopist. METHOD: Scanning ion conductance microscopy (SICM), which uses a glass nanopipette as a sensitive probe, has been shown to be suitable for imaging non-conducting surfaces bathed in electrolytes. A specialized version of this microscopy has been developed by our group and has been applied to image live cells at high-resolution for the first time. This method can also be used in conjunction with patch clamping to study both anatomy and function and identify ion channels in single cells. RESULTS: This new microscopy provides high-resolution images of living renal cells which are comparable with those obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Continuous 24h observations under normal physiological conditions showed how A6 kidney epithelial cells changed their height, volume, and reshaped their borders. The changes in cell area correlated with the density of microvilli on the surface. Surface microvilli density ranged from 0.5 microm(-2) for extended cells to 2.5 microm(2) for shrunk cells. Patch clamping of individual cells enabled anatomy and function to be correlated. CONCLUSIONS: Scanning ion conductance microscopy provides unique information about living cells that helps to understand cellular function. It has the potential to become a powerful tool for research on living renal cells.

Animals↗

PubMed2004

Confocal microscopy: a report by the American Academy of Ophthalmology.

OBJECTIVE: To review the available evidence for the use of confocal microscopy in diagnosing infectious keratitis and for other applications for ophthalmic practice. METHODS: A MEDLINE search of the peer-reviewed literature for the years 1990 to 2001 yielded 94 citations. The search was limited to studies of human subjects published in English with abstracts. The Ophthalmic Technology Assessment Committee Cornea Panel evaluated these 94 articles for possible clinical relevance and selected 51 (54%) for content review by the panel members. Of these 51 articles, 24 were selected for the panel methodologist to review and rate according to the strength of evidence. RESULTS: Of the 24 articles, 21 (87.5%) were classified as case reports or case series and were rated as level III evidence. Three articles were classified as independent, masked, or objective comparisons performed in a narrow spectrum of patients or in a nonconsecutive series of patients and were rated as level II evidence. No studies were rated as level I evidence, defined as an independent masked comparison of an appropriate spectrum of consecutive patients. CONCLUSION: Confocal microscopy is a new technology with clinical applications in ophthalmology. Although confocal microscopy has been used in other fields of medicine, the optical transparency of the cornea and other structures of the eye provides a unique opportunity to apply this technology. The targeted literature review of 24 articles found no level I studies to support the use of confocal microscopy in the management of eye disorders. Three level II studies pertained to promising clinical applications of the confocal microscope and provided evidence that supports the use of confocal microscopy as an adjunctive modality for diagnosing Acanthamoeba keratitis. The remaining 21 articles, rated as level III evidence, focus on the use of confocal microscopy to facilitate the diagnosis of infectious keratitis, including amoebic and fungal, but currently there are no definitive studies of its role in the differential diagnosis of this condition. There are also level III studies that support the use of the confocal microscope in refractive surgery. Facilitating the diagnosis of infectious keratitis and applying the confocal microscope to refractive surgery may hold the greatest promise of this new technology.

Diagnostic Techniques, Ophthalmological↗

PubMed1996

Confocal microscopy findings of Acanthamoeba keratitis.

PURPOSE: Tandem scanning confocal microscopy was performed on two patients with Acanthamoeba keratitis to provide images detailing characteristic findings of the disease. Although tandem scanning confocal microscopy of Acanthamoeba has been described in previous reports, Acanthamoeba keratitis has not been fully characterized with this instrument. In vivo confocal micrographs showed the double-walled structure of the Acanthamoeba cyst and associated radial keratoneuritis (perineuritis). METHODS: We reviewed the records of two patients with a clinical diagnosis of Acanthamoeba keratitis, one with culture-proven Acanthamoeba and the other with a suspected Acanthamoeba infection. Slit-lamp biomicroscopy and in vivo tandem scanning confocal microscopy were performed. The images obtained were compared with images from patients without corneal disease. RESULTS: High-contrast round bodies suggestive of Acanthamoeba cysts, as previously described, and irregular forms suggestive of Acanthamoeba trophozoites were found by tandem scanning confocal microscopy. Additionally, we showed conclusively that under certain circumstances (that is, corneal scarring) tandem scanning confocal microscopy can resolve the double-walled structure of the Acanthamoeba ectocyst surrounding the endocyst. Furthermore, radial keratoneuritis was demonstrated, consisting of an irregularly swollen nerve fiber with probable amoebic infiltration. CONCLUSIONS: Confocal microscopy can be a useful, noninvasive imaging technique helpful in the study, diagnosis, and treatment of Acanthamoeba keratitis.

Acanthamoeba↗

PubMed1990

Comparison of specular microscopy and examination of aspirate in phacolytic glaucoma.

Although phacolytic glaucoma is well known and the clinical picture is well recognized, the nature of the polychromatic hyperrefringent granules is less well understood. A series of five cases was examined by specular microscopy, and the anterior chamber aspirate obtained at operation was subsequently examined by direct microscopy of the wet aspirate. Direct microscopy showed that the macrophages identified histologically in the aspirate corresponded to regular round cells about three times the size of an erythrocyte seen in the relief mode of specular microscopy. This finding aided in the interpretation of cellular deposits seen on specular microscopy. One hyperrefringent granule was seen on the posterior endothelium on specular microscopy, and results of subsequent examination of the aspirate showed this and the crystals in another patient to be morphologically identical with cholesterol crystals. The findings confirm that cholesterol crystals are present in the exudate in phacolytic glaucoma and are responsible for hyperrefrigent granules.

Adult↗

PubMed2003

Comparison of central corneal thickness measurements by specular microscopy, ultrasound pachymetry, and ultrasound biomicroscopy.

PURPOSE: To compare the reproducibility and mean values of central corneal thickness (CCT) obtained by specular microscopy, ultrasound pachymetry, and ultrasound biomicroscopy (UBM). SETTING: Department of Ophthalmology, University of Toronto, Toronto, Ontario, Canada. METHODS: Thirty-one healthy volunteers were recruited for a sample size of 62 eyes. All subjects had pachymetric measurements by specular microscopy, ultrasound pachymetry, and UBM. Three separate measurements meeting criteria for centrality and perpendicularity were recorded for each eye. RESULTS: The mean CCT by specular microscopy was 572 microm (95% confidence interval (CI), 566-578 microm), which was significantly greater than 550 microm (95% CI, 545-556 microm) (P<.001) and 555 microm (95% CI, 550-560 microm) (P<.001) by ultrasound pachymetry and UBM, respectively. The mean standard deviation (SD) of repeated measurements by specular microscopy was 7.82 microm, which was significantly greater than the mean SDs of 4.14 microm (P<.001) and 3.90 microm (P<.001) by ultrasound pachymetry and UBM, respectively. There was no statistically significant difference between the mean SDs by ultrasound pachymetry and UBM (P=.156). CONCLUSIONS: Although the CCT measurements by specular microscopy were significantly less reproducible than those by ultrasound pachymetry and UBM, the error levels were clinically acceptable. Both ultrasound pachymetry and UBM produced similar CCT measurements, which were significantly less than those generated by specular microscopy. One should be aware of the advantages and limitations of each machine and of possible differences in the CCT measurements by optical and ultrasound pachymetry.

Body Weights and Measures↗

PubMed2005

In vivo and ex vivo virtual biopsy of the liver with near-infrared, reflectance confocal microscopy.

The assessment of liver architecture is an essential part of the understanding of its physiology and pathology. Current fluorescence confocal microscopy methods face numerous drawbacks, such as cytotoxicity, quenching effect, potential negative ino- and chrono-tropic effects and leaking of fluorescent agents through the sinusoid fenestrations. The recently developed, near-infrared reflectance confocal microscopy allows high-resolution optical sectioning through intact tissues, without employing fluorescent stains, while contrast between structures is provided by the natural refractivity of the tissue. The aim of this study is to assess the utility of near-infrared reflectance confocal microscopy in the evaluation of the hepatic microscopic architecture in vivo and ex vivo. Rat livers were noninvasively examined in vivo and ex vivo with near-infrared reflectance confocal microscopy. Two experimental contrast agents were subsequently used to enhance particular structures. Parenchymal and vascular structures are readily identified, as well as some intracellular details. Differences between in vivo and ex vivo states were also observed. The use of contrast agents also highlights certain morphologic structures. In conclusion, near-infrared reflectance confocal microscopy stands as a useful adjunct technique to the study of hepatic parenchyma offering details equivalent to, if not surpassing traditional light microscopy.

Animals↗

PubMed2026

Cumulative microscopy reveals cellular states in fibroblasts from patients with genetic disorders.

Analysis of cellular states and signaling trajectories can provide insights into causes of disease. We developed cumulative microscopy, a method to perform cyclical imaging without elution or quenching steps. Cumulative microscopy computationally extracts individual signals from accumulating fluorescence during sequential imaging. We use cumulative microscopy to quantitatively assess cell cycle and stress markers in individual primary fibroblasts from patients with rare genetic proliferative disorders with increased cancer risk. Neural network-based analysis of cumulative microscopy data suggests that cells from patients with Cartilage-hair hypoplasia (CHH), but not Mulibrey Nanism (MUL), show replication stress. We analyze cell states and cell trajectories and find that a subset of cells from patients with CHH show spontaneous replication stress, followed by cell cycle exit in both G1 and G2 phases. We note that replication stress potentially could underlie both proliferative defects and increased cancer risk in CHH patients and conclude that cumulative microscopy is an efficient, quantitative, and generalizable approach to multiplex microscopy.

Humans↗

PubMed2006

[Second- and third-harmonic generation microscopies for the structural imaging of intact tissues].

One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system ; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy.

Histological Techniques↗

PubMed2005

Four-dimensional ultrafast electron microscopy.

Electron microscopy is arguably the most powerful tool for spatial imaging of structures. As such, 2D and 3D microscopies provide static structures with subnanometer and increasingly with angstrom-scale spatial resolution. Here we report the development of 4D ultrafast electron microscopy, whose capability imparts another dimension to imaging in general and to dynamics in particular. We demonstrate its versatility by recording images and diffraction patterns of crystalline and amorphous materials and images of biological cells. The electron packets, which were generated with femtosecond laser pulses, have a de Broglie wavelength of 0.0335 angstroms at 120 keV and have as low as one electron per pulse. With such few particles, doses of few electrons per square ångstrom, and ultrafast temporal duration, the long sought after but hitherto unrealized quest for ultrafast electron microscopy has been realized. Ultrafast electron microscopy should have an impact on all areas of microscopy, including biological imaging.

Animals↗

PubMed2000

The role of electron microscopy in gynecological pathology.

Electron microscopy, as a diagnostic method, has been available to pathologists for about half a century. Its use in studying normal and abnormal gynecological tissues has been applied during the second half of that period, and many works on specific female genital topics have been published. Several of those subjects are worthy of citing in a review of the present type. Clear cell carcinoma has been revealed to be a mullerian, rather than a wolffian, derivative. Small cell carcinoma of the ovary with hypercalcemia is comprised of cells shown ultrastructurally to be epithelial, but unlike surface epithelial cells, germ cells, sex-cord cells, or neuroendocrine cells. Further electron microscopic studies provided evidence that these small cell tumors are not adult diffuse granulosa cell tumors, endometrioid stromal tumors, primitive neuroectodermal tumors, or numerous other primary and metastatic small cell tumors. Electron microscopy has also been useful in determining that not all signet-ring cell tumors of the ovary are stromal, and that there are multiple types of signet-ring (vacuolated) cells in ovarian tumors. Smooth muscle tumors are well known to have multiple light microscopic phenotypes, and electron microscopy has proven to be diagnostic in many of these cases, especially in epithelioid smooth muscle tumors. A number of other gynecological neoplasms that have been better defined by electron microscopic studies are described. Embryology and histogenesis are other areas of study in which electron microscopy has been a major contributor of new information at the subcellular level. Electron microscopy, solely or in harmony with clinical information, light microscopy, and immunohistochemistry, has been and is a valuable tool for the pathologist in the study of histogenesis and accurate diagnosis of gynecological lesions.

Adenocarcinoma, Clear Cell↗