Deep stroma investigation by confocal microscopy

Application of Atomic Force (AFM), Environmental Scanning Electron (ESEM) and Confocal Laser Scanning Microscopy (CLSM) in bitumen: A review of the ageing effect

Investigations of mass movements of the Messel oil shale in Germany presumed that swell- and especially shrink-deformations of the organic-rich clay were among the factors triggering initial displacements. Within the scope of the present studies, delicate clay/organic microstructures had to be deciphered. Reactions of clay minerals and microstructures upon dehydration were observed by environmental scanning electron microscopy (ESEM) and the distribution of organic matter was studied by confocal laser scanning microscopy (CLSM) in fluorescence mode. ESEM and CLSM have been demonstrated to be valuable tools for the microstructural characterization of laminated organic-rich sediments. The application of these analytical and imaging techniques will be of great interest to a wide range of geological disciplines such as palaeontology, organic petrology, engineering geology and global change studies.

The alkaliphilic cyanobacterium Limnospira fusiformis is an integral part in food webs of tropical soda lakes. Recently, sudden breakdowns of Limnospira sp. blooms in their natural environment have been linked to cyanophage infections. We studied ultrastructural details and prophage components in the laboratory by means of confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). For a comparison at the subcellular level, we included transmission electron microscopy (TEM) material of infected cells collected during a field survey. Compared to TEM, CLSM has the advantage to rapidly providing results for whole, intact cells. Moreover, many cells can be studied at once. We chemically induced lysogenic cyanophages by means of mitomycin C (MMC) treatments and studied the ultrastructural alterations of host cells. In parallel, the number of cyanophages was obtained by flow cytometry. After treatment of the culture with MMC, flow cytometry showed a strong increase in viral counts, i.e., prophage induction. CLSM reflected the re-organization of L. fusiformis with remarkable alterations of thylakoid arrangements after prophage induction. Our study provides a first step towards 3D visualization of ultrastructure of cyanobacteria and showed the high potential of CLSM to investigate viral-mediated modifications in these groups.

Quantitative Measurement of Acute Corneal Injury in Rabbits with Surfactants of Different Type and Irritancy

The efficacy of conventional high vacuum scanning electron microscopy (SEM), environmental SEM (ESEM), and confocal laser scanning microscopy techniques in the assessment of cell-material interactions is compared. Specific attention is given to the application of these techniques in the assessment of the early morphological response of human osteoblast-like cells cultured on titanium dioxide. The processing of cells cultured for conventional high vacuum SEM leads to the loss of morphological features that are retained when using ESEM. The use of cytoskeletal labeling, viewed with confocal laser scanning microscopy, in conjunction with ESEM gives an indication of the changes to cell morphology as a consequence of incubation time in response to interactions at the biological/material interface.

Enamel and dentin of human teeth irradiated by CO2 laser were investigated by confocal laser scanning microscopy (CLSM), low vacuum scanning electron microscopy (WET-SEM) and atomic force microscopy (AFM). Optical tomographic imaging by CLSM, compositional imaging based on atomic number effect of reflected electrons by WET-SEM, high resolution observation of surface morphology by AFM were done for both the irradiated and non-irradiated area of the same specimen throughout. The crystals of about 50 microm length and the bright spots were observed by CLSM at the bottom of the cavity induced by laser irradiation. They turned out from the observation by WET-SEM as the acicular crystals with the cross section of an irregularly hexagonal shape situated parallel and perpendicular, respectively, to the inner surface of the cavity. The thickness of the thermally deteriorated zone of the cavity was about 25 microm. The crystals unidirectionally grown up to the size of several hundreds nm were also observed by AFM, while the apatite crystallites of 50-150 nm were recognized all over in non-irradiated area. All the results suggest that after instantaneous melting at the surface of teeth by CO2 laser shot the crystals of calcium phosphate were recrystalized and grown to a large size. The compositional imaging in addition to morphological observation was useful to obtain the information of the change in materials induced by laser irradiation.

Centractin (Arp 1) is an actin-related protein that shares 53% sequence identity with conventional actin and is predicted to have a similar core structure for nucleotide binding. Biochemical studies of centractin, named for its apparent localization to the centrosome, reveal that it is a member of a stable macromolecular complex, dynactin. The dynactin complex consists of at least 9 polypeptides and has been implicated in activating dynein mediated vesicle transport along microtubules. Ultra-structural analysis, by rotary shadowing electron microscopy with antibody decoration, reveals that within the context of the dynactin complex, centractin forms a 37 nm filament resembling the structure of an actin filament with capping protein and p62 localizing to opposite ends.We have used transient transfection assays to investigate the effects of overexpression of centractin in cells. Mammalian PtK2 cells were transiently transfected with centractin cloned in the pcDNA3 vector under the control of a CMV promoter. The cells were transfected for 24 hours using Ca2+/DNA co-precipitates. The transfection efficiency was as high as 41 % as determined by direct counting of immunostained cells. Cells were fixed at time points ranging from 18 to 24 hours post washing using 1% formaldehyde plus 1% Triton X-100 (5 min), 0.05% glutaraldehyde (5 min), 1 % formaldehyde (5 min), and 0.05% sodium borohydride to block free aldehyde groups (3×5 min). Immunofluorescence employed an affinity-purified, rabbit polyclonal antibody to centractin followed by a Texas Red-labeled secondary antibody. Epifluorescence microscopy revealed that all of the cells overexpressing centractin contained centractin structures with novel morphology. Two distinct patterns were observed consistently: filamentous centractin localized throughout the cytoplasm (Figure la) and perinuclear centractin accumulations (Figure lb). The perinuclear accumulations appear to be highly three dimensional and curved in structure (confirmed by confocal microscopy), and often have associated filamentous structures (Figure lb). The addition of 33 micromolar nocodozole (30 min. at 4°C) to the transfected cells results in thinner centractin filaments (Figure lc).

The therapeutic efficacy of antitumor drugs is often limited by non-specific, systematic delivery. Here we report a novel targeted delivery platform designed by coupling liposomal nanobins (NB) with the urokinase plasminogen activator receptor (u-PAR) antibody (ATN-658). The urokinase system is overexpressed in epithelial ovarian cancer (OvCa) cells and is expressed only at low levels in normal cells. Doxorubicin (Dox), a FDA approved antitumor drug, had been loaded into the nanobins as a payload. The size and surface charge of the nanobins were optimized to facilitate specific binding to u-PAR expressing OvCa cells. Confocal and transmission electron microscopy showed that ATN-658-NB(Dox) was internalized in OvCa cells in a receptor-dependent manner and was released from endosomes as a function of time. This uptake could be blocked by stably down-regulating u-PAR expression in the OvCa cells using shRNA. In an orthotopic ovarian cancer model, athymic mice treated with ATN-658-NB(Dox) had a significantly greater reduction in tumor burden (0.06±0.01g versus 0.1±0.01g; p<0.001) than mice treated with NB(Dox), which was corroborated by in vivo fluorescence imaging results. A survival study showed that targeted nanobins prolonged the survival of mice by 3 days (43 days versus 40 days, median survival) as compared to untargeted nanobins. Immunoblot analysis of tumor sections showed that targeted nanobins induced apoptosis (cleaved caspase 3) and inhibited proliferation (Ki-67) more efficiently when compared to untargeted nanobins. Furthermore, tumor-targeted delivery of ATN-658-ND(Dox) was confirmed by ex vivo fluorescence imaging, FACS analysis, and confocal microscopy. Taken together, our findings show that receptor-mediated delivery of targeted nanobins to cancer cells could serve as a novel approach to ovarian cancer treatment. Citation Format: Yilin Zhang, Elden P. Swindell, Patrick L. Hankins, Hilary A. Kenny, Thomas V. O’Halloran, Andrew P. Mazar, Ernst Lengyel. Targeted delivery of doxorubicin loaded nanobins to ovarian cancer cells through the urokinase plasminogen activator system [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1440.

To evaluate the efficacy of topical voriconazole in patients with fungal keratitis caused by different fungal species. Interventional case series. Eighty-four patients aged 18 years or older from central China with confirmed fungal keratitis who presented at the outpatient department of Henan Eye Institute were enrolled in the study. The patients underwent in vivo confocal scanning laser microscopy examination, coneal scraping and microscopic examination and fungal culture, and then received topical voriconazole, closed curative effects were conducted. The sensitivity of three different diagnostic techniques, spectrum of the fungi, cure rate for fungal corneal infection related to each species were analyzed. In our study, 84 patients were diagnosed with fungal keratitis based on clinical symptoms, and results of at least one of the examinations of in vivo confocal scanning laser microscopy, conventional smear, and corneal scraping culture and the sensitivity were 92.85%(78/84), 85.71%(72/84), 84.52%(71/84)respectively. In viro confocal scanning laser microscopy. Successful management was achieved in 83.33% of the patients. Topical voriconazole treatment failed in 14 patients(16.67%), who required surgical treatment. In 36 patients with Fusarium corneal infections, 26(72.22%)were successfully treated with topical voriconazole; however, in 18 patients with Aspergillus corneal infections and 7 patients with Alternaria corneal infections, 94.44% and 100.00% of them were successfully treated, respectively. In a case-based dynamic tracking study, in vivo confocal microscopy provided real-time dynamic detection of surviving hyphae. The existence of hyphae was(38.35±17.32)days for Fusarium,(25.00±16.11)days for Aspergillus,(21.00±4.36)days for Alternaria, and(41.50±31.68)days for the focus in the deep stroma. The duration of treatment was similar for all four groups. Topical application of voriconazole is on effective method for the freatment of fungal keratitis Topical voriconazole is highly effective for fungal keratitis caused by Alternaria spp. and Aspergillus, however, for Fusarium, it is relatively low effective. (Chin J Ophthalmol, 2016, 52: 657-662).

The present investigation provides novel information on the topographical distribution of macrophages and dendritic cells (DCs) in normal meninges and choroid plexus of the rat central nervous system (CNS). Whole-mounts of meninges and choroid plexus of Lewis rats were incubated with various anti-leucocyte monoclonal antibodies and either visualised with gold-conjugated secondary antibody followed by silver enhancement and subsequent examination by environmental scanning electron microscopy or by the use of fluorochromes and confocal microscopy. Large numbers of MHC class II(+) putative DCs were identified on the internal or subarachnoid aspect of dural whole-mounts, on the surface of the cortex (pia/arachnoid) and on the surface of the choroid plexus. Occupation of these sites would allow DCs access to cerebrospinal fluid (CSF) and therefore allow antigens into the subarachnoid space and ventricles. By contrast, macrophages were less evident at sites exposed to CSF and were more frequently located within the connective tissue of the dura/arachnoid and choroid plexus stroma and also in a sub-pial location. The present data suggest that DC may be strategically located within the CNS to sample CSF-borne antigens. Furthermore, the data suggest that CNS tissue samples collected without careful removal of the meninges may inadvertently be contaminated by DCs and meningeal macrophages.

The ocular irritation responses to 11 different surfactants and two concentrations of acetic acid and sodium hydroxide have been shown to depend on the extent of initial injury, despite marked differences in the processes leading to tissue damage. The purpose of these studies was to determine the extent to which this fundamental relationship applies to other nonsurfactants. Ten microl of acetone (ACT). cyclohexanol (CY), parafluoroaniline (PF), or 37% formaldehyde (FA) was directly applied to the cornea of the right eye of each rabbit. Eyes and eyelids were macroscopically scored for signs of irritation beginning 3 hours after dosing and periodically until recovery or 35 days. Tissues were obtained for light microscopic examination after 3 hours and on days 1, 3, and 35. Initial corneal injury was characterized quantitatively at 3 hours and I day using in vivo confocal microscopy (CM) and by postmortem quantitation of dead corneal epithelial cells and keratocytes using a Live Dead Assay (L/D, Molecular Probes) and scanning laser CM. Corneal changes over time were characterized quantitatively using in vivo CM performed at 3 hours and 1, 3, 7, 14, and 35 days. The changes with ACT were consistent with mild irritation. Corneal injury was limited to the epithelium and superficial stroma, with the mean normalized depth of injury (NDI) being less than 10% with the majority of regions showing no stromal injury. Changes with CY and PF were consistent with moderate to severe irritation, and FA caused severe irritation. Specifically, corneal injury by CY and PF tended to involve the epithelium and anterior stroma, with the mean NDI being 10.4% to 23.8%, while injury with FA involved the epithelium, deep stroma, and at times the endothelium. Interestingly, with FA significantly less injury was observed at 3 hours with a dramatic increase in injury observed at 1 day and thereafter. In conclusion, these results continue to support and extend our hypothesis that ocular irritation is principally defined by the extent of initial injury despite clear differences in the means by which irritants cause tissue damage. We believe this approach can be applied to developing alternative assays based on injury to ex vivo eyes or injury to an in vitro corneal equivalent system.

Correlative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.

Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

Galloyl flavonoids from Acacia farnesiana pods possess potent anthelmintic activity against Haemonchus contortus eggs and infective larvae.

Актуальність. Біомікроскопічні зміни рогівки у пацієнтів, хворих на кератоконус, після кросслінкінгу (CLX) можливо виявити за допомогою конфокальної мікроскопії (КМ). В літературі зустрічаються поодинокі повідомлення про морфологічні зміни рогівки після CLX. Метою нашого дослідження було виявлення структурних змін рогівки за допомогою КМ після прискореного CLX у хворих на кератоконус. Матеріал та методи. В нашому дослідженні на 167 очах хворих на кератоконус після прискореного CLX виявленні структурні зміни рогівки за допомогою КМ в терміни спостереження до 12 міс після операції. Процедура прискоренного CXL виконувалася за допомогою приладу UV-X ™ 2000 з потужністю випромінювання 9 mW/см². Конфокальна біомикроскопія проводилась на приладі «Confoscan 4» (Nidek, Японія). Результати. Процедура акселерованого (10 хвилин) CLX при прогресуючому кератоконусі 2-3 стадій являється безпечною та приводить до стабілізації кератоконуса в терміни спостереження 12 місяців після операції. Через 3 місяці після процедури прискореного CLX зафіксовано активне відновлення кератоцитів в глибоких і поверхневих шарах строми з розсмоктуванням вогнищ фіброзу. Відновлення нормальної архітектоніки рогівки починається через 6 міс після прискореного кросслінкінгу та репополюзація кератоцитів завершається через 12 місяців за даними КМ.