Confocal Laser Research Articles

Cellular organisation in meiotic and early post-meiotic rice anthers

We have used fluorescent, confocal laser and transmission electron microscopy (TEM) to examine cellular organisations, including callose (1,3-beta-glucan) behaviour, in meiotic and early post-meiotic rice anthers. These features are critical for pollen formation and provide information to better understand pollen sterility caused by abiotic stress in rice and other monocotyledonous species. Among organelles during meiosis, abundant plastids, mitochondria and nuclei of the anther cells show distinctive features. Chloroplasts in the endothecium store starch and indicate a potential for photosynthetic activity. During meiosis, the middle layer cells are markedly compressed and at the tetrad stage are either vacuolated or filled with degenerating electron-opaque organelles. Viable mitochondria, stained with Rhodamine 123, are seen in the endothecium and tapetum, but the mitochondria in the middle layer are not stained during meiosis. The radial walls of the tapetum are disorganised and degenerating, indicating the formation of a syncytium; pro-orbicules are located at the locular walls at the tetrad stage. Immunohistochemical studies show that the sporogenous cells are entirely enveloped by a thick callosic layer at early meiosis. Cell plate callose was assembled in a plane between the dyad cells. In the tetrads, however, callose formed only at the centre, showing that the tetrad microspores are not enveloped but separated by callose walls. Thick, undulating electron-opaque walls around the tetrads indicate the beginning of exinous microspore wall differentiation.

Sequential confocal and multiphoton laser scanning microscopy using a single photonic crystal fibre based light source

A novel approach that simplifies the laser source requirements for confocal and multiphoton laser scanning (CLSM and MPLSM) using the novel dispersion properties of photonic crystal fibre (PCF) is reported. By tuning the fs-pulsed Ti:Sapphire laser to the zero dispersion wavelength of the PCF, the spectral and temporal properties of the source are largely unaffected and hence this source can easily be used for MPLSM. Conversely, by tuning the Ti:Sapphire laser emission wavelength by less than 10 nm to anomalously pump the PCF, the resultant white-light supercontinuum source can perform CLSM. Sequential CLSM and MPLSM of a double-labelled guinea pig detrusor (smooth muscle layer) specimen is described.

The Role of Grain Boundary Diffusion in Initial Selective Oxidation Kinetics of a Manganese-Aluminum TRIP Steel

Confocal scanning laser microscopy (CSLM) was used in real-time observation of alloy element oxidation of a manganese/aluminum transformation induced plasticity (TRIP) steel. CSLM images reveal a marked role of grain boundaries in the overall initial oxidation kinetics of the alloy and consequently in the morphology of the initial surface oxide. Changing the grain size and the oxidation temperature results in a change in the overall kinetics of selective oxidation and in the importance of oxidation at grain boundaries, respectively. Electron backscattered diffraction mapping was used to characterize the grain boundaries intersecting the surface over an area of the alloy. Subsequent CSLM observation of selective oxidation over the same area allowed quantification of oxidation kinetics as a function of individual grain boundary misorientation.

Study of a new energy efficient process for French fries production

A new, energy efficient production process for French fries was developed and evaluated. Superheated steam (SHS) was used for evaporation of water instead of pre-drying with air and par-frying with oil. The product was frozen by vacuum cooling. Unfortunately, with this process it was not possible to reach the quality of conventional French fries. Sensory analysis indicated that the main quality defect was a tough crust with a fatty appearance. Confocal Scanning Laser Microscopy showed that this was caused by skin formation on the surface during both SHS drying and vacuum cooling. A frying step was necessary to obtain a porous crust. A satisfactory product quality was feasible after drying with SHS instead of air. Due to the concessions made for the product quality, the final energy reduction was limited. Nevertheless, this study has gained more insight into how processing affects potato tissue on micro-scale and it has shown that a porous structure is essential for good quality French fries.

Improved cavitation resistance of structural materials in pulsed liquid metal targets by surface hardening

Cavitation damage of structural materials due to pressure waves is expected to be one of the major life-time limiting factors in high power liquid metal spallation targets under pulsed operation. Two methods are developed for the European Spallation Source (ESS) to mitigate this damage: Introduction of gas bubbles to suppress the pressure pulse, and surface hardening of structural materials to reduce their vulnerability. Surface hardening of four 8–13% Cr martensitic steels was performed by thermal treatment with pulsed or scanned electron- and laser beams as well as by nitriding in a r.f. plasma. Parametric dependences of these processes were investigated and partially optimised. Thermal treatment with electrons reached HV-hardness values up to about 600, laser treatment up to 710, and nitriding above 1200. After mechanical and metallographical characterisation, specimens of 12% Cr steel were tested in liquid mercury under pulsed proton irradiation, and under mechanical pulse-loading. Surface damage was analysed by optical, confocal laser, and scanning-electron microscopy, showing in all tests much better resistance of the hardened material compared to standard condition. Application of these techniques to a spallation target is outlined.

Observations and analysis of the peritectic reaction for Fe-Co Alloys

Abstract The thermodynamics and kinetics associated with the peritectic transition have become greatly discussed topics. A high-temperature confocal scanning laser microscope has been used to observe the growth of γ-austenite along the pro-peritectic δ-ferrite/liquid-Fe boundary and to measure the rate at which this occurs. It was found, for Fe-18 wt.% Co, Fe-19 wt.% Co, and Fe-20 wt.% Co alloys, that the reaction rate increased with increasing undercooling over the temperature range observed and that there was no discernible difference in rate between the three alloys. The growth rate dependence on undercooling was found to be v = (62.82 ΔT2-70.90 ΔT + 149.04) ± 15.61 μm s−1. This rate compared well with the growth model proposed by Bosze and Trivedi when the uncertainties in available phase diagram information were considered.

Evaluation of Surgical Tissue From Patients with Crohnʼs Disease for the Presence of Mycobacterium avium Subspecies paratuberculosis DNA by In Situ Hybridization and Nested Polymerase Chain Reaction

Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) with tissue granuloma and histopathological alteration that resembles aspects in tuberculosis, leprosy, and paratuberculosis. Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of paratuberculosis, with a suspected role in the etiology of CD. We investigated the presence of MAP DNA in 31 surgical tissue samples from 20 subjects using fluorescence in situ hybridization (FISH) with the aid of confocal scanning laser microscopy and nested polymerase chain reaction (PCR) using the IS900 sequence unique to MAP. MAP DNA was detected by PCR in tissue from 10 of 12 (83%) patients with CD: 7/12 (58%) in inflamed, 6/11 (55%) in noninflamed and in 10 (83%) of either tissue and by FISH in 8 of 12 (67%) patients with CD: 7 of 12 (58%) in inflamed, 4 of 11 (36%) in noninflamed, and in 8(67%) of either tissue. In non-IBD subjects, MAP DNA was detected in the tissue of only 1 of 6 patients (17%) by PCR and 0 of 6 patients (0%) by FISH. MAP DNA was identified by PCR in inflamed tissue from 2 of 2 patients with ulcerative colitis. The detection of MAP DNA by either technique in tissue from subjects with CD is significant compared with non-IBD subjects (P < 0.005). Identification of MAP DNA in both inflamed and noninflamed tissue by both techniques suggests that MAP infection in patients with CD may be systemic. The data add more evidence toward a possible association of MAP in the pathogenesis of CD.

Luminescent nanostructures based on colour centres produced in LiF films by direct writing with an X‐ray microprobe

Low-dimensionality photoluminescent patterns based on broad-band visible-emitting F2 and F3+ colour centres have been realised in Lithium Fluoride films by direct writing with a focused soft X-ray beam provided at the ELETTRA synchrotron radiation facility. Permanent fluorescent lines, with typical width from few hundreds of nanometers to tens of micrometers, have been prepared by scanning the LiF specimen with respect to the X-ray microprobe and imaged by confocal laser and near-field optical microscopy in fluorescence configuration. The use of this versatile lithographic technique offers the opportunity to integrate active and passive optical components, like waveguides and gratings, in this promising material, grown by simple and well assessed film deposition methods on different substrates. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The Effect of the Menstrual Cycle on the Optic Nerve Head Analysis of Migrainous Women

Purpose: To determine the effect of the menstrual cycle on the optic nerve head topographic analysis of normally menstruating migrainous women.Material and Methods: Randomly selected one eye of 44 migrainous and 49 healthy control women with regular menstrual cycles were included in the study. All subjects underwent complete ocular examination. Optic nerve head topographic analysis were performed using a confocal scanning laser ophthalmoscope, HRT II (Heidelberg Retinal Tomograph II, software version 1.6; Heidelberg Engineering, Heidelberg, Germany). They were repeated for two times during the menstrual cycle: in follicular phase (7th to 10th day of the cycle) and in the luteal phase (days 3 to 4 before the menstrual bleeding). Serum estradiol, progesterone, and luteinizing hormone measurements were repeated at each menstrual phase. Results: The mean age of migrainous and control subjects were 31.5 ± 5.1 years and 33.4 ± 3.7 years, respectively (P > 0.05). Their mean disc areas were 2.26 ± 0.46 mm2 and 1.95 ± 0.39 mm2, respectively (P < 0.05). Control subjects did not demonstrate any difference in the disc topography (P > 0.05). The parameter rim volume decreased, while the parameters cup volume and cup shape measure increased significantly in the luteal phase of the migrainous women (all P values < 0.05). Mean intraocular pressure of the migrainous women decreased significantly in luteal phase (P < 0.05). Conclusion: Significant differences exist in the optic rim and cup parameters during the menstrual cycle of the migrainous women. Further clinical trials on ocular blood flow changes during the menstrual cycle of the migrainous women may highlight the role of sex steroids in the optic nerve head of the migrainous women.

Macular pigment density in age-related maculopathy

Macular pigment (MP) is believed to have a protective role in the development and progression of age-related maculopathy (ARM). We present results of measurements of macular pigment density (MPD) in patients with ARM at different stages with a modified confocal scanning laser ophthalmoscope (HRA, Heidelberg Engineering, Heidelberg, Germany). ARM patients were categorized into four groups. Staging depended upon the size of drusen, geographic atrophy, and loss of visual acuity due to ARMD in the fellow eye; 1: drusen size <125 microm, 2: drusen size >125 microm, 3: drusen size <125 microm + ARMD in fellow eye, and 4: drusen size >125 microm + ARMD in fellow eye. We took autofluorescence images at 488 nm and 514 nm with a modified HRA. MP density was evaluated within 2 degrees around the center of the fovea. In this study, we included 146 patients with ARM: 26 in group 1, 19 in group 2, 80 in group 3, and 21 in group 4. The mean MPD of the different stages of ARM was: in group 1, 0.233 +/- 0.091 DU; group 2, 0.218 +/- 0.102 density units (DU); group 3, 0.252 +/- 0.085 DU; and group 4, 0.208 +/- 0.100 DU. Statistical analysis (Kruskal-Wallis test) showed no differences between groups. Recent studies in ARM did not result in significant differences of MPD between the various stages of ARM. Further longitudinal studies are requested to compare the incidence of ARMD in eyes with high and low MPD in order to provide definite evidence of the influence of MPD on the progression of ARMD.

Determination of Diffusion Coefficients of Fluorescently Labelled Molecules in Lipid Membranes Using Confocal Scanning Laser Microscopy

A series of liposomes has been studied by the Fluorescence Recovery after Photobleaching technique using confocal scanning laser microscopy. Both mobile and immobile specimens of lipid membranes have been examined. The specimens consisted of liposomal suspensions incorporating mixtures of the phospholipids dioleoyl phosphatidylcholine (DOPC) and distearoyl phosphatidylcholine (DSPC) with the aminolipid dihexadecanol glycerophospho-ethanolamine (DHPE). The measurements reported provide information about the effective diffusion coefficients, which vary from 0.2 · 10−10 cm2s−1 to 3.2 · 10−10 cm2s−1. They were highly dependent on the ratio DSPC:DOPC. A simple theoretical model of 1-D diffusion was applied for the calculation of the effective diffusion coefficients. The measurements show that it is possible to use straightforward procedures with an unmodified commercial confocal microscope to determine diffusion coefficients of biological specimens.

Transverse mode competition in pulsed-laser pumped solid-state lasers: generation of a second pulse

Effects of transverse mode competition in macroscopic nanosecond pulsed-laser-pumped confocal solid-state lasers have been observed and measured. The effect of the alignment of the resonator on the temporal structure of the laser output has been measured. For some angular positions of the mirror a second laser output pulse has been observed with a different spatial structure from the first pulse. In order to explain this phenomenon a diffraction theory which includes gain in the pumped sample and misalignment effects has been developed. Using this theory it is possible to explain the generation of the second pulse and the temporal dynamics of the laser. The fact that transverse modal competition is the cause of the effect is clearly established.

Arachidonic Acid Release from Mammalian Cells Transfected with Human Groups IIA and X Secreted Phospholipase A2 Occurs Predominantly during the Secretory Process and with the Involvement of Cytosolic Phospholipase A2-α

Stable expression of human groups IIA and X secreted phospholipases A(2) (hGIIA and hGX) in CHO-K1 and HEK293 cells leads to serum- and interleukin-1beta-promoted arachidonate release. Using mutant CHO-K1 cell lines, it is shown that this arachidonate release does not require heparan sulfate proteoglycan- or glycosylphosphatidylinositol-anchored proteins. It is shown that the potent secreted phospholipase A(2) inhibitor Me-Indoxam is cell-impermeable. By use of Me-Indoxam and the cell-impermeable, secreted phospholipase A(2) trapping agent heparin, it is shown that hGIIA liberates free arachidonate prior to secretion from the cell. With hGX-transfected CHO-K1 cells, arachidonate release occurs before and after enzyme secretion, whereas all of the arachidonate release from HEK293 cells occurs prior to enzyme secretion. Immunocytochemical studies by confocal laser and electron microscopies show localization of hGIIA to the cell surface and Golgi compartment. Additional results show that the interleukin-1beta-dependent release of arachidonate is promoted by secreted phospholipase A(2) expression and is completely dependent on cytosolic (group IVA) phospholipase A(2). These results along with additional data resolve the paradox that efficient arachidonic acid release occurs with hGIIA-transfected cells, and yet exogenously added hGIIA is poorly able to liberate arachidonic acid from mammalian cells.

The Hippocampal Cholinergic Neurostimulating Peptide, the N-terminal Fragment of the Secreted Phosphatidylethanolamine-binding Protein, Possesses a New Biological Activity on Cardiac Physiology

Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.

Peripapillary atrophy in the unilateral exfoliation syndrome

To compare the areas of peripapillary atrophy between fellow eyes in patients with unilateral exfoliation syndrome. Topographic measurements for peripapillary atrophy and the optic nerve head with confocal scanning laser ophthalmoscopy, using the Heidelberg Retina Tomograph were performed on 45 nonglaucomatous patients with unilateral exfoliation syndrome. The area of peripapillary atrophy was divided into an alpha and a beta zone. The areas of alpha and beta peripapillary atrophy and their angular extents around the disc were measured. The frequency distributions of the alpha and beta areas and their largest radial extents at different positions around the disc were calculated. An alpha area was detected in 84% of the exfoliative and 89% of the nonexfoliative fellow eyes, and a beta area in 31% and 42% of eyes, respectively. The exfoliative and the fellow nonexfoliative eyes did not differ in the sizes of the alpha (0.43+/-0.46 vs 0.33+/-0.18 mm(2), P=0.68) and beta (0.14+/-0.30 vs 0.17+/-0.34 mm(2), P=0.96) areas of peripapillary atrophy. The angular extents, the locations of the largest radial extent, and the frequency distributions of the alpha and beta peripapillary atrophy areas were similar in fellow eyes. Exfoliation syndrome itself is not a risk factor for peripapillary atrophy.

Measurement procedures in confocal choroidal laser Doppler flowmetry

Purpose. To estimate a practical number of measurements within a series of recordings with a confocal choroidal laser Doppler flowmeter, capable of providing an adequate statistical power for future studies. Methods. Ten independent measurements of choroidal blood flow were obtained within 30 minutes in one randomly selected eye of 24 subjects. In 12 subjects, subsequent series of 10 measurements were also obtained 2 and 7 days after baseline assessment. Average and median values of these series were calculated for the choroidal blood flow parameters velocity, volume, and flux, using either the first 3, the first 4, the first 5, the first 6, the first 7, the first 8, the first 9, or all 10 measurements. The variance between the series was analyzed using non-parametric tests. Results. The average values of a series of independent measurements decreased with an increasing number of independent measurements. More than 5–7 independent recordings did not markedly influence the estimate of the average value of choroidal blood flow parameter for an individual. Base-line measurements showed markedly higher values than subsequent series. Average values obtained with 6 independent measurements seem to provide a reasonable level of statistical power for paired comparisons, 13 subjects providing a 80% power at a probability level of 0.05 to find a significant difference of 15% between two means. Conclusion. It seems recommendable that experimental measurements with a confocal choroidal laser Doppler flow-meter are preceded by a session of familiarization and that average values of series of at least 6 measurements are used.

In-situ Observation of Surface Relief Formation and Disappearance during Order-Disorder Transition of Equi-atomic CuAu alloy using Laser Scanning Confocal Microscopy

ABSTRACTIn-situ observation of the formation and disappearance of the surface relief associated with the twinning during the order-disorder transitions among CuAu-I (L10), CuAu-II (PAP) and disordered fcc phases was conducted using Confocal Scanning Laser Microscopy equipped with a gold image furnace. The Retro effect was confirmed in poly-crystal samples, however no evidence was found in single-crystal samples. Also observed in poly-crystal samples are that the disordering temperature detected by the disappearing of relieves is different from grain to grain, and that grain boundary cracking alleviates the Retro effect. The observed phenomena were explained based on the crystallographic orientation relationship among grains investigated by FESEM/EBSD in terms of the elastic strain effect around grain boundaries induced by transition. It was confirmed that in each grain the surface relieves correspond to a set of two {011} planes having a &lt;100&gt; axis perpendicular to both planes in common. It was also found that the larger the average strain of two neighboring grains is, the lower the transition temperature. This observation was explained by the stress effect on the stability of a phase.

Substrata effects on bacterial biofilm development in a subsurface flow dairy waste treatment wetland

Biofilm development on two distinct rock substrata was investigated both in vitro and in a subsurface flow wastewater treatment wetland in order to determine the effect of hydrophobicity on initial bacterial adsorption, tertiary biofilm development and microbial population structure. Two commonly used wetland rock types, slag (a hydrophobic by-product of the steel smelter industry) and greywacke (a more hydrophilic sedimentary rock) were evaluated. In vitro investigations of initial microbial adsorption trends showed that the more hydrophobic slag displayed rapid bacterial adsorption rates compared to greywacke. Mean microbial adsorption rates of a mixed wetland bacterial population over 5 hours, described using a first order kinetics model, were 1.3 x 10(-12) m/sec for slag and consistently lower at 8.7 x 10(-13) m/sec for greywacke. Pristine rock studs of the two substrata were also exposed to wetland microbial communities during a six week field trial using confocal scanning laser microscopy to determine tertiary biofilm structure and fluorescent in situ hybridisation to investigate bacterial populations. During the first five weeks of growth CSLM analysis revealed that 75% of biofilms on slag were thicker and had greater coverage compared with those grown on greywacke. After six weeks of growth over 50% of the tertiary biofilms were structurally very similar on both rock types and only 25% of those grown on slag were larger than those on greywacke. In situ hybridisation analysis of bacterial populations revealed very little difference in population structure between biofilms grown on slag and those grown on greywacke. Eubacteria were present as a very high proportion of total bacteria throughout biofilm development (74.3%). The beta subgroup was the most populous of the Proteobacteria (31.4%) followed by the gamma subgroup (13.4%) and the alpha subgroup (1.3%). The results of this study suggest that slag, as a more hydrophobic substratum, promotes the initial adsorption of bacteria during early biofilm growth and better supports mature biofilm structures when used in wetlands. This study has implications for the design and construction of wastewater treatment wetlands.

Correlation of cyclooxygenase-2 pathway and VEGF expression in head and neck squamous cell carcinoma

The purpose of this study was to determine whether cyclooxygenase-2 (COX-2) was overexpressed in head and neck squamous cell carcinoma (HNSCC) in relation to vascular endothelial growth factor (VEGF) expression and lymph node metastasis. COX-2 has an important role in the promotion of carcinogenesis, tumor invasiveness and angiogenesis. VEGF is a proangiogenic factor that is up-regulated in various tumors. VEGF has been shown to interact with COX-derived prostaglandins in angiogenesis. To better understand the roles of these genes in HNSCC, we evaluated the immunohistochemical expression pattern of COX-2 and VEGF in relationship with histologic differentiation, clinical stage, and nodal status in 146 HNSCC. The COX-2/VEGF double immunofluorescein staining was evaluated with confocal scanning laser microscope. A positive expression was seen in 96% (140/146: 98 strong positive, 42 weakly positive) for COX-2 and in 98% (143/146: 63 strong positive, 80 weakly positive) for VEGF in HNSCC. A correlation was present in the positive expression of COX-2 with histologic differentiation, clinical stage, and nodal status. VEGF expression was also correlated with nodal status. A significant relation was observed between COX-2 and VEGF expressions. The simultaneous expression of COX-2 and VEGF was statistically significant (p<0.05). These results indicate that elevated COX-2 or VEGF expression is more common in HNSCC with poor prognostic characteristics. The present findings support the efforts to initiate clinical trials on the efficacy of COX-2 inhibitors in adjuvant treatment of HNSCC.

Ischemic preconditioning preserves connexin 43 phosphorylation during sustained ischemia in pig hearts in vivo.

During myocardial ischemia, connexin 43 (Cx43) is dephosphorylated in vitro, and the subsequent opening of gap junctions formed by two opposing Cx43 hexamers was suggested to propagate ischemia/reperfusion injury. Reduction of infarct size (IS) by ischemic preconditioning (IP) involves activation of protein kinase C (PKC) and p38 mitogen activated protein kinase (MAPK), both of which can phosphorylate Cx43. We now studied in anesthetized pigs whether IP impacts on Cx43 phosphorylation by measuring the density of non-phosphorylated and total Cx43 (confocal laser) during normoperfusion and 90-min ischemia in non-preconditioned and preconditioned hearts. Co-localization of PKCalpha, p38MAPKalpha, and p38MAPKbeta with Cx43 and the activity of p38MAPK were assessed. IP by 10 min ischemia and 15 min reperfusion reduced IS. Non-phosphorylated Cx43 remained unchanged during ischemia in preconditioned hearts, while it increased from 35+/-3 to 75+/-8 AU (P<0.05) in non-preconditioned hearts. Co-localization of PKCalpha, p38MAPKalpha, and p38MAPKbeta with Cx43 during ischemia increased only in preconditioned hearts. While the ischemia-induced increase in p38MAPKalpha activity was comparable in preconditioned and non-preconditioned hearts, p38MAPKbeta activity was increased only in preconditioned hearts. Blockade of p38MAPK by SB203580 attenuated the IS-reduction and the increased p38MAPK-Cx43 co-localization by IP. We conclude that IP increases co-localization of protein kinases with Cx43 and preserves phosphorylation of Cx43 during ischemia.