Dual-channel Confocal Laser Scanning Microscopy Research Articles

Direct Three-Dimensional Visualization of Membrane Fouling by Confocal Laser Scanning Microscopy.

Membrane-based separation is an important technique for removing emulsified oil from water. However, the mechanisms of fouling are complex because of the deformability and potential for coalescence and break-up of the oil droplets. Here, we report for the first time direct, three-dimensional (3D) visualization of oil droplets on electrospun fiber microfiltration membranes after a period of membrane-based separation of oil-in-water emulsions. High-resolution 3D images were acquired by a dual-channel confocal laser scanning microscopy (CLSM) technique in which both the fibers and the oil (dodecane) were fluorescently labeled. The morphology of dodecane as the foulant was observed for two different types of fibers, an oleophobic nylon (PA6(3)T), and oleophilic polyvinylidene fluoride (PVDF). Through direct visualization, the rejected oil was found to form droplets of clam-shell shape on the PA6(3)T fibers, whereas the oil tended to wet the PVDF fibers and spread across the membrane. The morphology was also analyzed as a function of separation time (i.e., "4D" imaging), as the oil accumulated within and upon the membranes. The observations are qualitatively consistent with a transition from blocking of individual pores in the membrane to coalescence of oil droplets into coherent liquid films with increasing filtration time. Analysis of permeate flux using blocking filtration models corroborate the transition of fouling modes indicated by the 3D images. This direct, 3D visualization CLSM technique is a powerful tool for characterizing the mechanisms of fouling in membranes used for liquid emulsion separations.

Quantitative analysis of synaptophysin immunoreactivity in human neocortex after cardiac arrest: confocal laser scanning microscopy study

Transient global ischaemia caused by cardiac arrest results in lesions that involve all brain structures. The aim of this study was to investigate the condition of synapses in patients surviving, but remaining in a persistent vegetative state, following resuscitation after cardiac arrest. We performed a quantitative analysis of the distribution and density of elements containing a synaptic vesicle protein-synaptophysin (p38)-in human neocortex in cases which survived for 1 week, 2 months, and 1 year after the cardiac arrest. Neurologically healthy cases that died following an accident served as control. Dual-channel confocal laser scanning microscopy (CLSM) was used to image p38-immunoreactivity (IR) and lipofuscin autofluorescence. In control cases no statistically significant differences were found for p38-IR between layers II–III and V–VII. However, the temporal cortex had a higher density of p38-immunoreactive structures than the motor cortex. In postischaemic cases a reduction in the density of p38-IR elements was apparent, mainly in the frontal and motor cortices and less pronounced in the temporal cortex. The least decrease compared with controls was observed in the visual cortex. In the 1 week survival case, a maximal decrease in p38-IR (35% below control) was found. In this case, the number of p38-IR elements per visual field was decreased, and big aggregates of p38-IR structures were observed. In general, the amounts of p38-IR structures were higher in all of the control cases compared with the postischaemic cases.

Molecular Dissection of the Mechanism by Which Potexvirus Triple Gene Block Proteins Mediate Cell-to-Cell Transport of Infectious RNA

The triple gene block (TGB; consisting of proteins TGB1–3) and coat protein (CP) of white clover mosaic potexvirus (WClMV) are required for cell-to-cell movement of viral RNA. Cell-to-cell spread of WClMV mutants in which the TGB open reading frames had been mutated was rescued in transgenic plants expressing specific TGB proteins (TGBPs). This indicated that there are no requirements for the synthesis in cis of viral TGBPs. These transgenic plants provided an experimental framework to explore the roles performed by the TGBPs and CP in cell-to-cell movement of WClMV RNA. Microinjection experiments established that TGB1 functions as the WClMV cell-to-cell movement protein (MP). Furthermore, combined microinjection and dual-channel confocal laser scanning microscopy provided direct evidence that infectious transcripts of WClMV move cell to cell as a ribonucleoprotein complex, consisting of single-stranded RNA, TGB1, and CP. Movement of this ribonucleoprotein complex displayed an absolute requirement for the presence of both TGB2 and TGB3. A model consistent with these findings is presented.

Structural and Functional Association between Substance P- and Calcitonin Gene-related Peptide-immunoreactive Nerves and Accessory Cells in the Rat Dental Pulp

Defense mechanisms of the dentin/pulp complex involve a variety of biological systems in which immunocompetent cells, the nervous system, and the vascular supply play important roles. In the present study, pulpal accessory cells were examined regarding (i) their structural relationship to nerves and (ii) how the functional capacities of these cells were affected by neuropeptides. Micro-anatomic association was investigated in the normal rat molar pulp with the use of double-immunofluorescence staining and dual-channel confocal laser scanning microscopy. Examinations of confocal laser scanning microscopic images from single focal planes revealed the presence of apparent contacts between thin, varicose nerve fibers and immunocompetent cells, indicating proximity between these two structures. The close associations were most frequently observed in the para-odontoblastic region of the coronal pulp, where more than 70% of class II antigen-expressing (OX6+) cells showed proximity to nerve fibers immunoreactive to calcitonin gene-related peptide. The corresponding figure for substance P was about 50%. ED2+ macrophages closely associated with nerves were less frequently observed. Functional studies conducted in vitro demonstrated that 10(-9) to 10(-7) mol/L of substance P significantly increased (p < 0.05), while 10(-7) to 10(-6) mol/L of calcitonin gene-related peptide suppressed (p < 0.01) proliferation of purified T-lymphocytes stimulated with sub-optimal concentrations of concanavalin A in the presence of rat incisor pulpal cells as accessory cells. These data suggest that pulpal sensory nerve fibers and their products may have an influence upon the immune defense of the dental pulp.

Morphological study of neocortical areas in Rett syndrome.

Various neocortical areas from four females aged 16-24 years with Rett syndrome (RS) were investigated and compared with brains of therapy-resistant partial epilepsy (TRPE) patients (18-25 years), infantile autism (IA), and control brains (24 and 58 years). The cytoarchitecture of area 10 (frontal), area 21 (temporal), area 4 (primary motor cortex), and area 17 (primary visual cortex) was studied by the combined Klüver-Barrera (luxol fast blue and cresyl violet) standard procedure. Autofluorescence of lipofuscin, immunofluorescence of synaptic vesicle proteins [synaptophysin (p38)] and lectin-stained (Wisteria floribunda agglutinin) perineuronal nets (PNs) were studied in the cortices using dual-channel confocal laser scanning microscopy. The brains of RS females show various types of morphological/cytoarchitectonical abnormalities of single pyramidal neurons in layers II-III, and V-VII of different cortical areas. The abnormalities include mild losses of pyramidal neurons, more pronounced in layers II and III than in layers V and VII, and more evident in frontal and temporal areas than in the visual cortex. Microdysgenesis, including abnormalities due to neuronal migration disorders, was not found in RS, in contrast to the observations in TRPE patients, strongly indicating that RS is not a neuronal migration disorder. Lipofuscin distribution was normal but amounts were lower in RS cases than in control and TRPE brains. PNs were less expressed in cortices of the IA case but were clearly overexpressed in the motor cortex of RS. Quantitative analysis of p38 showed a decrease in the area occupied by p38 immunoreactivity by 20-40% in RS compared with controls. It is concluded that RS could best be explained by a postnatal synaptogenic developmental deficiency; the basic defect, however, is still completely unknown.

Quantitative analysis of immunofluorescence and lipofuscin distribution in human cortical areas by dual-channel confocal laser scanning microscopy

A method for quantitative analysis of confocal laser scanning microscopy (CLSM) images of immunofluorescence and lipofuscin in human brain cortical areas is described. Indirect immunofluorescence was used to show the distribution and density of a synaptic vesicle protein — synaptophysin (p38). Dual channel CLSM was used for imaging immunofluorescence and autofluorescence of lipofuscin granules. Special software was developed for quantitative analysis of the CLSM images. The method has been tested in studies of frontal, temporal, motor, visual, and entorhinal cortices in normal and pathological human brains (Rett syndrome and autism). Using this technique the intensity and amount of p38 immunoreactivity was quantified after subtraction of lipofuscin fluorescence. In normal cortex no statistically significant differences were found for p38 antigen between layers II, III and V. The highest concentration of p38 immunoreactivity was found in the frontal cortex. In Rett syndrome and autistic patients, the p38 immunofluorescence in the investigated cortex areas appeared reduced compared with normal. The software is also suitable for quantitative analysis of double-labelled immunofluorescent specimens in animals without lipofuscin.

Local Ca2+ transients (Ca2+ sparks) originate at transverse tubules in rat heart cells.

1. The origins of local [Ca2+]i transients (Ca2+ sparks) were studied using dual-channel confocal laser scanning microscopy. Line scan images showing [Ca2+]i (as fluo-3 fluorescence) and the transverse tubule membranes (as Di-8 fluorescence) were obtained simultaneously in single rat cardiac ventricular cells. 2. Line scan images of Di-8 fluorescence showed peaks regularly spaced at intervals of 1.83 +/- 0.30 microns (mean +/- S.D.). These peaks corresponded to the transverse tubules (T-tubules) in cross-section. 3. Line scan images of fluo-3 fluorescence showed local [Ca2+]i transients (LCTs or Ca2+ sparks) evoked by electrical stimulation. 4. Eighty-five per cent (85%) of all Ca2+ sparks evoked by electrical stimulation (n = 138, in 5 cells) occurred within 0.5 micron of a T-tubule. Thirty per cent (30%) occurred within 1 pixel (0.20 micron) of a T-tubule. 5. In some cells studied (3 out of 5), certain T-tubules had a higher probability of being sites of origin of Ca2+ sparks than others. 6. These results support local control theories of excitation-contraction coupling in which Ca2+ release from the sarcoplasmic reticulum (SR) is triggered by a high local [Ca2+]i established between the L-type Ca2+ channels in the T-tubules and associated ryanodine receptor(s) in the junctional SR.

Confocal microscopy analysis of NPY and TH immunoreactivities in the hypothalamo-hypophysial system of the frog.

Co-expression of tyrosine hydroxylase (TH) and neuropeptide Y (NPY) in local circuits innervating the hypothalamo-pituitary complex of the green frog, Rana ridibunda, was investigated using simultaneous double immunohistochemical technique, aided by dual-channel confocal laser scanning microscopy. NPY and TH immunoreactivities were observed co-occurring within a discrete neuronal population located in the suprachiasmatic region. In other hypothalamic areas, NPY-immunoreactive (IR) perikarya were generally codistributed, but distinct from TH-IR cells. In the adenohypophysial pars intermedia, the overlap between the two markers was partial, demonstrating the existence of multiple neuronal sources for the inputs to the gland.

Micromapping of the human brain: Three‐dimensional imaging of immunofluorescence and dendritic morphology using dual‐channel confocal laser scanning microscopy

A strategy for investigating neuronal networks at the microscopic level is described. The Lucifer Yellow microinjection technique was combined with immunofluorescence on human brain material, which was then studied in a confocal laser scanning microscope with dual-channel scanning and equipped with an argon/krypton laser. The three-dimensional architecture of the Lucifer Yellow-injected neurons was investigated after transfer of the scanned frames in file format to a Silicon Graphics IRIS computer, using VoxelView software from Vital Images Inc. Microinjection of Lucifer Yellow revealed the dendritic morphology of various types of cells in different brain areas. Indirect immunofluorescence, with Texas Red as the secondary label, was used to determine the distribution of various categories of macromolecules (enzymes, receptor protein, and synaptic vesicle proteins) in the brain slices. We used single- as well as dual-channel confocal laser scanning microscopy for imaging these double-stained fluorescent specimens. Using these techniques in combination, we have created and saved three-dimensional confocal images of detailed morphology (axons and dendrites with spines and varicosities) of individual cells, together with the localization of immunofluorescence. These three-dimensional confocal images will be collected in a database for probable future use in human brain mapping. © 1994 Wiley-Liss, Inc.