Microtubule treadmilling in vitro investigated by fluorescence speckle and confocal microscopy.

Whether polarized treadmilling is an intrinsic property of microtubules assembled from pure tubulin has been controversial. We have tested this possibility by imaging the polymerization dynamics of individual microtubules in samples assembled to steady-state in vitro from porcine brain tubulin, using a 2% glycerol buffer to reduce dynamic instability. Fluorescence speckled microtubules were bound to the cover-glass surface by kinesin motors, and the assembly dynamics of plus and minus ends were recorded with a spinning-disk confocal fluorescence microscopy system. At steady-state assembly, 19% of the observed microtubules (n = 89) achieved treadmilling in a plus-to-minus direction, 34% in a minus-to-plus direction, 37% grew at both ends, and 10% just shortened. For the population of measured microtubules, the distribution of lengths remained unchanged while a 20% loss of original and 27% gain of new polymer occurred over the 20-min period of observation. The lack of polarity in the observed treadmilling indicates that stochastic differences in dynamic instability between plus and minus ends are responsible for polymer turnover at steady-state assembly, not unidirectional treadmilling. A Monte Carlo simulation of plus and minus end dynamics using measured dynamic instability parameters reproduces our experimental results and the amount of steady-state polymer turnover reported by previous biochemical assays.     

Localization of cerium-based reaction products by scanning laser reflectance confocal microscopy

Scanning laser confocal microscopy was utilized to visualize sites of hydrogen peroxide release from stimulated neutrophils and lysosomal acid phosphatase in these and other cells using cerium in the detection systems. Imaging of the cerium-containing reactions was achieved by employing the reflectance mode of this instrument. Localization of these products at the light microscope level was direct and did not require other reactions to generate a visible product. This new approach to cerium cytochemistry should prove useful for many applications.     

Quantitative imaging of intact cells and tissues by multi-dimensional confocal fluorescence microscopy

Confocal fluorescence microscopy enables visualisation and quantitation of fluorescent probes at high resolution deep within intact tissues, with minimal disturbance both of cell–cell interactions and the mechanical, ionic and physiological effects of the extracellular matrix. We illustrate the principles of multiple-parameter 3-D (x,y,z) imaging using reconstruction of nuclear channels in mammalian cells. Repeated sampling in time generates 4-D (x,y,z,t) images which can be used to follow dynamic changes, such as blue-light-dependent chloroplast re-orientation, in intact tissues. Quantitative measurements from multi-dimensional images require calibration of the spatial dimensions of the image and the fluorescence intensity response. This must be determined throughout the volume, which must be sampled to correct for geometric distortion as well as photometric errors arising from the complete optical system, including the specimen. The effects of specimen calibration are illustrated for morphological analysis of stomatal closing responses to abscisic acid in Commelina from 4-D images. Calibrated 4-D imaging allows direct volume measurements and we have followed volume regulation of chondrocytes in cartilage explants during osmotic perturbation. In intact cartilage, unlike in isolated cells, the chondrocytes exhibit volume regulatory mechanisms. In other cases, the fluorescence intensity of the probe may be related to a physiological parameter of interest and changes in its distribution within the cell. Optical sectioning permits discrimination of signal in separate compartments within the cell and can be used to follow transport events between different organelles. We illustrate 3-D (x,y,t) measurements of vacuolar glutathione conjugate pump activity in intact roots of Arabidopsis by following the sequestration of a fluorescent conjugate between glutathione and monochlorobimane. Dynamic measurements of protein localisation are now possible following the introduction of chimeric fusion proteins with green fluorescent protein (GFP) from Aequoria victoria. We have analysed the disposition of heterochromatin in nuclei of living Schizosaccharomyces pombe cells expressing a chimeric construct between Swi6 and GFP. Heterochromatin dynamics can be followed throughout mitosis in 4-D (x,y,z,t) images. Statistical analysis of the fluorescence histograms from each nucleus over time provides quantitative support for aggregation and dispersion of Swi6-GFP clusters during mitosis, rather than dissociation of Swi6 from the heterochromatin. A wide range of single-wavelength and ratio probes are available for imaging different ion activities. We compare 3-D (x,y,t) measurements of ion activities made using single-wavelength (Fluo-3 for calcium) and ratio (BCECF for pH) measurements, using stomatal responses in Vicia faba to peptides from the auxin-binding protein of maize and tip growth in pollen tubes of Lilium longiflorum as examples. Ratioing techniques have many advantages for quantitative fluorescence measurements and we conclude with a discussion of techniques to develop ratioing of single-wavelength probes against alternative references, such as DNA, protein or cell wall material.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Three-dimensional imaging of monogenoidean sclerites by laser scanning confocal fluorescence microscopy

A nondestructive protocol for preparing specimens of Monogenoidea for both alpha-taxonomic studies and reconstruction of 3-dimensional structure is presented. Gomori's trichrome, a stain commonly used to prepare whole-mount specimens of monogenoids for taxonomic purposes, is used to provide fluorescence of genital spines, the copulatory organ, accessory piece, squamodisc, anchors, hooks, bars, and clamps under laser scanning confocal microscopy.     

Tissue localization of phenolic compounds in plants by confocal laser scanning microscopy

Phenolic compounds are involved in many interactions of plants with their biotic and abiotic environment. These substances accumulate in different plant tissues and cells during ontogenesis and under the influence of various environmental stimuli, respectively. Studies on the tissue localization of phenolic compounds provide a fundamental prerequisite for understanding the ecological functions of these compounds. The present work shows the localization of various phenolics in cell walls, vacuoles, and associated with cell nuclei, in leaves of a monocotyledonous and a dicotyledonous plant, in a gymnosperm as well as in rhizomes of a horsetail by confocal laser scanning microscopy (CLSM). Using fresh plant material, it compares in detail the tissue localization of autofluorescent styrylpyrones and hydroxycinnamic acids and the visualization of epidermal flavonoid compounds using shift reagents like ammonia, and fluorescence-inducing reagents like Naturstoffreagenz A (diphenyl-boric acid 2-aminoethyl ester). The comparison of microscopic data obtained from different plant species shows the advantages and limitations of confocal laser scanning microscopy in ecological biochemistry of phenolic plant metabolites.     

Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy

Endothelial cells are covered by a surface layer of membrane-associated proteoglycans, glycosaminoglycans, glycoproteins, glycolipids, and associated plasma proteins. This layer may limit transendothelial solute transport. We determined dimension and transport properties of this endothelial surface layer (ESL) in isolated arteries. Rat mesenteric small arteries (diameter approximately 150 microm) were isolated and cannulated with a double-barreled -pipette on the inlet side and a regular pipette on the outlet side. Dynamics and localization of intra-arterial fluorescence by FITC-labeled dextrans (FITC-Deltas) and the endothelial membrane dye DiI were determined with confocal microscopy. Large FITC-Delta (148 kDa) filled a core volume inside the arteries within 1 min but was excluded from a 2.6 +/- 0.5-microm-wide region on the luminal side of the endothelium during 30 min of dye perfusion. Medium FITC-Delta (50.7 kDa) slowly penetrated this ESL within 30 min but did not permeate into the arterial wall. Small FITC-Delta (4.4 kDa) quickly passed the ESL and accumulated in the arterial wall. Prolonged luminal fluorochrome illumination with a bright mercury lamp destroyed the approximately 3-microm exclusion zone for FITC-Delta 148 within a few minutes. This study demonstrates the presence of a thick ESL that contributes to the permeability barrier to solutes. The layer is sensitive to phototoxic stress, and its damage could form an early event in atherosclerosis.     

Use of UV excitation in confocal laser scanning fluorescence microscopy

By making only minor modifications, we adapted a conventional confocal beam-scanning laser microscope for the recording of UV-excited fluorescence. The major, and most expensive, change is that we coupled an external UV argon ion laser, providing the wavelengths 334, 351 and 364 nm, to the microscope scanner. We also replaced some optical components to obtain improved transmission and reflection properties in the UV. Only easily obtainable and inexpensive off-the-shelf components were used. The most serious problem encountered was the chromatic aberration of the microscope objective when using both UV and visible wavelengths. This is of no consequence in conventional microscopy where good imaging properties are important only in the visible region. In confocal microscopy on the other hand, good imaging properties are necessary for both the exciting and fluorescent light. Rather than having new optics designed, we tried with simple means to reduce the effects of the chromatic aberration to a tolerable level. This was done by mechanical adjustments in the ray-path. In addition we also tested two mirror objectives, which are inherently free from chromatic aberrations. However, such objectives have rather limited numerical apertures and are not of the immersion type. Their value in biomedical applications is therefore limited.The objective most frequently used in our experiments was a 63/1.25 oil-immersion fluorite. Without any compensation this objective had a depth resolution in UV-excited confocal fluorescence that was an order of magnitude worse than when using visible-light excitation. The useful field of view was also very small due to lateral chromatic aberration. By simple means we managed to improve the depth resolution by a factor of 4.4, and at the same time increase the useful field of view substantially. Still, the depth resolution was worse than what is obtained using visible light excitation. We think this is due to the fact that after compensation the objective is working with an incorrect tube length.Using the modified instrument, we recorded specimens labelled with AMCA and Fluoro-Gold, obtaining 1.5 μm thick optical sections.     

Fluid phase and receptor-mediated endocytosis in Paramecium primaurelia by fluorescence confocal laser scanning microscopy

In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.     

Tracking viral movement in plants by means of chlorophyll fluorescence imaging

Many techniques have been applied to understand viral cell-to-cell movement in host plants, but little progress has been made in understanding viral vascular transport mechanisms. We propose the use of chlorophyll fluorescence imaging techniques, not only to diagnose the viral infection, but also to follow the movement of the virus through the vascular system and its subsequent spread into the leaves. In Nicotiana benthamiana plants, imaging of chlorophyll fluorescence parameters such as Ф_PSII and NPQ proved useful to follow infections with Pepper mild mottle virus. The results demonstrate a correlation between changes in the chlorophyll fluorescence parameters and the viral distribution analyzed by tissue printing.     

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

We present a multimodal technique for measuring the integral refractive index and the thickness of biological cells and their organelles by integrating interferometric phase microscopy (IPM) and rapid confocal fluorescence microscopy. First, the actual thickness maps of the cellular compartments are reconstructed using the confocal fluorescent sections, and then the optical path difference (OPD) map of the same cell is reconstructed using IPM. Based on the co‐registered data, the integral refractive index maps of the cell and its organelles are calculated. This technique enables rapidly measuring refractive index of live, dynamic cells, where IPM provides quantitative imaging capabilities and confocal fluorescence microscopy provides molecular specificity of the cell organelles. We acquire human colorectal adenocarcinoma cells and show that the integral refractive index values are similar for the whole cell, the cytoplasm and the nucleus on the population level, but significantly different on the single cell level.     

Antigen receptor-mediated calcium signals in B cells as revealed by confocal fluorescence microscopy

A confocal fluorescence microscope was used to study the antigen receptor-mediated calcium signals in B cells. Anti-IgD binding to B lymphoma cells (BAL17) increased the intracellular calcium concentration with short lag times. Confocal fluorescence images of the fluo-3-loaded BAL17 cells showed that the intracellular calcium ion concentrations increased non-homogeneously, suggesting that the calcium signals transferred not only to the cytoplasm but also to the nucleus.     

Localization of telomeres in plant interphase nuclei by in situ hybridization and 3D confocal microscopy

We investigated the three-dimensional (3D) arrangement of telomeres in structurally well preserved, interphase nuclei of Pisum stativum and Vicia faba root tips using in situ hybridization of a probe to telomeric sequences. The probe was labelled with either digoxygenin or biotin and hybridized sequences were detected by immunofluorescence. Three-dimensional data sets were collected by confocal optical microscopy or using a cooled CCD camera. Twelve stacks of optical sections of P. sativum nuclei and nine of V. faba nuclei were studied in detail. Projections through the stacks of optical sections revealed that, in both species, most of the telomeres were adjacent to the nuclear envelope except for a small number next to the nucleolar periphery. In V. faba nuclei, the telomeres were clearly clustered at one pole while in P. sativum there was only a slight tendency for clustering. In V. faba, clusters were found at opposite poles in pairs of sister nuclei rather than at adjacent poles as would be expected if the arrangement at telophase were maintained into interphase.by D. Bazett-Jones     

Unsupervised noise removal algorithms for three-dimensional confocal fluorescence microscopy

Algorithms are presented for effective suppression of the quantum noise artifact that is inherent to three-dimensional confocal fluorescence microscopy images of extended spatial objects such as neurons. The specific advances embodied in these algorithms are as follows: (i) they incorporate an automatic and pattern-constrained three-dimensional segmentation of the image field, and use it to limit any smoothing to the interiors of specified image regions and hence avoid the blurring that is inevitably associated with conventional noise removal algorithms; (ii) they are ‘unsupervised’ in the sense that they automatically estimate and adapt to the unknown spatially and temporally varying noise level in the microscopy data. Fast computation is achieved by parallel computation methods, rather than by algorithmic or modelling compromises.The quantum noise artifact is modelled using a mixture of spatially non-homogeneous Poisson point processes. The intensity of each component process is constrained to lie in specific intervals. A set of segmentation and edge-site variables are used to determine the intensity of the mixture process. Using this model, the noise-removal process is formulated as the joint optimal estimation of the segmentation labels, edge-sites and intensity of the mixture Poisson point process, subject to a combination of stochastic priors and syntactic pattern constraints. The computations proceed iteratively, starting from a set of approximate user-supplied, or default initial guesses of the underlying random process parameters. An Expectation Maximization algorithm is used to obtain a more precise characterization of these parameters, that are then input to a joint estimation algorithm.Stereoscopic renderings of processed three-dimensional images of murine hippocampal neurons are presented to demonstrate the effectiveness of the method. The processed images exhibit increased contrast and significant smoothing and reduction of the background intensity while avoiding any blurring of the foreground neuronal structures.     

Novel dye for detection of callus embryo by confocal laser scanning fluorescence microscopy

In the present study a new luminescent dye 3‐N‐(2‐pyrrolidinylacetamido)benzanthrone (AZR) was synthesized. Spectroscopic measurements of the novel benzanthrone 3‐aminoderivative were performed in seven organic solvents showing strong fluorescence. The capability of the prepared dye for visualization has been tested on flax, red clover and alfalfa to determinate the embryo in plant callus tissue cultures. Callus cells were stained with AZR and further analysed utilizing confocal laser scanning fluorescence microscopy. Performed experiments show high visualization effectiveness of newly synthesized fluorescent dye AZR that is efficient in fast and relatively inexpensive diagnostics of callus embryos that are problematic due to in vitro culture specificity.     

Living sieve cells of conifers as visualized by confocal,laser-scanning fluorescence microscopy

Summary Confocal laser scanning microscopy (CLSM) and fluorochromes were used to visualize the assimilate-conducting sieve cells of conifers in vivo. When still nucleate, the cytoplasm of these cells shows streaming and occupies the cell periphery including the pitlike, thin wall regions where sieve areas would develop. During differentiation the nuclear fluorescence and the central vacuoles disappear. At maturity and after ER-specific staining the sieve areas are the most conspicuous character of sieve cells. Those linking two sieve cells are covered on either side with prominent amounts of ER, while those leading to a Strasburger (=albuminous) cell show fluorescence on the sieve-cell side only. Within the sieve-area wall fluorescence appears also in the common median cavity which is part of the symplastic path between sieve cells. Electron microscopy (EM) depicts the ER as complexes of densely convoluted tubules of smooth ER, equally on either side of a sieve area, provided that the fixation of this sensitive tissue is appropriate. Purposeful wounding causes a swelling and vesiculation of the ER-tubules which is visible in both CLSM and EM. Electron micrographs of ER-complexes at sieve areas -in this paper demonstrated in vivo -have often been argued to be artefacts, since they should raise flow resistance considerably and are not consistent with the Münch hypothesis on phloem transport. The implications of this location for phloem transport are discussed.Abbreviations CLSM confocal laser scanning microscopy - DiOC 3,3-dioxacarbocyanine iodide - EM electron microscopy - ER endoplasmic reticulum - FDA fluorescein diacetate     

An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy

Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.     

Determination and localization of oil components in living benthic organisms by fluorescence microscopy

Microspectrofluorometric measurements are made to determine uptake and distribution of oil in marine organisms after exposure to crude oil. Equipment combining fluorescence microscopy with spectral analysis of the fluorescence emission is described. After contamination with oil, the intestine content ofLumbricillus lineatus, Nereis diversicolor andAnaitides mucosa shows a fluorescence emission at long wavelengths with a maximum at about 550 nm; this is in contrast to the fluorescence emission of these organisms without oil contamination. There is evidence that aromatic hydrocarbons are metabolized in the intestine of the worms studied.     

Copulatory organ musculature in Childia (Acoela) as revealed by phalloidin fluorescence and confocal microscopy

Copulatory organs of eight species of the monophyletic taxon Childia were investigated in detail, using phalloidin fluorescence method and confocal microscopy. Childia species were shown to have one, two or several tubular stylets, conical to cylindrical in shape, composed of few to numerous needles. The musculature varied greatly, from the absence of seminal vesicle to extensively developed seminal vesicles with several additional types of specialized muscles. Ten copulatory organ characters were coded and mapped on the total evidence tree. The data obtained permitted to follow the evolution of the Childia stylet and to demonstrate that the structure of the stylet apparatus is largely consistent with the phylogeny of the group (CI=0.75). Possible function of different muscle specializations was discussed.