The Fluorescent Dye 3, 3′ Dihexyloxacarbocyanine Iodide Selectively Stains the Midpiece and Apical Region of the Heads of Murid Rodent Spermatozoa

Fluorescence microscopy of caudal epididymal spermatozoa stained with 3, 3′ dihexyloxacarbocyanine iodide (DiOC₆(3)) showed intense fluorescence along the concave surface of the apical hook of spermatozoa of Rattus species and along the upper concave margin of the sperm head in Mus musculus In the spermatozoa of Hydromys chrysogaster, Melomys cervinipes, and Pseudomys australis, the two ventral processes also fluoresced brightly. In P. australis, fluorescence in the apical hook of sperm heads was largely localized to its upper and lower surfaces. The sperm of N. alexis did not show consistent positive fluorescence. The localization of fluorescence in these spermatozoa after staining with DiOC₆(3) was mainly restricted to regions where a large accumulation of perinuclear theca material lies beneath the plasmalemma. The reason for this remains to be determined, but DiOC₆(3) may be useful for quickly demonstrating areas of abundant perinuclear thecal material in sperm heads of eutherian mammals by light microscopy.     

Imaging of the Blue,Green, and Red Fluorescence Emission of Plants: An Overview

An overview is given on the fluorescence imaging of plants. Emphasis is laid upon multispectral fluorescence imaging in the maxima of the fluorescence emission bands of leaves, i.e., in the blue (440 nm), green (520 nm), red (690 nm), and far-red (740 nm) spectral regions. Details on the origin of these four fluorescence bands are presented including emitting substances and emitting sites within a leaf tissue. Blue-green fluorescence derives from ferulic acids covalently bound to cell walls, and the red and far-red fluorescence comes from chlorophyll (Chl) a in the chloroplasts of green mesophyll cells. The fluorescence intensities are influenced (1) by changes in the concentration of the emitting substances, (2) by the internal optics of leaves determining the penetration of excitation radiation and partial re-absorption of the emitted fluorescence, and (3) by the energy distribution between photosynthesis, heat production, and emission of Chl fluorescence. The set-up of the Karlsruhe multispectral fluorescence imaging system (FIS) is described from excitation with UV-pulses to the detection with an intensified CCD-camera. The possibilities of image processing (e.g., formation of fluorescence ratio images) are presented, and the ways of extraction of physiological and stress information from the ratio images are outlined. Examples for the interpretation of fluorescence images are given by demonstrating the information available for the detection of different developmental stages of plant material, of strain and stress of plants, and of herbicide treatment. This novel technique can be applied for near-distance screening or remote sensing.     

On the role of ß-carotene in the reaction center chlorophyll a antennae of photosystem I

Absorption and low temperature fluorescence emission spectra were measured on chloroplast thylakoids and on purified reaction center chlorophyll a-protein complexes of photosystem I, CP-a₁. A clear association between the presence of ß-carotene and the occurrence of far red absorbing and emitting chlorophyll a components of the reaction center antennae of photosystem I was demonstrated. For this study chloroplasts and CP-a₁ were obtained from normal and carotenoid deficient plant material of various sources. The experimental material included 1) lyophilized pea chloroplasts extracted with petroleum ether, 2) the carotenoid deficient mutant C-6E of Scenedesmus obliquus and 3) wheat chloroplasts derived from normal and SAN-9789 treated plants. Removal of carotenoids, most likely principally ß-carotene, caused a loss of long wavelength absorbing chlorophylls in chloroplasts and purified CP-a₁, and the loss or diminution of the long wavelength peak seen in the low temperature fluorescence emission spectrum. This association between ß-carotene and special chlorophyll a forms may explain both the photoprotective and antenna functions ascribed to ß-carotene. In the absence of carotenoids in wheat and in the Scenedesmus mutant, the chlorophyll a antenna of photosystem I was extremely photosensitive. A triplet-triplet resonance energy transfer from chlorophyll a to ß-carotene and a singlet-singlet energy transfer from excited ß-carotene to chlorophyll would explain the photoprotective and antenna functions, respectively. The role of this association in determining some of the fluorescence properties of photosystem I is also discussed.     

Characterization of two fluorescent tryptophans in recombinant human granulocyte-colony stimulating factor: Comparison of native sequence protein and tryptophan-deficient mutants

In order to probe the role of the individual tryptophans of granulocyte-colony stimulating factor (G-CSF) inpH and guanidine HCl-induced fluorescence changes, site-directed mutagenesis was used to generate mutants replacing Trp¹¹⁸, Trp⁵⁸, or both with phenylalanine. Neither Trp to Phe mutation affected the folding or activity of the recombinant G-CSF, and the material expressed in yeast behaved identically to that expressed inEscherichia coli. All of the G-CSF species responded topH and guanidine HCl in qualitatively the same manner. Trp⁵⁸ has a fluorescence maximum at 350 nm and is quenched to a greater extent by the addition of guanidine HCl, indicating that it is fully solvent-exposed. Trp¹¹⁸ has a fluorescence maximum at 344 nm, and is less solvent-accessible than Trp⁵⁸. The analog in which both tryptophans have been replaced with phenylalanine shows only tyrosine fluorescence, with a peak at 304 nm which decreases with increasingpH. The intensity of the tyrosine fluorescence in this analog is much greater than that of the native sequence protein or single tryptophan mutants, indicating that energy transfer is taking place from tyrosine to tryptophan in these molecules. Below neutralpH the tyrosine fluorescence is much greater in the [Phe⁵⁸]G-CSF than in the [Phe¹¹⁸]G-CSF, indicating that Trp⁵⁸ might be a more efficient recipient of energy transfer from the tyrosine(s).     

Multiphoton microscopy for the in-situ investigation of cellular processes and integrity in cryopreservation

In this study we demonstrate a new noninvasive imaging method to monitor freezing processes in biological samples and to investigate life in the frozen state. It combines a laser scanning microscope with a computer-controlled cryostage. Nearinfrared (NIR) femtosecond laser pulses evoke the fluorescence of endogenous fluorophores and fluorescent labels due to multiphoton absorption.The inherent optical nonlinearity of multiphoton absorption allows 3D fluorescence imaging for optical tomography of frozen biological material in-situ. As an example for functional imaging we use fluorescence lifetime imaging (FLIM) to create images with chemical and physical contrast.     

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.     

Mild temperature “stress” and callose synthesis

Seedlings of Zea mays L., Sorghum vulgare, Pisum sativum L., Phaseolus aureus, Glycine max L. and Lycopersicum esculentum were grown at 20°C and at 26°C. The seedlings were fixed in glutaraldehyde and sections were examined for aniline-blue-induced fluorescence, which is supposedly indicative of -1,3-glucans or callose. There was much more aniline-blue fluorescence in Zea, Glycine and Phaseolus seedlings grown at 20°C compared with 26°C whereas Pisum and Lycopersicum seedlings grown at 26°C showed more fluorescence than those grown at 20°C. In Zea, large deposits of fluorescent material were particularly noticeable in the walls of elongating cells around the shoot apex and in root-cap cells, and appeared to be closely associated with a few of the pitfields. The remaining pitfields showed the normal, low level of aniline-blue fluorescence.     

综述——基于纳米材料的生物检测与医学诊断技术：贵金属团簇探针用于细胞成像及体外检测

贵金属团簇(noble metal clusters)是近年来新兴的一类荧光标记材料．由于具有物理尺寸小、荧光可调及生物相容性等优异的性能使得其在生物成像及检测领域都有着广泛的应用前景．本文讨论了贵金属团簇的制备和荧光特性,重点论述了其作为标记材料在细胞成像方面及体外检测应用中的研究进展．     

Comparative morphology of nucleolar DNA in Drosophila. II. Drosophila fulvimaculoides and drosophila tumiditarsus

This paper reports the demonstration, using fluorescence microscopy, of nucleolar DNA in two species of Drosophila. In Drosophila fulvimaculoides, the nucleolar DNA presents a variable morphology, suggestive of puffing activity. This material, which sometimes shows a banded structure like that of the polytene chromosomes, is shown not to be coextensive with the Y chromosome. Nucleolar DNA is demonstrated in Drosophila tumiditarsus also, and previous reports of an association of the dot chromosome with the nucleolus in this species are confirmed. The special usefulness of these two species for various sorts of investigation in pointed out.     

Photochemical and Thermal Phases of Chlorophyll a Fluorescence

The measurement of variable chlorophyll (Chl) a fluorescence is widely used as a convenient and versatile tool in photosynthesis research. In many applications empirical correlations and simplified models of Chl a fluorescence are used with success. Nevertheless, variable Chl a fluorescence provides only indirect and complex image of processes occurring within photosynthetic membranes and such simplifications have only limited validity. In this review we elucidate some controversial and still unresolved questions about the origin and interpretation of the variable Chl a fluorescence induction and the proper use of variable Chl a fluorescence for studies of photochemical events in photosystem 2 (PS2). Although the major part of variable Chl a fluorescence reflects the photochemical closure of the PS2 reaction centers (RCs) and can be considered as a function of the redox state of the primary acceptor Q_A, up to 50 % of the change in the Chl a fluorescence yield can be of secondary, nonphotochemical origin. We review the possible sources of the inherent heterogeneity in the origin of variable Chl a fluorescence. We also comment on the practical implications this bears for the use of variable Chl a fluorescence. This revised version was published online in August 2006 with corrections to the Cover Date.     

Reflection versus fluorescence

Summary Bright microscopic images against a dark background can be originating not only from fluorescence, but also from selective reflection. Selective reflection or scattering of visible light in microscopic preparations can be used for the visualization of sometimes otherwise barely distinguishable material. The images obtained superficially resemble those from fluorescence microscopy. They do not, howeverm result from luminescence but from selectively reflected light with wavelengths in the region of the absorbance peak of the chromophore present in the stained biological material. The respective backgrounds of the underlying physical phenomena and the conditions under which selective reflection can occur are discussed.     

Investigation on the infection mechanism of the fungus <Emphasis Type="Italic">Clonostachys rosea</Emphasis> against nematodes using the green fluorescent protein

The fungus Clonostachys rosea (syn. Gliocladium roseum) is a potential biocontrol agent. It can suppress the sporulation of the plant pathogenic fungus Botrytis cinerea and kill pathogenic nematodes, but the process of nematode pathogenesis is poorly understood. To help understand the underlying mechanism, we constructed recombinant strains containing a plasmid with both the enhanced green fluorescent protein gene egfp and the hygromycin resistance gene hph. Expression of the green fluorescent protein (GFP) was monitored using fluorescence microscopy. Our observations reveal that the pathogenesis started from the adherence of conidia to nematode cuticle for germination, followed by the penetration of germ tubes into the nematode body and subsequent death and degradation of the nematodes. These are the first findings on the infection process of the fungal pathogen marked with GFP, and the developed method can become an important tool for studying the molecular mechanisms of nematode infection by C. rosea. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lin Zhang and Jinkui Yang contributed equally to this work.     

Preparation of carbon dot‐based ratiometric fluorescent probes for cellular imaging from Curcuma longa

This work derived biocompatible and stable probes based on fluorescent nanoparticles (FNPs) from a natural source, Curcuma longa. The multi‐color fluorescence emissions from carbonized Curcuma longa (C‐FNPs) obtained through defined dehydration conditions are soluble in water and have a small particle size (~17 nm). The surface passivation with polyethylene glycol (PEG) capped with amine groups in FNPs (P‐FNPs) generated a probe with a higher quantum yield and longer fluorescence lifetime than obtained with C‐FNPs. The X‐ray photoelectron spectroscopy and X‐ray diffraction spectra confirmed the associated chemical moieties of C‐FNPs and P‐FNPs. Furthermore, the prepared material showed non‐toxic effects with almost 100% cell viability, even at high concentrations. In conclusion, fluorescence sensors from natural sources may be useful for numerous biomedical research applications.     

Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression

The effects of six different plant viral suppressors of gene silencing were compared using an automated image collection and analysis system developed for continual monitoring of GFP expression. Suppressors were introduced into lima bean cotyledonary tissues either as 3′-GFP translational fusions or as co-introductions with the GFP gene on a separate plasmid. The resultant transient expression profiles for each suppressor depended on whether the suppressor was introduced as a fusion or co-introduced on separate plasmids. As co-introductions, the silencing suppressors HCPro (from Tobacco etch virus), p19 (from Tomato bushy stunt virus), γb (from Barley stripe mosaic virus) and p21 (from Beet yellows virus) led to an almost twofold increase in initial GFP expression levels, followed by a rapid decline. In contrast, fusions of HCPro, p19, and γb to the 3′-end of GFP resulted in slightly lower but more prolonged GFP expression. Compared with the co-introductions, all GFP::Suppressor translational fusions gave reduced GFP fluorescence levels, suggesting interference of the fusion partner with GFP fluorescence. Regardless of the configuration, introductions of the silencing suppressors AL2 (from Tomato golden mosaic virus) and 126-kDa protein (from Tobacco mosaic virus) resulted in very low GFP fluorescence. This is the first report that directly compares the effects of a large number of viral suppressors of silencing on transient transgene expression using both translational fusions and co-introductions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Recombinant peptide fusion construction for protein-templated catalytic palladium nanoparticles

Although peptide-enabled synthesis of nanostructures has garnered considerable interest for use in catalytic applications, it has so far been achieved mostly via Fmoc based solid phase peptide synthesis. Consequently, the potential of longer peptides in nanoparticle synthesis have not been explored largely due to the complexities and economic constraints of this chemical synthesis route. This study examines the potential of a 45-amino acid long peptide expressed as fusion to green fluorescence protein (GFPuv) in Escherichia coli for use in palladium nanoparticle synthesis. Fed-batch fermentation with E. coli harboring an arabinose-inducible plasmid produced a product containing three copies of Pd4 peptide fused to N-terminus of GFPuv ((Pd4)₃-GFPuv). Using the intrinsic fluorescence of GFPuv, expression and enrichment of the fusion product was easily monitored. Crude lysate, desalted lysate, and an ion-exchange enriched fraction containing (Pd4)₃-GFPuv were used to test the hypothesis that high purity of the biologic material used as the nanoparticle synthesis template may not be necessary. Nanoparticles were characterized using a variety of material science techniques and used to catalyze a model Suzuki–Miyaura coupling reaction. Results demonstrated that palladium nanoparticles can be synthesized using the soluble cell extract containing (Pd4)₃-GFPuv without extensive purification or cleavage steps, and as a catalyst the crude mixture is functional.     

Light,fluorescence and electron microscopic studies on “neurosecretion” in tritonia diomedia bergh (Mollusca,Nudibranchia)

Summary Membrane-limited electron-dense inclusions designated as elementary neurosecretory granules have a characteristic distribution in cerebropleural ganglia of the nudibranch snail Tritonia diomedia. They occur in the neuropile and also in individual nerve fibres, connectives and commissures. These granules have been found neither in perikarya of nerve cells nor in proximal segments of their processes.Specific fluorescence obtained in Tritonia preparations with Sterba's pseudoisocyanin method for neurosecretory products has the same pattern of location.The distribution of stainable material in preparations prepared with ordinary neurosecretory procedures (chrome haematoxylin-phloxin after Gomori-Bargmann and paraldehydefuchsin after Gomori-Gabe) is similar to that described by different authors in other gastropods, but strongly differs from the locationof elementary neurosecretory granules and of pseudoisocyanin-positive material. The adequacy of different histological methods for studying neurosecretion in gastropods is discussed.     

ASSESSING PHYSIOLOGICAL STRESS IN THALASSIOSIRA FLUVIATILIS (BACILLARIOPHYTA) AND DUNALIELLA TERTIOLECTA (CHLOROPHYTA) WITH DCMU-ENHANCED FLUORESCENCE1

Exponentially growing cultures of Thalassiosira fluviatilis Hustedt and Dunaliella tertiolecta Butcher were exposed to 4 min temperature shocks of 5° to 20°C above ambient (20°C). Photosynthetic carbon fixation, changes in in vivo fluorescence and fluorescence on the addition of the herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) were measured over the subsequent 24 h. The fluorescence ratio (R, DCMU-enhanced fluorescence/in vivo fluorescence) paralleled changes in photosynthesis over this period; both were significantly reduced (P < 0.05) by temperature shocks of +15° and +20° C, but +5° and +10° C treatments had no inhibitory effect on either relative to the control. The instantaneous response obtained with the fluorescence ratio indicates that the technique might be applicable to routine bioassay procedures and thus replace the time consuming methods now used for the estimation of ¹⁴C-incorporation and growth rates.     

A dissolved oxygen sensor based on composite fluorinated xerogel doped with platinum porphyrin dye

A new functional fluorinated material taking n‐propyltrimethoxysilicane (n‐propyl‐TriMOS) and 3,3,3‐trifluoropropyltrimethoxysilicane (TFP‐TriMOS) as precursors was applied to construct a novel dissolved oxygen sensing film. The sensing film was fabricated by dip‐coating the functional fluorinated material‐doped [meso‐tetrakis(pentafluorophenyl) porphyinato] platinum(II) (PtF₂₀TPP) onto a glass slide. The oxygen sensing film exhibited a good linear relationship, fast response time, long stability and high sensitivity to dissolved oxygen. In the developed optical oxygen sensor, an LED and a photodiode were composed to construct a back‐detection optical system not needing an optical fiber based on fluorescence intensity detection. The smart optical oxygen sensor based on the PtF₂₀TPP fluorescence quenching possesses the advantages of portability and low cost and can be applied to the dissolved oxygen in situ monitoring in seawater. Copyright © 2009 John Wiley & Sons, Ltd.     

Monoclonal antibodies to a fern spermatozoid detect novel components of the mitotic and cytokinetic apparatus in higher plant cells

Summary The aim of this study was to search for uncharacterized components of the plant cytoskeleton using monoclonal antibodies raised against spermatozoids of the fernPteridium (Marc et al. 1988). The cellular distribution of crossreacting immunoreactive material during the division cycle in wheat root tip cells was determined by immunofluorescence microscopy and compared to the fluorescence pattern obtained with antitubulin. Five antibodies are of special interest. Pas1D3 and Pas5F4 detect a diffuse cytoplasmic material, which, during mitosis, follows the distribution of microtubules (MTs) at the nuclear surface and in the preprophase band (PPB), spindle and phragmoplast. The immunoreactive material codistributes specifically with MT arrays of the mitotic apparatus and does not associate with interphase cortical MTs. Pas5D8 is relevant to the PPB and spatial control of cytokinesis. It binds in a thin layer at the cytoplasmic surface throughout the cell cycle, except when its coverage is transiently interrupted by an exclusion zone at the PPB site and later at the same site when the phragmoplast fuses with the parental cell wall.Pas2G6 reacts with a component of basal bodies and the flagellar band in thePteridium spermatozoid and recognizes irregularly shaped cytoplasmic vesicles in wheat cells. During interphase these particles form a cortical network.Pas6D7 binds to dictyosomes and dictyosome vesicles. At anaphase the vesicles accumulate at the equator and subsequently condense into the cell plate.Abbreviations MT microtubule - PPB preprophase band     

Evidence for a coelomic maturation factor controlling oocyte maturation in the polychaete Arenicola marina (L.)

Summary

Previous studies on Arenicola marina suggested that oocyte maturation was induced by a single maturation hormone from the prostomium. This maturation hormone was thought to act directly on the oocyte (Meijer and Durchon, 1977), A recently described species, Arenicola defodiens (Cadman and Nelson-Smith, 1993), morphologically very similar to A. marina, has been found at the sampling sites described by Meijer and Durchon (1977). Results presented here from studies on British populations of Arenicola marina show that in this species, oocyte maturation is controlled by two hormonal steps. The first step involves the prostomial maturation hormone. The second step depends on a maturation inducing substance in the coelomic fluid. We will refer to this as the coelomic maturation factor (CMF). A reliable in vitro assay for oocyte maturation in the lugworm Arenicola marina has been adopted. It utilizes fluorescence staining of the chromosome material with DNA labelling dyes (Hoechst 33342 and 33258). Maturation of oocytes in A. marina involves germinal vesicle breakdown (GVBD). This is accompanied by the movement of chromosomes from late prophase to metaphase of meiosis I and chromosome condensation. The chromosomes are stained brightly by the dyes and their relative positions can be easily identified so that mature and immature eggs can be distinguished by the differences in chromosome position and form. The development of the in vitro fluorescence assay has enabled us to demonstrate that there are two endocrine steps involved in the induction of oocyte maturation. We have begun the characterization of CMF, and data show this to be a thermolabile molecule with a molecular mass greater than 10 kd.