An in vitro preparation to access cellular and neuronal components in the mouse inner ear

An in vitro mouse temporal bone preparation has been developed in order to allow the investigation of structures and functions in a living hearing organ. Fluorescent vital probes (a potentiometric styryl dye, RH 795, and a vital dye staining cellular cytoplasm, calcein) were perfused through the scala tympani to stain cellular components of the cochlea. Observations of the cochlear apical turn were performed using a confocal microscope. In spite of the anatomical constraints due to the small size of the mouse cochlea, detailed images were obtained. The sensory cells as well as their innervating nerve fibres were clearly seen. Nerve fibres crossing the tunnel of Corti and projecting to the outer hair cells could also be visualised.     

An NIR emitting styryl dye with large Stokes shift to enable co-staining study on zebrafish neuromast hair cells

Hearing loss is a significant public health problem, and the “loss of sensory hair cells” is one of two leading causes in humans. Advanced imaging reagents are desirable for understanding the role of the surrounding support cells in the loss or regeneration of the hair cells. A styryl dye was found to exhibit NIR emission (λ_em ≈ 684 nm) with a very large Stokes shift (Δν ≈ 9190 cm⁻¹), due to the incorporation of excited state intramolecular proton transfer (ESIPT) mechanism. When used to stain live zebrafish embryos, the probe was found to exhibit good selectivity in targeting neuromasts, which are sensory organs on the surface of the fish’s body. The finding was verified by direct comparison with the known neuromast-labeling reagent, 4-Di-2-ASP. In contrast to the existing styryl dyes that label neuromast hair cells, the new probe labeled both neuromast hair cells and the surrounding support cells, while giving discernable signals. The study thus illustrated a useful tool to aid the developmental study of two closely related cell types on the mechanosensory sensory organ of zebrafish, which is a powerful animal model for hearing loss research.     

Visualization of plastids and lipophilic components in living colonies of a wild strain of the hydrocarbon-forming, green alga Botryococcus by laser scanning confocal microscopy

Colonies of a wild strain from Lake Burley-Griffin, Australia, of the hydrocarbon-producing green alga Botryococcus were examined by confocal laser scanning microscopy. The microscope was fitted with a dual wavelength krypton-argon laser, which permitted simultaneous detection of chlorophyll autofluorescence and lipophilic dye fluorescence. This quick and simple technique revealed the precise structural conformation of the autofluorescing plastids in living cells and their 3-dimensional spatial arrangement within the dense globular colonies. Cells stained with the lipophilic carbocyanine dye, DIOC6(3) contain an apical array of intensely staining granules as well as a more diffuse internal cisternal system thought to be endoplasmic reticulum. The cationic lipophilic dye rhodamine123 revealed a finer reticulate system in the outermost cytoplasm partially overlaying the plastid. Both dyes revealed the lipophilic nature of the extracellular matrix and enabled the secretion of lipid globules exuded from the colonies to be visualized. It is suggested that confocal laser scanning microcopy would make an ideal tool to screen isolates for their potential to form and secrete hydrocarbon, processes which are still far from clearly understood in this potentially commercially important alga. This revised version was published online in June 2006 with corrections to the Cover Date.     

Wavelength- and Time-Dependence of Potentiometric Non-linear Optical Signals from Styryl Dyes

Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, complementing existing one- and two-photon fluorescence (2PF) methods. A non-linear phenomenon employing light from mode-locked Ti:sapphire or fiber-based lasers, SHG results in intrinsic optical sectioning without the need for a confocal aperture. Furthermore, as a second-order process SHG is confined to loci lacking a center of symmetry, a constraint that is readily satisfied by lipid membranes with only one leaflet stained by a dye. Of particular interest is “resonance-enhanced” SHG from styryl dyes in cellular membranes and the possibility that SHG is sensitive to transmembrane potential. We have previously confirmed this, using simultaneous voltage-clamping and non-linear imaging of cells to find that SHG is up to four times more sensitive to potential than fluorescence. In this work, we have extended these results in two directions. First, with a range of wavelengths available from a mode-locked Ti:sapphire laser and a fiber-based laser, we have more fully investigated SHG and 2PF voltage-sensitivity from ANEP and ASTAP chromophores, obtaining SHG sensitivity spectra that are consistent with resonance enhancements. Second, we have modified our system to coordinate the application of voltage-clamp steps with non-linear image acquisition to more precisely characterize the time dependence of SHG and 2PF voltage sensitivity, finding that, at least for some dyes, SHG responds more slowly than fluorescence to changes in transmembrane potential.     

Concentration-dependent staining of lactotroph vesicles by FM 4-64

Hormones are released from neuroendocrine cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. An elegant way to study this process at the single-vesicle level is to use styryl dyes, which stain not only the membrane, but also the matrix of individual vesicles in some neuroendocrine cells. However, the mechanism by which the vesicle matrix is stained is not completely clear. One possibility is that molecules of the styryl dye in the bath solution dissolve first in the plasma membrane and are then transported into the vesicle by lateral diffusion in the plane of the membrane, and finally the vesicle matrix is stained from the vesicle membrane. On the other hand, these molecules may enter the vesicle lumen and reach the vesicle matrix by permeation through an open aqueous fusion pore. To address these questions, we exposed pituitary lactotrophs to different concentrations of FM 4-64 to monitor the fluorescence increase of single vesicles by confocal microscopy after the stimulation of cells by high K(+). The results show that the membrane and the vesicle matrix exhibit different concentration-dependent properties: the plasma membrane staining by FM 4-64 has a higher affinity in comparison to the vesicle matrix. Moreover, the kinetics of vesicle loading by FM 4-64 exhibited a concentration-dependent process, which indicates that FM 4-64 molecules stain the vesicle matrix by aqueous permeation through an open fusion pore.     

The apical sensory organ of a gastropod veliger is a receptor for settlement cues

On the basis of anatomy and larval behavior, the apical sensory organ (ASO) of gastropod veliger larvae has been implicated as the site of perception of cues for settlement and metamorphosis. Until now, there have been no experimental data to support this hypothesis. In this study, cells in the ASO of veliger larvae of the tropical nudibranch Phestilla sibogae were stained with the styryl vital dye DASPEI and then irradiated with a narrow excitatory light beam on a fluorescence microscope. When its ASO cells were bleached by irradiation for 20 min or longer, an otherwise healthy larva was no longer able to respond to the usual metamorphic cue, a soluble metabolite from a coral prey of the adult nudibranch. The irradiated cells absorbed the dye acridine orange, suggesting that they were dying. When larvae not stained with DASPEI were similarly irradiated, or when stained larvae were irradiated with the light beam focused on other parts of the body, there was no loss of ability to metamorphose. Together these data provide strong support for the hypothesis. Potassium and cesium ions, known to induce metamorphosis in larvae of many marine-invertebrate phyla, continue to induce metamorphosis in larvae that have lost the ability to respond to the coral inducer due to staining and irradiation. These results demonstrate that (1) the ASO-ablated larvae have not lost the ability to metamorphose and (2) the ions do not act only on the metamorphic-signal receptor cells, but at other sites downstream in the metamorphic signal transduction pathway.     

Multi-photon microscopy of cell types in the viable taste disk of the frog

The morphology of viable taste disks of the frog was explored with multi-photon microscopy. In order to identify single sensory or supporting cells within the tissue, we searched for fluorescent dyes that stained subsets of the cell population or possibly cell types. Some cell types indeed stained preferentially with certain fluorescent dyes. A subset of glia-like cells (type Ic) stained with BCECF, a H⁺-sensitive dye, and indo-1, a Ca²⁺-sensitive dye, both presented in the membrane-permeant ester form. BCECF-ester also stained the dendrites of type III receptor cells, but indo-1 ester did not. Receptor cells of type II stained with MQAE, a positively charged Cl^–-sensitive dye. A subset of type II cells accumulated amiloride, a positively charged fluorescent diuretic. Certain supporting cells, i.e., wing cells (type Ib) and glia-like cells (type Ic), were labeled by negatively charged dyes, e.g., calcium green-1 dextran. Mucus cells (type Ia) were stained with only two of the 19 dyes examined, and Merkel-like basal cells (type IV) were stained only with a membrane-labeling voltage-sensitive dye, presumably by endocytosis. No dye was found which would stain all types of cells or all receptor cells. This finding reveals a potential problem for future functional imaging aiming at population responses, as the responses of unstained cells then would remain unobserved. Specificity of dyes with respect to cell types was sufficient to identify supporting cells and receptor cells. Cell shape could then be reconstructed, using optical slicing and rendering techniques. Thus populations of dye-loaded elongated cells, especially types Ic, II and III, could for the first time be visualized in three dimensions.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 530, project B2)     

Endocytosis against high turgor: intact guard cells of Vicia faba constitutively endocytose fluorescently labelled plasma membrane and GFP-tagged K-channel KAT1

The relevance of endocytosis in plants against high turgor pressure has frequently been questioned on the basis of energetic considerations. Here, we examine the dynamics of the plasma membrane (PM) in turgid guard cells of Vicia faba by monitoring with confocal microscopy the fate of fluorescent styryl dyes (FM1-43, FM2-10 and FM4-64). As a second marker, we also observe the retrieval of a fluorescent chimaera of the K(+)-inward rectifying channel from Arabidopsis thaliana and the green fluorescent protein (KAT1::GFP). Analysis of cytoplasmic structures, which became labelled by the different styryl dyes, revealed that only FM4-64, the most hydrophobic dye, was a reliable marker of endocytosis, whereas the two other styryl dyes resulted also in an unspecific labelling of different cytoplasmic structures including mitochondria. Over some minutes of incubation in continuous presence of these dyes, endocytic vesicles in the cortical cytoplasm beneath the PM were fluorescently labelled. The identification is based on the observation that the size distribution of these structures is very similar to that of endocytic vesicles obtained from patch-clamp capacitance recordings. Also, these structures are frequently co-labelled with KAT1::GFP. Taken together, the data show that turgid guard cells undergo vigorous constitutive endocytosis and retrieve membrane including the K(+)-channel KAT1 from the PM via endocytic vesicles.     

Synthesis and properties of three fluorescent derivatives of gastrin

Summary As a first step in the study of hormone interaction with gastrin receptor-expressing cells, three fluorescent derivatives of heptagastrin were synthesized, characterized and tested for specificity and affinity towards gastrin/CCK_B receptor by means of confocal laser scanning microscopy (CLSM). Cyanine dye Cy3.29 and borfluoropyrromethene (BODIPY) derivatives of the hormone were found to be absorbed into the cells and concentrated in perinuclear organelles by a non-receptor mediated process. The BODIPY derivative turned out to be chemically unstable and was bleached by the laser beam very rapidly. Rhodamine Green-heptagastrin retained a high affinity toward the gastrin receptor (K_d=45 nm in displacement of ¹²⁵I-labeled cholecystokinin-8) and showed specific binding to NIH/3T3 cells stably transfected with human gastrin/CCK_B receptor cDNA, but not to nontransfected 3T3 cells. The fluorescent signal of all three dyes was sufficiently intense for localization of the compounds in cells by means of CLSM. Rhodamine Green derivative was found to be a useful tool for the study of endocytosis of the hormone. It can also be utilized for quantitative estimation of binding and determination of K_d instead of the traditionally used radiolabeled derivatives of gastrin.Abbreviations BODIPY borfluoropyrromethene - CCK cholecystokinin - CCK-8 CCK octapeptide - RG-7G Rhodamine Green heptagastrin - BSA bovine serum albumin - DMEM Dulbecco's modified Eagle's medium - TFA trifluoroacetic acid - DMSO dimethylsulfoxide - EDTA ethylenediamino tetraacetic acid - CLSM confocal laser scanning microscopy     

Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging

Phytoplasmas are cell wall-less bacteria inhabiting the phloem and utilizing it for their spread. Infected plants often show changes in growth pattern and a reduced crop yield. A quantitative real-time polymerase chain reaction (Q-PCR) assay and a bioimaging method were developed to quantify and localize phytoplasmas in situ. According to the Q-PCR assay, phytoplasmas accumulated disproportionately in source leaves of Euphorbia pulcherrima and, to a lesser extent, in petioles of source leaves and in stems. However, phytoplasma accumulation was small or nondetectable in sink organs (roots and sink leaves). For bioimaging, infected plant tissue was stained with vital fluorescence dyes and examined using confocal laser scanning microscopy. With a DNA-sensitive dye, the pathogens were detected exclusively in the phloem, where they formed dense masses in sieve tubes of Catharanthus roseus. Sieve tubes were identified by counterstaining with aniline blue for callose and multiphoton excitation. With a potentiometric dye, not all DNA-positive material was stained, suggesting that the dye stained metabolically active phytoplasmas only. Some highly infected sieve tubes contained phytoplasmas that were either inactive or dead upon staining.     

Direct monitoring of vesicular release and uptake in brain slices by multiphoton excitation of the styryl FM 1-43

Fluorescence imaging using FM 1-43 and related styryl dyes has provided invaluable insights into presynaptic function of synapses in culture preparations, but has been limited in use for studying central synapses in vivo or in brain slices, because of excessive fluorescence background due to nonspecific membrane binding of dye. We demonstrate here that focal excitation of FM dyes using two-photon laser-scanning microscopy (TPLSM) provides high resolution of FM 1-43-labeled nerve terminals in brain slices by suppressing out-of-focus background and that a readily releasable pool of vesicles can be selectively and stably labeled by hypertonic shock despite slice diffusion barriers. We find direct TPLSM of FM 1-43-labeled nerve terminals to be superior to treatment of slices with either the fluorescent quencher sulforhodamine 101 or dye scavenger ADVASEP-7 in resolving nerve terminal against background fluorescence, enabling continuous monitoring of vesicular uptake, and release of styryl dyes from individual nerve terminals in brain slices.     

Head sensory organs of Dactylopodola baltica (Macrodasyida, Gastrotricha): a combination of transmission electron microscopical and immunocytochemical techniques

The anterior and posterior head sensory organs of Dactylopodola baltica (Macrodasyida, Gastrotricha) were investigated by transmission electron microscopy (TEM). In addition, whole individuals were labeled with phalloidin to mark F-actin and with anti-alpha-tubulin antibodies to mark microtubuli and studied with confocal laser scanning microscopy. Immunocytochemistry reveals that the large number of ciliary processes in the anterior head sensory organ contain F-actin; no signal could be detected for alpha-tubulin. Labeling with anti-alpha-tubulin antibodies revealed that the anterior and posterior head sensory organs are innervated by a common stem of nerves from the lateral nerve cords just anterior of the dorsal brain commissure. TEM studies showed that the anterior head sensory organ is composed of one sheath cell and one sensory cell with a single branching cilium that possesses a basal inflated part and regularly arranged ciliary processes. Each ciliary process contains one central microtubule. The posterior head sensory organ consists of at least one pigmented sheath cell and several probably monociliary sensory cells. Each cilium branches into irregularly arranged ciliary processes. These characters are assumed to belong to the ground pattern of the Gastrotricha.     

Optical Saturation as a Versatile Tool to Enhance Resolution in Confocal Microscopy

One of the most actively developing areas in fluorescence microscopy is the achievement of spatial resolution below Abbe's diffraction limit, which restricts the resolution to several hundreds of nanometers. Most of the approaches in use at this time require a complex optical setup, a difficult mathematical treatment, or usage of dyes with special photophysical properties. In this work, we present a new, to our knowledge, approach in confocal microscopy that enhances the resolution moderately but is both technically and computationally simple. As it is based on the saturation of the transition from the ground state to the first excited state, it is universally applicable with respect to the dye used. The idea of the method presented is based on a principle similar to that underlying saturation excitation microscopy, but instead of applying harmonically modulated excitation light, the fluorophores are excited by picosecond laser pulses at different intensities, resulting in different levels of saturation. We show that the method can be easily combined with the concept of triplet relaxation, which by tuning the dark periods between pulses helps to suppress the formation of a photolabile triplet state and effectively reduces photobleaching. We demonstrate our approach imaging GFP-labeled protein patches within the plasma membrane of yeast cells.     

Flow-cytometric determination of fluorescence ratios between differently stained particles is dependent on excitation intensity

By use of a flow cytometer, the fluorescence of cells stained with hematoporphyrin derivative and the fluorescence of plastic beads stained with different dyes were analysed as a function of the intensity of the exciting laser light. The ratios of the fluorescence values of stained and unstained cells as well as of stained cells and beads were sensitively dependent on excitation intensities. As a consequence of this finding, the normalization of cellular fluorescence by use of reference particles needs to be made on a well-defined and reproduced intensity of the exciting laser light.     

Computer-Assisted Laser Scanning and Video Microscopy for Analysis of Cryptosporidium parvum Oocysts in Soil, Sediment, and Feces

A computer-assisted laser scanning microscope equipped for confocal laser scanning and color video microscopy was used to examine Cryptosporidium parvum oocysts in two agricultural soils, a barnyard sediment, and calf fecal samples. An agar smear technique was developed for enumerating oocysts in soil and barnyard sediment samples. Enhanced counting efficiency and sensitivity (detection limit, 5.2 x 10(sup2) oocysts(middot)g [dry weight](sup-1)) were achieved by using a semiautomatic counting procedure and confocal laser scanning microscopy to enumerate immunostained oocysts and fragments of oocysts in the barnyard sediment. An agarose-acridine orange mounting procedure was developed for high-resolution confocal optical sectioning of oocysts in soil. Stereo images of serial optical sections revealed the three-dimensional spatial relationships between immunostained oocysts and the acridine orange-stained soil matrix material. In these hydrated, pyrophosphate-dispersed soil preparations, oocysts were not found to be attached to soil particles. A fluorogenic dye permeability assay for oocyst viability (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992) was modified by adding an immunostaining step after application of the fluorogenic dyes propidium iodide and 4(prm1),6-diamidino-2-phenylindole. Comparison of conventional color epifluorescence and differential interference contrast images on one video monitor with comparable black-and-white laser-scanned confocal images on a second monitor allowed for efficient location and interpretation of fluorescently stained oocysts in the soil matrix. This multi-imaging procedure facilitated the interpretation of the viability assay results by overcoming the uncertainties caused by matrix interference and background fluorescence.     

Flow cytometry of DNA content using oxazine 750 or related laser dyes with 633 nm excitation

The laser dyes oxazine 750 (OX750), LD700, and rhodamine 800 (R800) can be used in an instrument employing a low-power helium-neon laser source for flow cytometry of DNA content in ethanol-fixed or detergent-permeabilized cells. Cells in near-isotonic medium are stained with 10-30 microM dye, and fluorescence excited at 633 nm is measured at wavelengths above 665 nm. The dyes do not appear to stain RNA, and the intensity of DNA staining is not changed when 2 microM Hoechst 33342 is added to cells simultaneously with a red-excited dye. The effects on fluorescence of addition of DNA to LD700 or R800 in aqueous solution are strongly influenced by the base composition of the DNA; binding mechanisms remain to be determined.     

DNA labeling in living cells.

BACKGROUND: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. METHODS: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin. RESULTS: From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several advantages, namely low photobleaching, labeling of the chromatin compartments comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission compatible with available genetically encoded fluorescent proteins such as C/G/YFP or mRFP. CONCLUSIONS: The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can easily be combined with other fluorophores with blue to orange emission.     

Survival of bundleless hair cells and subsequent bundle replacement in the bullfrog's saccule.

Our senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Millions of people suffer from hearing and balance deficits caused by damage to hair cells as a result of exposure to noise, aminoglycoside antibiotics, and antitumor drugs. In some species such damage can be reversed through the production of new cells. This proliferative response is limited in mammals but it has been hypothesized that damaged hair cells might survive and undergo intracellular repair. We examined the fate of bullfrog saccular hair cells after exposure to a low dose of the aminoglycoside antibiotic gentamicin to determine whether hair cells could survive such treatment and subsequently be repaired. In organ cultures of the bullfrog saccule a combination of time-lapse video microscopy, two-photon microscopy, electron microscopy, and immunocytochemistry showed that hair cells can lose their hair bundle and survive as bundleless cells for at least 1 week. Time-lapse and electron microscopy revealed stages in the separation of the bundle from the cell body. Scanning electron microscopy (SEM) of cultures fixed 2, 4, and 7 days after antibiotic treatment showed that numerous new hair bundles were produced between 4 and 7 days of culture. Further examination revealed hair cells with small repaired hair bundles alongside damaged remnants of larger surviving bundles. The results indicate that sensory hair cells can undergo intracellular self-repair in the absence of mitosis, offering new possibilities for functional hair cell recovery and an explanation for non-proliferative recovery.     

Dye Exchange in Bacterial Cells, and the Theory of Staining

Bacterial cells were stained in sequence, and at various pH's, by 22 different basic dyes. It was found that any dye could replace another already present in the bacterial cell. This replacement was shown to act according to mass action laws for reversible reactions, and hence was influenced by concentration of reagent and time of application. Since basic dyes are also known to react at carboxyl group sites, the phenomena of staining of bacterial cells by ordinary basic dyes must be a chemical adsorption exchange reaction.     

Endocytosis and Exocytosis Events Regulate Vesicle Traffic in Endothelial Cells

We used water-soluble styryl pyridinium dyes that fluoresce at the membrane-water interface to study vesicle traffic in endothelial cells. Cultured endothelial cells derived from bovine and human pulmonary microvessels were incubated in styryl probes, washed to remove dye from the plasmalemmal outer face, and observed by digital fluorescence microscopy. Vesicles that derived from plasmalemma by endocytosis were filled with the styryl dye. These vesicles were distributed throughout the cytosol as numerous particles of heterogeneous diameter and brightness. Vesicle formation was activated 2-fold following addition of extracellular albumin whereas a control protein, immunoglobulin G, had no effect. Dye uptake was abrogated by labeling at low temperatures and inhibitors of phosphoinositide-3-kinase (PI 3-kinase). Tyrosine kinase inhibitors (genistein and herbimycin A) prevented the albumin-induced vesicle formation. Cytochalasin B prevented vesicle redistribution indicating involvement of actin filaments in translocation of endosomes away from sites of vesicle formation. Styryl dye was lost from cells by exocytosis as evident by the disappearance of discrete fluorescent particles. N-ethylmaleimide and botulinum toxin types A and B caused cells to accumulate increased number of vesicles suggesting that exocytosis was regulated by NSF-dependent SNARE mechanism. The results suggest that phosphoinositide metabolism regulates endocytosis in endothelial cells and that extracellular albumin activates endocytosis by a mechanism involving tyrosine phosphorylation, whereas exocytosis is a distinct process regulated by the SNARE machinery. The results support the hypothesis that albumin regulates its internalization and release in vascular endothelial cells via activation of specific endocytic and exocytic pathways.