Cycloheptaamylose-dansyl chloride complex as a fluorescent label of surface membranes in living ciliates

Labelling of surface membrane of living ciliates: Paramecium aurelia and Tetrahymena pyriformis with fluorescent compound--cycloheptaamylose-dansyl chloride complex (CDC) has been achieved. Fluorescence micrographs of the dried samples showed specific localization of CDC on the cell membrane without any intracellular penetration. On the contrary the ciliates which have been dead during labelling revealed a non-specific fluorescence of their whole bodies. Microspectrofluorimetric analysis of labelled Paramecium cells was performed with Leitz microspectrograph. Spectrum of fluorescence emission measured over the cell membrane level had maximum at 450 nm. Strikingly, the emission maximum of the cells dead at the moment of labelling was shifted 10 nm to a longer wavelength. The rate of photofading measured in this case was almost 3-fold higher than for the ciliates labelled as living ones. Fluorescence excitation spectra did not show any difference in the peak position. Thus CDC staining appears to be an useful method of supravital labelling of cell surface enabling also to distinguish--on the basis of spectral characteristics--the ciliates being alive from those dead at the moment of fluorochrome binding.     

Enhanced membrane fluorescence of CDC-labelled paramecium subsequent to removal of surface components

Cytofluorimetric analysis of cycloheptaamylose-dansyl chloride (CDC) labelled Paramecium indicates that after mild trypsin removal of surface components the localization of CDC on the outer surface of living cells was not modified by the treatment. After such treatment the intensity of fluorescence emission was found about 3-fold higher in treated single cell than in the untreated one. These findings indicate that CDC labelling can be used to follow alteration occurred on the membrane of the living cell prior to labelling.     

Localization of polyphosphates in Saccharomyces fragilis, as revealed by 4′,6-diamidino-2-phenylindole fluorescence

The fluorescent dye 4′,6-diamidino-2-phenylindole has its emission maximum at 456 nm. Fluorescence intensity at this wavelength is significantly increased by various negatively-charged polyelectrolytes. Among several polyelectrolytes tested, polyphosphates appeared to be unique in the sense that they shifted the emission maximum from 456 to 526 nm. Addition of Saccharomyces fragilis cells to a diamidinophenylindole solution caused an immediate shift of the emission maximum to 526 nm, followed by a gradual increase of fluorescence at 456 nm. The 526 nm, but not the 456 nm fluorescence was instantly quenched by non-penetrating cations, like UO²⁺₂. These results suggest a momentary interaction of diamidinophenylindole with polyphosphate, localized outside the plasma membrane, followed by a slow penetration of the dye into the cells, yielding increased fluorescence at 456 nm by interaction of the dye with e.g., nucleic acids. This was confirmed by fluorescence microscopy. After addition of diamidinophenylindole the yeast cells exhibited an immediate green-yellow fluorescence of the membrane, that was suppressed by UO²⁺₂. After longer incubation times the cytoplasm and nucleus developed a blue fluorescence.     

Fluoreszenzmikroskopischer Nachweis des Schreckstoffes in den Schreckstoffzellen der Elritze,Phoxinus phoxinus (L.) (Cyprinidae,Ostariophysi, Pisces)

Zusammenfassung Die Schreckstoffzellen der Elritze,Phoxinus phoxinus (L.), zeigen nach Gefriertrocknung der Haut eine Eigenfluoreszenz. Diese wird bei 360–380 nm maximal angeregt; das Emissionsmaximum liegt bei ca. 515 nm. Die Fluoreszenz einer Schreckstoffzelle wird mit der Fluoreszenz des isolierten Schreckstoffes mikrospektralphotofluorimetrisch verglichen: die Emissions-maxima liegen nur 10 nm voneinander entfernt. Die Kurven relativer Fluoreszenzintensität verlaufen weitgehend gleichartig. Dies zeigt, daß der Schreckstoff tatsächlich den Schreckstoffzellen entstammt.

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Fluorescence microscopical demonstration of the alarm substance in the alarm substance cells of the European minnow,Phoxinus phoxinus (L.) (cyprinidae, ostariophysi, pisces)

Summary The alarm substance cells of the European minnow,Phoxinus phoxinus (L.), are autofluorescent after freeze-drying of the skin (Fig. 1). Their autofluorescence is maximally excited at 360–380 nm; the maximum of emission lies at about 515 nm. The fluorescence of an alarm substance cell is compared with the autofluorescence of the isolated alarm substance by means of a microspectro-photofluorometer. The maxima of emission are only about 10 nm apart. The curves of relative fluorescence intensity are almost identical (Fig. 3). These results show that the alarm substance actually comes from the alarm substance cells.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells

Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of ¹H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae.     

Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry

Summary Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG—gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase—isomaltase. The labelling efficiency of RPGG was compared to that of protein A—gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling intensity was dependent on the size of the marker as well as on the kind of protein used for complex formation. The distributions for RPGG and PAG were respectively: for the 12nm particles, 10.3 and 6.2 particles/µm of length of microvillar membrane, 3.5 and 1.0 particles/µm² of Golgi profile and 5.9 and 2.0 particles/µm² of multivesicular body profile; and for the 6nm particles, 49.6 and 15.7 particles/µm of length of microvillar membrane, 24.4 and 5.0 particles/µm² of Golgi profile and 25.4 and 3.4. particles/µm² of multivesicular body profile. Controls showed very little non-specific gold labelling (<0.02 gold particles/µm² of section).     

Polarized site-selective fluorescence spectroscopy of the long-wavelength emitting chlorophylls in isolated Photosystem I particles of Synechococcus elongatus

Isolated trimeric Photosystem I complexes of the cyanobacterium Synechococcus elongatus have been studied with absorption spectroscopy and site-selective polarized fluorescence spectroscopy at cryogenic temperatures. The 4 K absorption spectrum exhibits a clear and distinct peak at 710 nm and shoulders near 720, 698 and 692 nm apart from the strong absorption profile located at 680 nm. Deconvoluting the 4 K absorption spectrum with Gaussian components revealed that Synechococcus elongatus contains two types of long-wavelength pigments peaking at 708 nm and 719 nm, which we denoted C-708 and C-719, respectively. An estimate of the oscillator strengths revealed that Synechococcus elongatus contains about 4–5 C-708 pigments and 5–6 C-719 pigments. At 4 K and for excitation wavelengths shorter than 712 nm, the emission maximum appeared at 731 nm. For excitation wavelengths longer than 712 nm, the emission maximum shifted to the red, and for excitation in the far red edge of the absorption spectrum the emission maximum was observed 10–11 nm to the red with respect to the excitation wavelength, which indicates that the Stokes shift of C-719 is 10–11 nm. The fluorescence anisotropy, as calculated in the emission maximum, reached a maximal anisotropy of r=0.35 for excitation in the far red edge of the absorption spectrum (at and above 730 nm), and showed a complicated behavior for excitation at shorter wavelengths. The results suggest efficient energy transfer routes between C-708 and C-719 pigments and also among the C-719 pigments.Abbreviations Chl chlorophyll - FWHM full width at half maximum - PS I Photosystem I     

Highly sensitive determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine by high-performance liquid chromatography using a new fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive pre-column HPLC method for simultaneous determination of prolyl dipeptides, Pro and Hyp in urine was developed. The analytes were labelled with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 20 min. The derivatives separated on tandem reversed-phase columns by a gradient elution and were monitored with fluorescence detection at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides, Pro and Hyp were 1–5 fmol/injection (S/N=3). Urine samples were treated with o-phthalaldehyde, followed by purification on a Bond Elut C₁₈ column before conducting the labelling reaction. Pro–Hyp, Pro–Gly and Pro–Pro were identified as prolyl dipeptides in urine. The within-day and between-day relative standard deviations were 1.5–4.8 and 1.7–5.8%, respectively. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human urine were 97.6±28.2, 2.74±1.48, 2.08±1.13, 6.71±3.34 and 2.30±1.59 nmol/mg creatinine, respectively.     

Aminomethyl coumarin acetic acid: A new fluorescent labelling agent for proteins

Summary A new fluorescent protein labelling agent, 7-amino-4-methyl coumarin-3-acetic acid (AMCA), emits in the blue region (440–460 nm) on activation with UV light (350 nm). The active reagent is theN-hydroxysuccinimide ester which reacts with lysine residues under mild conditions to form photostable amide links.The Stokes shift of 100 nm compared to 30 nm for Fluorescein isothiocyanate (FITC) allows easy filter discrimination of exciting and emitting radiation. The agent has been demonstrated in use for fluorohistochemical examination of human kidney glomeruli, using the sandwich technique and compared with the same procedure using FITC-labelled antibodies. The good quantum yield coupled with convenient emission lines in the mercury spectrum allows photographic exposure time of fluorescent labelled sections to be reduced to a quarter of that required for a corresponding FITC conjugate.AMCA—immunoglobulin conjugates were not susceptible to photobleaching and have a storage life at – 20° C of more than two years.To whom correspondence and reprint requests should be addressed.     

A CCD-OMA device for the measurement of complete chlorophyll fluorescence emission spectra of leaves during the fluorescence induction kinetics

Summary A new device for the measurement of complete laser induced fluorescence emission spectra (maxima near 690 and 735 nm) of leaves during the induction of the chlorophyll fluorescence is described. In this the excitation light (cw He/Ne laser, 632.8 nm) is switched on by a fast electro-mechanical shutter which provides an opening time of 1 ms. The emitted fluorescence is imaged onto the entrance slit of a multichannel spectrograph through a red cut-off filter (> 645 nm). A charge coupled device (CCD) sensor with 2048 elements simultaneously detects the complete chlorophyll fluorescence emission spectrum in the 650–800 nm wavelength range. Scanning is accomplished electronically and the integration time for a complete fluorescence emission spectrum can be selected from 10 ms up to 260 ms. Shutter, detector system and data acquisition are controlled by an IBM-PC/AT compatible computer. A maximum of 32 spectra can be measured at selected times during the fluorescence induction kinetics with the shortest time resolution of 10 ms. The instrument permits the determination of various fluorescence parameters:a) the rise-time of the fluorescence to the maximum level fm,b) the changes in the shape of the fluorescence emission spectra during the induction kinetics,c) the induction kinetics in the fluorescence ratio F690/F735 as well asd) the fluorescence decrease ratio Rfd at any wavelength between 650 to 800 nm. These fluorescence parameters provide information about the functioning of photosynthesis. The ratio F690/F735 allows the non-destructive determination of the chlorophyll content of leaves. The application of this instrument in ecophysiological research and stress physiology of plants is outlined.     

Isolation and Characterization of the Thylakoid Membranes from the NaCl-Resistant (NaClr) Mutant Strain of the Cyanobacterium Anabaena variabilis

NaCl-induced changes in the thylakoid membrane of wild-type Anabaena variabilis and its NaCl^r mutant strain have been studied. Biochemical characterization of the thylakoid membrane was done by taking its absorption and fluorescence spectra at different wavelength. The thylakoid membranes of both strains were isolated by mechanical disruption of the freeze-dried and lysozyme-treated cells, followed by differential and density gradient centrifugation. The light absorption spectra of the thylakoid membrane showed three and two peaks in NaCl^r mutant strain and its wild-type counterpart respectively at wavelengths of 400–850 nm. These peaks revealed that the thylakoid membrane contains a large amount of carotenoid and chlorophyll a. Fluorescence emission spectra of thylakoid membrane of NaCl^r mutant and its wild-type strain at excitation wavelength of 335 nm showed two different peaks, one at 340 nm and the other at 663 nm respectively. The light absorption and fluorescence spectra of the thylakoid membrane also revealed that the membrane contained carotenoid pigment, chlorophyll (Chl) a, and a pigment with an emission peak at 335 nm. The HPLC analysis of the pigments of the thylakoid membrane indicates that the NaCl^r mutant strain under NaCl stress contained an additional peak for the carotenoid pigment, which was lacking in its wild-type counterpart. The major peak in thylakoid membrane was that of echinenone and β-carotene. Whereas the polypeptide composition of thylakoid membrane differed in the wild-type and its NaCl^r mutant strain, no difference in the cell wall protein pattern was observed in both strains. The thylakoid membrane of NaCl^r mutant strain contained two additional protein bands that were absent in its wild-type counterpart. The thylakoid membrane of the wild-type and its NaCl^r mutant strain also showed morphological variations under NaCl stress. Received: 14 April 2000 / Accepted: 23 May 2000     

Fluorescence staining of living cells with fluorescamine

Summary The interaction of fluorescamine with living plant and animal cells was investigated to determine which subcellular structures and molecular species might react with the dye and to assess its effects on cell viability and function.Plasma and nuclear membranes ofXenopus erythrocytes, mitochondria of sea urchin sperm, growing apices of Timothy root hairs, and various organelles ofNitella andEuglena were labelled as judged by fluorescence microscopy. Cytoplasmic fluorescence was particulate inNitella and easily displaced by moderate centrifugal fields in sea urchin eggs. Chloroplasts and nuclei isolated from cells labelledin vivo exhibited fluorescamine dependent fluorescence.Reaction seemed to have little or no effect on cell viability (Euglena) photoautotrophic growth (Euglena), cell motility (sperm), fertilizability (sperm or egg), embryonic development (sea urchin), or cytoplasmic streaming (Nitella, Timothy).Quantitative fluorometric analysis of thein vivo reactants in sperm indicated a reaction preference for phospholipid over protein compared to control cells dissociated in SDS prior to labelling. The bulk of labelled lipid was phosphatidylethanolamine.These results suggest that fluorescamine is a true vital dye which can label the cell surface as well as penetrate deeply within cells to label a variety of organelles. The distribution of fluorescence and results of chemical analysis suggest thatin vivo the dye may preferentially react with membrane.     

Extracellular protein fibrils in Chrysochromulina breviturrita (Prymnesiophyceae) and their serological relationship to fungal fimbriae

An extensive network of extracellular fibrils was revealed by negative staining in the greenish gold algal flagellate, Chrysochromulina breviturrita. These fibrils were of uniform diameter (4–5 nm), sometimes exceeding 5 m in length. In addition there were short, narrower fibrils (2–3 nm) on the surface of the flagella. Six protein bands were isolated from spent culture medium by SDS-PAGE and one of 80,000 Da was found to polymerize after dialysis into 4–5 nm fibrils identical to those found on the cell surface. Two other proteins of 58,000 Da and 65,000 Da also formed 4–5 nm fibrils but these were either rare or of a shorter length and different appearance. An antiserum directed against the surface 7 nm fibrils (fimbriae) of fungi agglutinated cells of C. breviturrita and some other Prymnesiophyceae and Chrysophyceae, but did not agglutinate cells of algal species in other groups. Immunofluorescence and protein A gold labelling confirmed that antigens related to fungal fimbriae were present on the surface of cells of C. breviturrita. Only the 80,000 and 58,000 Da proteins labelled heavily following protein A gold labelling. Some individual 4–5 nm fibrils labelled with gold were observed in the material prepared from the 80,000 Da band. These results therefore establish that C. breviturrita produces a surface network of fibrils that are serologically related to the fimbriae of fungi, and suggest a previously unrecognized relationship between members of the Prymnesiophyceae, Chrysophyceae and fungal groups.     

Molecular-cytogenetic characterization of a higher plant centromere/kinetochore complex

The centromeric region of a telocentric field bean chromosome that resulted from centric fission of the metacentric satellite chromosome was microdissected. The DNA of this region was amplified and biotinylated by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)/linker-adapter PCR. After fluorescence in situ hybridization (FISH) the entire chromosome complement of Vicia faba was labelled by these probes except for the nucleolus organizing region (NOR) and the interstitial heterochromatin, the chromosomes of V. sativa and V. narbonensis were only slightly labelled by the same probes. Dense uniform labelling was also observed when a probe amplified from a clearly delimited microdissected centromeric region of a mutant of Tradescantia paludosa was hybridized to T. paludosa chromosomes. Even after six cycles of subtractive hybridization between DNA fragments amplified from centromeric and acentric regions no sequences specifically located at the field bean centromeres were found among the remaining DNA. A mouse antiserum was produced which detected nuclear proteins of 33 kDa and 68 kDa; these were predominantly located at V. faba kinetochores during mitotic metaphase. DNA amplified from the chromatin fraction adsorbed by this serum out of the sonicated total mitotic chromatin also did not cause specific labelling of primary constrictions. From these results we conclude: (1) either centromere-specific DNA sequences are not very conserved among higher plants and are — at least in species with large genomes — intermingled with complex dispersed repetitive sequences that prevent the purification of the former, or (2) (some of) the dispersed repeats themselves specify the primary constrictions by stereophysical parameters rather than by their base sequence.     

DPH标记细胞膜的动力学与膜脂流动性的荧光偏振校正测量

用稳态荧光技术测得经过校正的荧光成分,由此算出用DPH标记的细胞膜的偏振度。方法是作荧光偏振值在随时间变化的曲线,将其外推至零标记时间求出该时间的荧光偏振值。用此法测定了艾氏腹水癌细胞的膜流动性。结果表明流动性比用整个细胞测得之值小,说明膜脂的有序程度和包装密度比胞浆中的脂大。实验结果和用三房空模型分析所得的理论值符合较好,提示荧光探剂的标记过程主要受分子扩散所控制。     

An approach to measure ciliate grazing on living heterotrophic nanoflagellates

The complicated routes by which organic material is channelled up to higher trophic levels via bacteria and protozoans is a major issue in aquatic microbial ecology. Because of the fragile nature of protists it is not straightforward to perform experimental studies of prey–predator interactions. Here we present an approach for the assessment of ciliate grazing on living heterotrophic nanoflagellates. Stationary phase cultures of a heterotrophic nanoflagellate (Cafeteria sp.) were live-stained by allowing them to take up fluorescently labelled macromolecules. Controls revealed that this label persisted for several hours. Fluorescently labelled living flagellates (FLLF) were added into enriched natural assemblages of marine oligotrich ciliates and uptake of FLLF was monitored over time. Oligotrich ciliates did not incorporate fluorescent-labelled macromolecules but a linear FLLF uptake over time was observed for 20–30 min at 20°C. Ingestion rates were 21–46 FLLF h^–1 at a concentration of about 2×10⁴ FLLF ml^–1, which corresponded to clearance rates of 0.7–0.8 l ciliate^–1 h^–1. These results are in the same order of reported ciliate grazing on phytoplankton of similar size. This method represents a direct approach to measure ciliate grazing specifically on living heterotrophic nanoflagellates.     

Fluorimetry of mitochondria in cells vitally stained with DASPMI or rhodamine 6 GO

The fluorescent dyes DASPMI and rhodamine 6 GO specifically stain mitochondria in living cells. Dye concentrations from 10^?8 to 5 × 10^?6 mole l^?1 can be used. Excitation and emission spectra, and quantum efficiency of DASPMI depend on solvent characteristics. Spectra of mitochondria in living cells correspond to those in phospholipids (excitation around 470 nm, emission 560–570 nm). Fluorescence intensity of DASPMI is a measure for the energization of mitochondria, as revealed by in vitro studies. In living cells uptake of the dye is strongly influenced by inhibitors of oxidative phosphorylation (i.e. by oligomycin, FCCP). Distribution of fluorescence intensity indicates an intracellular gradient in energy load of endothelial cells. Single mitochondria exhibit oscillations in fluorescence. Mitochondria loaded with DASPMI release the dye suddenly into the cytoplasm on treatment with FCCP. Cyanide and antimycin however, do not diminish fluorescence in vivo under optimal nutritional conditions, while they do so in mitochondrial suspension, indicating different mitochondrial behaviour in vivo and in suspension.     

Fluorescence and Phosphorescence Study of Tet Repressor–Operator Interaction

Fluorescence and phosphorescence measurements have been carried out on single-p tryptophan (Trp 43 or Trp 75)-containing mutants of Tet repressor (Tet R). Tet R containing Trp 43, the residue localized in the DNA recognition helix of the repressor, has been used to observe the binding of Tet R to two 20-bp DNA sequences of tet O₁ and tet O₂ operators. Binding of Tet R to tet O₁ operator leads to a 78% decrease of the repressor fluorescence intensity, with an accompanying 20-nm blue shift of its fluorescence emission maximum to 330 nm. Upon binding of Tet R to tet O₂ operator, the Trp 43 fluorescence intensity is quenched by 60%, and a 10-nm shift of its emission maximum to 340 nm occurs. Solute fluorescence quenching studies, using acrylamide, performed at low ionic strength indicate that in both the complex of Tet R with the O₁ and that with the O₂ operator, Trp 43 is moderately buried, as indicated by a bimolecular rate quenching constant of about 1.8 × 10⁹ M^–1 sec^–1. In contrast to the Tet R–tet O₂ complex, the Stern–Volmer acrylamide quenching constant K _sv of the complex with tet O₁ operator changes from 7.5 M^–1 at 5 mM NaCl to 22 M^–1 at 200 mM NaCl, indicating different exposures of Trp 43 in the two complexes in solutions of higher ionic strength. Phosphorescence studies showed a 0–0 vibronic transition at 408 and 403 nm for Trp 43 and Trp 75, respectively. Upon binding of Tet R to the tet operators, we observed red shifts of 0–0 vibronic bands of Trp 43 to 413 and 412 nm for tet O₁ and tet O₂ operator, respectively, and the phosphorescence triplet lifetime of Trp 43 at 75 K was quenched from 6.0–5.5 to 3.5–3.3 sec. The thermal phosphorescence quenching profile ranged from –200°C to –20°C, and differed drastically for the two complexes, suggesting different dynamics of the microenvironment of the Trp 43 residue. The luminescence data for Trp 43 of Tet R suggest that the recognition helix of Tet R interacts in different fashions with the tet O₁ and tet O₂ operators.     

Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining

The fluorescence properties of one chemically and seven biologically produced polyhydroxyalkanoic acids were investigated as film castings and in living cells respectively after staining with Nile red. All these polyesters show a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm. This could be shown by the use of two-dimensional fluorescence spectroscopy and flow cytometry. The examination of native poly(3-hydroxybutyric acid), poly(3HB), granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 μg Nile red is necessary for total staining of 1.0 mg granules. The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the poly(3HB) concentration of grana suspensions at different grana concentrations. These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a high potential for the quantitative determination of hydrophobic bacterial polyhydroxyalkanoic acids. Received: 13 November 1998 / Received revision: 4 February 1999 / Accepted: 12 February 1999     

Dual‐color‐emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy

We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long‐pass emission filter with single‐wavelength excitation, which is technically easier to use than dual‐emission fluorescent proteins that require dual‐wavelength excitation. Copyright © 2013 John Wiley & Sons, Ltd.