Single cell measurement of micro-viscosity by ratio imaging of fluorescence of styrylpyridinium probe

In aqueous solution, compounds containing the styrylpyridinium group showed dual fluorescence, in which excitation at either 469 or 360 nm each produced an emission band around 600 nm. The ratio of fluorescence intensities of the two bands (R = I469/I360) was sensitive to local viscosity. The N-carboxymethyl butyl ester of DMASP was found to be able to irreversibly load into a living cell; presumably by hydrolysis involving cellular lipases it was transformed to a membrane-impermeable fluorescent carboxylate. A map of the ratio, R, from a single cell was generated using fluorescence imaging microscopy with a spectrofluorimeter in dual-excitation single-emission mode. After calibrating the ratio for the probe in water/glycerol solutions, the intracellular viscosities were obtained for a single cell of smooth muscle of a rat embryonic thoracic aorta. The intracellular viscosity is differentiated inside the cell and the obtained values 18-7 cP obey all the values reported by other laboratories. Fluorescence emission of the probe (500-650 nm) is in a very favourable region for its use with visible fluorescence microscopy, without interferences from cell or tissue auto-fluorescence. The results present ability to detect and follow small changes in the ratio of fluorescence intensities, and apparently of the micro-viscosity.     

The two-photon laser-induced fluorescence of the tumor-localizing photosensitizer hematoporphyrin derivative. Resonance-enhanced 750 nm two-photon excitation into the near-UV Soret band

The tumor-localizing photosensitizer hematoporphyrin derivative (HPD) is shown to undergo a simultaneous two-photon excitation into the near-ultraviolet Soret band system upon intense laser irradiation at 750 nm, a spectral region where there is no significant HPD one-photon absorbance in aqueous solution. Subsequent to this excitation, internal conversion and vibrational relaxation occur, resulting in the population of the vibrationless level of the first electronically excited singlet state. This state relaxes by two channels, the emission of fluorescence in the spectral region 600-700 nm and intersystem crossing into the triplet manifold, followed by near-resonant electronic energy transfer with surrounding oxygen to result in the generation of highly reactive singlet molecular oxygen (1 delta g). Evidence for the two-photon excitation consists in the observation both of the HPD fluorescence spectrum in the region of 615 nm as a result of 750 nm excitation and the quadratic dependence of this fluorescence emission intensity upon the excitation laser intensity. Since, in general, the penetration depth of ultraviolet and visible light into tissue varies directly with wavelength (red penetrating more deeply than blue), these studies suggest the possibility that two-photon-induced localization of tumor-bound HPD might facilitate the detection of deeper lying tumors than allowed by the current one-photon photolocalization method.     

Fluorescein conjugates of 9- and 10-hydroxystearic acids: synthetic strategies, photophysical characterization, and confocal microscopy applications

Different strategies are presented to conjugate a fluorescein moiety to 9- and 10-hydroxystearic acids (HSAs). 5-Amino-fluorescein (5-AF) was used as a starting reagent. When reacted with acyl-chloride-modified HSAs, 5-AF gave rise to stable amide derivatives with a 75% reaction yield. These products exhibited the typical steady-state and time-resolved fluorescence properties of the fluorescein chromophore with absorption at 494 nm and emission at 519 nm. Flow cytometry studies confirmed the distinct proapoptotic effect of underivatized 9-HSA on Jurkat cells and revealed a comparable ability of its amide derivative. Confocal microscopy imaging studies showed that green fluorescence could stain intracellular membranous structures. Moreover, dual-dye labeling with Mito Tracker Red, followed by colocalization analysis, revealed that HSA can move to the mitochondria. Thus, fluorescent derivatives of HSA can be used to monitor the localization of these biologically active molecules in living cells and can provide a useful tool for linking biochemical investigation with optical visualization methods. In contrast, when unmodified HSAs were used, the reaction gave monoesterified and diesterified fluorescein derivatives. These products exhibited unusual steady-state and time-resolved fluorescence properties with the excitation wavelength at 342 nm and the emission wavelength at 432 nm. It is shown that the synthesized HSA amides of fluorescein provide all of the typical photophysical and instrumental advantages of this popular dye, whereas the unusual luminescence and excitation properties of the monoester and diester of the 5-aminofluorescein would make these dyes interesting to explore as potential candidates for two photon excitation applications.     

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     

Comparison of chlorophyll fluorescence emission characteristics of wheat leaf tissue and isolated thylakoids as a function of excitation wavelength

Abstract. Chlorophyll fluorescence emission spectra and the kinetics of 685 mm fluorescence emission from wheat leaf tissue and thylakoids isolated from such tissue were examined as a function of excitation wavelength. A considerable enhancement of fluorescence emission above 700 nm relative to that at 685 nm was observed from leaf tissue when it was excited with 550 nm rather than 450 nm radiation. Such excitation wavelength dependent changes in the emission spectrum occurred over an excitation spectral range of 440–660 nm and appeared to be directly related to the total quantity of radiation absorbed at a given excitation wavelength. Experiments with isolated thylakoid preparations demonstrated that changes in the fluorescence emission spectrum of the leaf were attributable to the optical properties of the leaf and were not due to the intrinsic characteristies of the thylakoid photochemical apparatus. This was not the case for the observed excitation wavelength dependent changes in the 685 nm fluorescence induction curve obtained from leaf tissue infiltrated with DCMU. Excitation wavelength dependent changes in the ratio of the variable to maximal fluorescence emission and the shape of the variable fluorescence induction were observed for leaf tissue. Isolated thylakoid studies showed that such changes in the leaf fluorescence kinetics were representative of the way in which the photochemical apparatus in vivo was processing the absorbed radiation at the different excitation wavelengths. The results are considered in the context of the use of fluorescence emission characteristics of leaves as non-destructive probes of the photochemical apparatus in vivo.     

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.     

Haematococcus pluvialis cell-mass sensing using ultraviolet fluorescence spectroscopy

A simple whole-cell-based sensing system is proposed for determining the cell mass of H. pluvialis using ultraviolet fluorescence spectroscopy. An emission signal at 368 nm was used to detect the various kinds of green, green-brown, brown-red, and red H. pluvialis cells. The fluorescence emission intensities of the cells were highest at 368 nm with an excitation wavelength of 227 nm. An excitation wavelength of 227 nm was then selected for cell-mass sensing, as the emission fluorescence intensities of the cell suspensions were highest at this wavelength after subtracting the background interference. The emission fluorescence intensities of HPLC-grade water, filtered water, and HPLC-grade water containing a modified Bold's basal medium (MBBM) were measured and the difference was less than 1.6 for the selected wavelengths. Moreover, there was no difference in the emission intensity at 368 nm among suspensions of the various morphological states of the cells. A calibration curve of the fluorescence emission intensities and cell mass was obtained with a high correlation (R(2)=0.9938) for the various morphological forms of H. pluvialis. Accordingly, the proposed method showed no significant dependency on the various morphological cell forms, making it applicable for cell-mass measurement. A high correlation was found between the fluorescence emission intensities and the dry cell weight with a mixture of green, green-brown, brown-red, and red cells. In conclusion, the proposed model can be directly used for cell-mass sensing without any pretreatment and has potential use as a noninvasive method for the online determination of algal biomass.     

Nile red: a selective fluorescent stain for intracellular lipid droplets

We report that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.     

Studies with a fluorescent vital probe for DNA in mammalian cells

Olivomycin is taken up efficiently by HeLa cells and by rat fibroblast cells at 38.5 °C, but not by BHK cells. On irradiation with light of 425 nm wavelength, the nuclei of living cells that have taken up olivomycin fluoresce. When olivomycin complexes with DNA in solution, the emission spectrum broadens and shifts, the excitation wavelength maximum shifts up 15 nm, and the fluorescence polarization increases. In HeLa and fibroblast cells, the fluorescence characteristics indicate that olivomycin is entirely complexed to DNA, and its rotational mobility indicates that it is complexed to DNA in regions where other components of the chromatin offer no steric hindrance.     

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin

Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.     

肽链及蛋白质N-末端喹喔啉类荧光衍生物的形成

蛋白质及肽链N-末端经Dixon转氨后的羰酰基与邻苯二胺作用可形成喹喔啉类荧光衍生物.形成这一发色团的过程较为缓慢;喹喔啉短肽的激发(Ex 293-305nm)与发射(Em 360-365nm)波长随肽链氨基酸残基组成不同而有一定变化;喹喔啉胰岛素荧光衍生物的激发光波长为Ex318nm,发射波长为Em 353nm.喹喔啉三肽的荧光在乙二醇溶液中的变化因其氨基酸残基组成的不同而有差异.以上结果提示:喹喔啉类荧光衍生物可能作为研究蛋白和肽结构与功能的一种手段.     

Fluorescein excitation and emission polarization spectra in living cells: changes during the cell cycle.

Changes in the fluorescein fluorescence emission and excitation polarization spectra in synchronized cultured S3 fibroblasts at G1, mid-S, and mitosis, as well as in human lymphocytes before and after stimulation with mitogens, were studied. In contrast to those measured in aqueous solutions the emission and excitation polarization spectra in living cells exhibit a wavelength dependence characteristic for the state of the cell cycle. Changes in the temperature and in the amount of intracellular water result in quantitative wavelength-independent changes in the polarization spectra. Possible mechanisms for the qualitative wavelength-dependent changes in the fluorescein emission and excitation polarization spectra during the cell cycle are discussed.     

Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors

The first step in the Visual Cycle, the series of reactions that regenerate the vertebrate visual pigment rhodopsin, is the reduction of all-trans retinal to all-trans retinol, a reaction that requires NADPH. We have used the fluorescence of all-trans retinol to study this reduction in living rod photoreceptors. After the bleaching of rhodopsin, fluorescence (excitation, 360 nm; emission, 457 or 540 nm) appears in frog and wild-type mouse rod outer segments reaching a maximum in 30-60 min at room temperature. With this excitation and emission, the mitochondrial-rich ellipsoid region of the cells shows strong fluorescence as well. Fluorescence measurements at different emission wavelengths establish that the outer segment and ellipsoid signals originate from all-trans retinol and reduced pyridine nucleotides, respectively. Using outer segment fluorescence as a measure of all-trans retinol formation, we find that in frog rod photoreceptors the NADPH necessary for the reduction of all-trans retinal can be supplied by both cytoplasmic and mitochondrial metabolic pathways. Inhibition of the reduction reaction, either by retinoic acid or through suppression of metabolic activity, reduced the formation of retinol. Finally, there are no significant fluorescence changes after bleaching in the rod outer segments of Rpe65(-/-) mice, which lack 11-cis retinal.     

Possible involvement of the lipid-peroxidation product 4-hydroxynonenal in the formation of fluorescent chromolipids.

The effects of the lipid-peroxidation product 4-hydroxynonenal on the formation of fluorescent chromolipids from microsomes, mitochondria and phospholipids were studied. Incubation of freshly prepared rat liver microsomes or mitochondria with 4-hydroxynonenal results in a slow formation of a fluorophore with an excitation maximum at 360 nm and an emission maximum at 430 nm. The rate and extent of the development of the 430 nm fluorescence can be significantly enhanced by ADP-iron (Fe3+). With microsomes, yet not with mitochondria. NADPH has a catalytic effect similar to that of ADP-iron. Fluorescent chromolipids with maximum excitation and emission at 360/430 nm are also formed during the NADPH-linked ADP-iron-stimulated lipid peroxidation. Phosphatidylethanolamine and phosphatidylserine react with 4-hydroxynonenal revealing a fluorophore with the same spectral characteristics as that obtained in the microsomal and mitochondrial system. The findings suggest that the fluorescent chromolipids formed by lipid peroxidation are not derived from malonaldehyde, but are formed from 4-hydroxynonenal or similar reactive aldehydes via a NADPH and/or ADP-iron-catalysed reaction with phosphatidylethanolamine and phosphatidylserine contained in the membrane.     

A novel fluorescent probe with large Stokes shift for the detection of viscosity changes and its imaging in living cells

As an important cellular microenvironmental parameter, viscosity could reflect the status of living cells. Small molecular fluorescent probes are a vital tool to measure the change of viscosity in living cells. A novel fluorescence probe ZL-1 with a large Stokes shift (in methanol it reached to 153 nm and in glycerol it reached to 125 nm) and excellent sensitivity toward viscosity was developed. The sharp enhancement of the emission intensity for the probe ZL-1 from low viscous methanol to high viscous glycerol indicated that the probe ZL-1 could respond to the viscosity variations. Moreover, the probe ZL-1 has been successfully utilized to detect of the viscosity variations in living cells.     

The ultraviolet fluorescence spectra of DPN-linked isocitrate dehydrogenase from bovine heart.

The emission maximum of DPN-linked isocitrate dehydrogenase from bovine heart shifted from 316 nm to 324 nm as the excitation wavelength was varied from 265 nm to 300 nm. This shift was accompanied by a nonproportional change in fluorescence intensity. Comparisons of the emission spectra of model compounds in aqueous buffer at pH 7.07 and n-butanol showed that lowered solvent polarity led to a blue shift of the peak of free tryptophan without significant change of fluorescence intensity, whereas the fluorescence intensity of tyrosine amide increased markedly without change in emission maximum. The emission peak of mixtures of tryptophan and tyrosine amide shifted to shorter wavelengths as the proportion of tyrosine amide increased. The results suggest a major contribution of tyrosine to the overall fluorescence of the dehydrogenase. DPNH caused quenching and a blue shift of the protein fluorescence maximum when excited between 270 nm and 290 nm, indicating that the two tryptophan residues per subunit of enzyme are located in different microenvironments of the protein and that DPNH may interact preferentially with the residue emitting at the longer wavelength.     

Rapid differentiation between short-chain-length and medium-chain-length polyhydroxyalkanoate-accumulating bacteria with spectrofluorometry

An approach for rapid differentiation between short-chain-length (scl) and medium-chain-length (mcl) polyhydroxyalkanoate (PHA) producers was developed. Polyhydroxyalkanoate-accumulated bacterial cells stained with Nile red were suspended in water and subjected to fluorescence spectroscopy at a fixed excitation wavelength of 488 nm. The scl-PHA-accumulated bacteria revealed a maximum emission wavelength at 590 nm, and for mcl-PHA producers were seen at a wavelength of 575 nm. Combining Nile red staining and fluorescence spectroscopy, the accumulated PHA granules could be rapidly differentiated into scl-PHA and mcl-PHA from the intact cells.     

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.     

Spectral resolution of low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 through direct excitation

We have measured fluorescence spectra from Photosystem I (PS I) on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803 at room temperature as a function of excitation wavelength. Our data show a gradual enhancement of long-wavelength fluorescence at 710 nm as the excitation wavelength is increased from 695 to 720 nm. This verifies the presence of low-energy chlorophylls (LE Chls), antenna Chls with energy levels below that of the primary electron donor, P700. The change in fluorescence with excitation wavelength is attributed to the finite time it takes for equilibration of excitations between the bulk and LE Chls. The spectra were deconvoluted into the sum of two basis spectra, one an estimate for fluorescence from the majority or bulk Chls and the other, the LE Chls. The bulk Chl spectrum has a major peak at 688 nm and a lower amplitude vibrational band around 745 nm and is assumed independent of excitation wavelength. The LE Chl spectrum has a major peak at 710 nm, with shoulders at 725 and 760 nm. The relative amplitude of emission at the vibrational side bands increases slightly as the excitation wavelength increases. The ratio of the fluorescence yields from LE Chls to that from bulk Chls ranges from 0.3 to 1.3 for excitation wavelengths of 695 to 720 nm, respectively. These values are consistent with a model where the LE Chls are structurally close to P700 allowing for direct transfer of excitations from both the bulk and LE Chls to P700.     

A fluorometric assay for cholesterol reductase activity.

A fluorometric method for the assay of cholesterol reductase activity from pea leaves (Pisum sativum) is presented. This method is based on the decrease in relative fluorescence occurring as a result of the oxidation of NADH when cholesterol is reduced catalytically to coprostanol by cholesterol reductase. The reaction mixture consisted of micellar cholesterol, NADH, and cytosol of pea leaves in a phosphate buffer. After incubation for 1 h, the reaction mixture were diluted with 2-(N-cyclohexylamino)ethanesulfonic acid buffer (50 mM, pH 10.0) to an appropriate concentration for NADH quantification. The relative fluorescence was measured at an excitation wavelength of 360 nm and at an emission wavelength of 460 nm. This fluorometric method is relatively rapid, simple, and inexpensive. The results obtained show close correlation (R = 0.997) with those obtained by the more time-consuming and expensive radiometric method for assay of cholesterol reductase activity. Results suggest that the fluorometric method is useful for the accurate determination of cholesterol reductase activity in biological specimens.