New indodicarbocyanine dyes for the biological microchip technology

New indodicarbocyanine dyes with the carboxybutyl group in position-3 of the indolenine fragment bearing methyl and sulfonic groups in positions 5 and 7 of the cycle were synthesized in order to find the most effective fluorescent labels for the biological microchip technology. The position of absorption and fluorescence maxima, the total charge of the dye molecule, and water solubility depend on the location and the total amount of methyl and sulfonic groups. The spectral characteristics of the dyes synthesized were determined. The relative fluorescence efficiencies of the dyes at equal concentrations were measured at excitation wavelengths of 635 and 655 nm and emission wavelengths of 670 and 690 nm, respectively.     

Transformation of Azo Dye Isomers by Streptomyces chromofuscus A11

Fourteen mono-azo dyes were used to study the effects of substitution patterns on the biodegradability of dimethyl-hydroxy-azobenzene 4(prm1)-sulfonic acids by Streptomyces chromofuscus A11. Two substitution patterns were analyzed: (i) all possible substitution patterns of the two methyl and hydroxy substitution groups, 2-hydroxy (3,5; 4,5; 5,6) dimethyl and 4-hydroxy (2,3; 2,5; 2,6; 3,5) dimethyl isomers of azobenzene 4(prm1)-sulfonic acid; and (ii) replacement of the sulfonic group with a carboxylic group in these sulfonated azo dyes. The structural pattern of the hydroxy group in para position relative to the azo linkage and of two methyl substitution groups in ortho position relative to the hydroxy group was the most susceptible to degradation. Replacement of the sulfonic group with a carboxylic group enhanced overall dye degradability by S. chromofuscus A11.     

Heterodimeric DNA-binding dyes designed for energy transfer: synthesis and spectroscopic properties.

Heterodimeric dyes are described which bind tightly to double-stranded (dsDNA) with large fluorescence enhancements. These dyes are designed to exploit energy transfer between donor and acceptor chromophores to tune the separation between excitation and emission wavelengths. The dyes described here absorb strongly at the 488 nm argon ion line, but emit at different wavelengths, and can be applied to multiplex detection of various targets. The chromophores in these dyes, a thiazole orange-thiazole blue heterodimer (TOTAB), two different thiazole orange-ethidium heterodimers (TOED1 and TOED2), and a fluorescein-ethidium heterodimer (FED), are in each case linked through polymethyleneamine linkers. The emission maxima of the DNA-bound dyes lie at 662 (TOTAB), 614 (TOED 2), and 610 nm (FED). The dyes showed a > 100 fold enhancement of the acceptor chromophore fluorescence on binding to dsDNA and no sequence selectivity. In comparison with direct 488 nm excitation of the constituent monomeric dyes, in the heterodimers the fluorescence of the acceptor chromophores was greatly enhanced and the emission of the donor chromophores quenched by over 90%. The acceptor emission per DNA-bound dye molecule was constant from 100 DNA bp:dye to 20 bp:dye and decreased sharply at higher dye:DNA ratios.     

Selective excitation of mithramycin or DAPI fluorescence on double-stained cell nuclei and chromosomes.

Fluorescence spectra of leukocytes stained by both mithramycin and DAPI showed that the fluorescence of the two dyes can be separated efficiently by using different excitation wavelengths, for instance the 435 nm and the 365 nm mercury lines. In human chromosomes the complementary ("reverse") banding pattern produced by these dyes may thus be observed on double stained chromosome spreads. In plants, for instance in Anemone blanda, the two dyes may reveal two different banding patterns. The results of absorption and fluorescence measurements suggest the existence of at least two binding sites, or types, for each dye, with different fluorescent yields and binding strengths.     

Fluorescent styryl dyes applied as fast optical probes of cardiac action potential

Several styryl dyes were tested as fast optical probes of membrane action potentials in mammalian heart muscle tissue. After staining, atrial specimens were superfused in physiological salt solution, and fluorescence was excited by an argon ion laser. Excitation spot size on the surface of the preparation was 60 m in diameter. Dyes RH 160, RH 237, and RH 421 performed excellently as fast fluorescent probes of cardiac membrane potential. Fractional fluorescence changes, F/F, due to the action potential were in the range 2 to 6% at 514.5 nm excitation. Rise times of the action potential onset detected with each of the dyes were less than 0.5 ms, which is as fast or even faster than microelectrode measurements (atria of the rat). Thus membrane potential changes could be monitored with high resolution in both time and space. Emission spectra from heart muscle preparations stained with these dyes were shifted to shorter wavelengths by 70 nm and more as compared to spectra of the dyes in ethanol solution. The fluorescence spectrum of RH 160 at resting potential and the spectrum recorded during the plateau phases of the action potential were measured and showed no difference within the spectral resolution. As can be concluded from measurements of fluorescence changes at different excitation wavelengths, electrochromism cannot be the only mechanism causing the potential response.     

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.     

Structure property analysis of pentamethine indocyanine dyes: identification of a new dye for life science applications

A collection of nine pentamethine indocyanine dyes was synthesized, and the photophysical characteristics relevant to applications in cell biology and single molecule detection were analyzed in detail. Substituents at the aromatic system covering the auxochromic series and substitutions in the polymethine chain were investigated with respect to absorption and emission spectra, fluorescence lifetimes, fluorescence quantum yields, and fluorescence autocorrelations. Substitutions in the polymethine chain increased the nonradiative energy dissipation of the excited singlet state and decreased the fluorescence quantum yield, relative to the unsubstituted compound. For substituents at the aromatic rings the fluorescence quantum yield negatively correlates with the position of the substituents in the auxochromic series -SO(3)(-), -H, -F, -CH(3). Compounds with sulfonic acid groups or halogen atoms attached to the indolenine systems had the highest fluorescence quantum yields. The compound S0387 had nearly 70% of the quantum yield of Cy5 and comparable photostability. The free carboxylic acid of S0387 was attached to peptides in high yield and purity by established procedures of solid-phase synthesis. The dye-labeled peptides did not aggregate or bind to tissue culture cells and proteins unspecifically. The indocyanine dye S0387 is therefore an attractive new fluorophore for in vitro and cell-based detection of receptor ligand interaction at nanomolar concentrations by flow cytometry, fluorescence correlation spectroscopy, and laser scanning microscopy.     

Selective excitation of mithramycin or DAPI fluorescence on double-stained cell nuclei and chromosomes

Summary Fluorescence spectra of leukocytes stained by both mithramycin and DAPI showed that the fluorescence of the two dyes can be separated efficiently by using different excitation wavelengths, for instance the 435 nm and the 365 nm mercury lines. In human chromosomes the complementary (reverse) banding pattern produced by these dyes may thus be observed on double stained chromosome spreads. In plants, for instance in Anemone blanda, the two dyes may reveal two different banding patterns. The results of absorption and fluorescence measurements suggest the existence of at least two binding sites, or types, for each dye, with different fluorescent yields and binding strengths.     

Dynamic fluorescence of extrinsic fluorophores as a tool for studying protein conformational substates

Summary The fluorescence lifetime distribution of 2-p-toluidinyl-6-naphthalene sulfonic acids (TNS) bound to the heme site of apomyoglobin has been examined. The results were compared to those observed for the free fluorophore in isotropic nonviscous solvent. Two different excitation wavelengths were used, i.e. 290 and 350 nm. The results showed that the distribution of TNS bound to apomyoglobin is wider than that of the free fluorophore, thus indicating the existence of a large number of conformational substates originating from the interaction between TNS and the protein matrix. The comparison of the distribution obtained at two different excitation wavelengths allowed the emission arising from conformational substates, in which the excited state of fluorophore moiety has a higher probability to be populated by Forster energy transfer mechanism, to be distinguished.     

New near-infrared optical probes of cardiac electrical activity

Styryl voltage-sensitive dyes (e.g., di-4-ANEPPS) have been widely and successfully used as probes for mapping membrane potential changes in cardiac cells and tissues. However, their utility has been somewhat limited because their excitation wavelengths have been restricted to the 450- to 550-nm range. Longer excitation/emission wavelength probes can minimize interference from endogenous chromophores and, because of decreased light scattering and lower absorption by endogenous chromophores, improve recording from deeper tissue layers. In this article, we report efforts to develop new potentiometric styryl dyes that have excitation wavelengths ranging above 700 nm and emission spectra extending to 900 nm. Three dyes for cardiac optical mapping were investigated in depth from several hundred dyes containing 47 variants of the styryl chromophores. Absorbance and emission spectra in ethanol and multilamellar vesicles, as well as voltage-dependent spectral changes in a model lipid bilayer, have been recorded for these dyes. Optical action potentials were recorded in typical cardiac tissues (rat, guinea pig, pig) and compared with those of di-4-ANEPPS. The voltage sensitivities of the fluorescence of these new potentiometric indicators are as good as those of the widely used ANEP series of probes. In addition, because of molecular engineering of the chromophore, the new dyes provide a wide range of dye loading and washout time constants. These dyes will enable a series of new experiments requiring the optical probing of thick and/or blood-perfused cardiac tissues.     

Simultaneously measuring pulse-amplitude-modulated (PAM) chlorophyll fluorescence of leaves at wavelengths shorter and longer than 700 nm

PAM fluorescence of leaves of cherry laurel (Prunus laurocerasus L.) was measured simultaneously in the spectral range below 700 nm (sw) and above 700 nm (lw). A high-sensitivity photodiode was employed to measure the low intensities of sw fluorescence. Photosystem II (PSII) performance was analyzed by the saturation pulse method during a light response curve with subsequent dark phase. The sw fluorescence was more variable, resulting in higher PSII photochemical yields compared to lw fluorescence. The variations between sw and lw data were explained by different levels of photosystem I (PSI) fluorescence: the contribution of PSI fluorescence to minimum fluorescence (F₀) was calculated to be 14% at sw wavelengths and 45% at lw wavelengths. With the results obtained, the validity of an earlier method for the quantification of PSI fluorescence (Genty et al. in Photosynth Res 26:133–139, 1990, https://doi.org/10.1007/BF00047085) was reconsidered. After subtracting PSI fluorescence from all fluorescence levels, the maximum PSII photochemical yield (F_V/F_M) in the sw range was 0.862 and it was 0.883 in the lw range. The lower F_V/F_M at sw wavelengths was suggested to arise from inactive PSII reaction centers in the outermost leaf layers. Polyphasic fluorescence transients (OJIP or OI₁I₂P kinetics) were recorded simultaneously at sw and lw wavelengths: the slowest phase of the kinetics (IP or I₂P) corresponded to 11% and 13% of total variable sw and lw fluorescence, respectively. The idea that this difference is due to variable PSI fluorescence is critically discussed. Potential future applications of simultaneously recording fluorescence in two spectral windows include studies of PSI non-photochemical quenching and state I–state II transitions, as well as measuring the fluorescence from pH-sensitive dyes simultaneously with chlorophyll fluorescence.

     

Fast, three-dimensional super-resolution imaging of live cells

We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolution using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast-switching probes enabled us to achieve 2D imaging at spatial resolutions of ～25 nm and temporal resolutions as fast as 0.5 s. We also demonstrated live-cell 3D super-resolution imaging. We obtained 3D spatial resolution of ～30 nm in the lateral direction and ～50 nm in the axial direction at time resolutions as fast as 1-2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also demonstrated two-color 3D super-resolution imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.     

The Changes in Fluorescence of Acridine Orange-Stained Hela Cells During Ultraviolet Illumination

HeLa cells were stained with a 1/12,000 concentration of acridine orange at pH 7.2 for 3 min and the fluorescence emission was measured quantitatively for effects of ultraviolet illumination with durations including intervals between 5 and 210 min. The total photometric fluorescence intensity increased for the first 30 min, then decreased with illumination time. The initial maximum fluorescence intensity occurred at 525 nm and shifted progressively to shorter wavelengths. Fluorescence intensity above 580 nm decreased with increasing duration of illumination time while that below 580 nm showed an initial increase in intensity followed by a gradual fading.     

Photophysical and laser emission studies of 8-polyphenylene-substituted BODIPY dyes in liquid solution and in solid polymeric matrices.

In our search for efficient and photostable laser dyes, four new dyes with the basic structure of the commercial BODIPY laser dye PM567, with either an 8-diphenylene or an 8-p-triphenylene group, both substituted at the terminal polyphenylene position with an acetoxymethyl (dyes P2Ar1Ac and P3Ar1Ac, respectively) or a methacryloyloxymethyl group (dyes P2Ar1MA and P3Ar1MA, respectively), have been synthesized. The photophysical and lasing properties of the dyes have been studied both dissolved in liquid solvents (acetoxymethyl dyes) and incorporated into solid polymeric matrices, in the latter case as solutions (acetoxymethyl dyes) or as copolymers with methyl methacrylate (methacryloyloxymethyl dyes). In liquid solution, the photophysics of P2Ar1Ac and P3Ar1Ac is scarcely affected by the number (two or three) of p-phenylene units. Quantum mechanical calculations reveal that the p-phenylene units in these dyes are twisted ca. 37 degrees each other, an that the first 8-p-phenylene group stands nearly perpendicular to the aromatic BODIPY plane, resulting in electronic decoupling of the two chromophores. P2Ar1Ac exhibits a somewhat lower photodegradation quantum yield under UV and visible irradiation, if compared with P3Ar1Ac or with PM567, likely because of its also lower rate constant for the reaction with in situ-generated singlet molecular oxygen. Both acetoxymethyl dyes emit laser radiation in solution in all the solvents tried, under transversal pumping at 532 nm. In ethyl acetate, with a dye concentration of 0.80 x 10(-3) M, laser efficiencies as high as 80% have been observed. When the 8-polyphenylene dyes were incorporated into solid poly(methyl methacrylate) (PMMA) matrices, as solutions or as copolymers, the fluorescence emission increased with respect to that of the parent PM567 dye dissolved in the same matrix, and lasing efficiencies in the range 18-31% were obtained, with good photostability. The dye P2Ar1Ac dissolved in PMMA was found to exhibit the best overall laser behavior, with a good balance between efficiency and photostability.     

Wavelength dependence of the time course of fluorescence enhancement and photobleaching during irradiation of ethidium bromide-stained nuclei

The variation of fluorescence during irradiation of ethidium bromide-stained nuclei with the 458 nm argon laser line was measured at different wavelengths throughout the emission spectrum. When glycerol was used as a mountant, photoenhancement of fluorescence was observed at all wavelengths, but was greater at the shorter wavelengths. Fluorescence increased by almost one order of magnitude at 500 nm after 40 s of irradiation, compared with only about 10% at wavelengths longer than 600 nm after 2-3 s. In nuclei mounted in phosphate buffer, an initial photoenhancement of fluorescence was detected only at the shorter wavelengths, while continuous photobleaching was observed in the rest of the emission spectrum. When the spectra are normalized to maximum, so as to eliminate the effect of the concurrent photobleaching, it appears that the difference between the time course of fluorescence variation in buffer and glycerol depends largely on the lower photobleaching rate in glycerol. The photoenhancement of fluorescence at shorter wavelengths was found to consist of a band peaking at 485-491 nm in glycerol and at 495-496 nm in buffer. Attenuation of the inner-filter effect contributes minimally to the enhancement of fluorescence at shorter wavelengths. Since the dimer is known to be non fluorescent, the light-induced disaggregation of dimers to monomers cannot be an explanation for the large increase of fluorescence at the shorter wavelengths. The same laser beam that was used to excite the fluorescence of stained nuclei was also used for monitoring the concomitant variation of transmitted light, from which the variation of absorptance during irradiation was computed. While the expected decrease of absorptance was observed in glycerol, reflecting the photodestruction of the fluorophore, in buffer solution an unexpected initial increase was found, which may reflect the accumulation of an absorbing photoproduct.     

Flow cytometric analysis of cell cycle-dependent changes in cell thiol level by combining a new laser dye with Hoechst 33342.

By halogenation of methylfluorescein-diacetate (MFDA) or eosin-diacetate, two new dyes for cellular thiol compatible with visible laser excitation have become available. These probes circumvent the use of an ultraviolet (UV)-excitation system as required by bimane-based dyes and allow combination with probes for other cellular parameters. The thiol dyes attain maximal staining after 10 min at 37 degrees C, and fluorescence is sensitive to pretreatment with diethylmaleate but not to buthionine sulfoximine. In a dual-laser system, analysis of the cellular thiol level as a function of cell cycle distribution can be achieved in viable cells by simultaneous staining with the bisbenzimidazole dye Hoechst 33342 and one of the halogenated dyes. Using this approach, we were able to show that cells in the G2 phase of the cell cycle were more sensitive to thiol depletion with diethylmaleate than were cells in the G1 compartment. The new thiol dyes allow a more flexible selection of wavelengths of excitation and emission for assessing changes in cellular thiol (glutathione and other thiol compounds) and allow this parameter to be examined as a function of cell cycle position.     

Effects of temperature on calcium-sensitive fluorescent probes

The effect of temperature on the binding equilibria of calcium-sensing dyes has been extensively studied, but there are also important temperature-related changes in the photophysics of the dyes that have been largely ignored. We conducted a systematic study of thermal effects on five calcium-sensing dyes under calcium-saturated and calcium-free conditions. Quin-2, chlortetracycline, calcium green dextran, Indo-1, and Fura-2 all show temperature-dependent effects on fluorescence in all or part of the range tested (5-40 degrees C). Specifically, the intensity of the single-wavelength dyes increased at low temperature. The ratiometric dyes, because of variable effects at the two wavelengths, showed, in general, a reduction in the fluorescence ratio as temperature decreased. Changes in viscosity, pH, oxygen quenching, or fluorescence maxima could not fully explain the effects of temperature on fluorescence. The excited-state lifetimes of the dyes were determined, in both the presence and absence of calcium, using multifrequency phase-modulation fluorimetry. In most cases, low temperature led to prolonged fluorescence lifetimes. The increase in lifetimes at reduced temperature is probably largely responsible for the effects of temperature on the physical properties of the calcium-sensing dyes. Clearly, these temperature effects can influence reported calcium concentrations and must therefore be taken into consideration during any investigation involving variable temperatures.     

Evaluation of six fluorescent protein stains for use in flow microfluorometry.

Flow microfluorometric (FMF) analysis of stained cells has provided protein distribution histograms for large populations of cells. Spectral data and staining protocols were evaluated for six fluorescent protein dyes suggested for staining cells in liquid suspension. The requirements for dyes and/or staining protocol included minimal cell clumping and cell loss, near-optimal dye excitation at existing laser wavelengths, and tenacity of the dye/protein interaction. These criteria were best satisfied by fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RITC). Both fluorescamine and 8-aniline-1-naphthalene sulfonic acid (ANSA) showed potential applicability for use in systems where excitation wavelengths in the ultraviolet range are available. Protein staining with fluorescamine was extremely rapid. Brilliant sulfaflavine and 1-dimethyl-aminonaphthalene-5-sulfonyl chloride (DANSYL) were found unsatisfactory in these studies, since the former dye tended to diffuse from the cells, while the latter induced excessive cell clumping and cell loss. These techniques have application to immunofluorescence analysis and can also be profitably employed in dual-parameter analysis systems in connection with double-staining techniques for simultaneous DNA and protein analysis.     

Optical Imaging and Functional Characterization of the Transverse Tubular System of Mammalian Muscle Fibers using the Potentiometric Indicator di-8-ANEPPS

Potentiometric dyes are useful tools for studying membrane potential changes from compartments inaccessible to direct electrical recordings. In the past, we have combined electrophysiological and optical techniques to investigate, by using absorbance and fluorescence potentiometric dyes, the electrical properties of the transverse tubular system in amphibian skeletal muscle fibers. In this paper we expand on recent observations using the fluorescent potentiometric indicator di-8-ANEPPS to investigate structural and functional properties of the transverse tubular system in mammalian skeletal muscle fibers. Two-photon laser scanning confocal fluorescence images of live muscle fibers suggest that the distance between consecutive rows of transverse tubules flanking the Z-lines remains relatively constant in muscle fibers stretched to attain sarcomere lengths of up to 3.5 μm. Furthermore, the combined use of two-microelectrode electrophysiological techniques with microscopic fluorescence spectroscopy and imaging allowed us to compare the spectral properties of di-8-ANEPPS fluorescence in fibers at rest, with those of fluorescence transients recorded in stimulated fibers. We found that although the indicator has excitation and emission peaks at 470 and 588 nm, respectively, fluorescence transients display optimal fractional changes (13%/100 mV) when using filters to select excitation wavelengths in the 530–550 nm band and emissions beyond 590 nm. Under these conditions, results from tetanically stimulated fibers and from voltage-clamp experiments suggest strongly that, although the kinetics of di-8-ANEPPS transients in mammalian fibers are very rapid and approximate those of the surface membrane electrical recordings, they arise from the transverse tubular system membranes.     

Correcting for artifacts in complex aqueous solutions when using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)- 5-(and -6)carboxyfluorescein

The pH-sensitive fluorescent indicator dye 2', 7'-bis-(2-carboxyethyl)-5-(and -6)carboxyfluorescein (BCECF) is routinely used to measure intracellular pH within cells. Surprisingly, no studies have been performed to see if various solution parameters modulate the fluorescence intensity of BCECF even though viscosity artifacts have been reported for particular Ca2+ selective dyes. In this report we demonstrate that even minor increases in the concentration of a number of different agents significantly decrease the excitation fluorescence intensity at two wavelengths routinely used to determine solution pH. Solution viscosity was varied using a number of different agents including glycerol, sucrose, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose. In general, there was a detectable and significant decrease in the maximum fluorescence excitation ratio as the viscosity was increased, although the effect was more dramatic with Newtonian solutions than with non-Newtonian solutions. This same general effect was seen at pH 6.5, 7.0, and 7.3, a range of pH levels where BCECF is found to be particularly sensitive. To correct for these artifactually low values we used different combinations of excitation wavelengths to determine which could be used to accurately measure pH while minimizing the artifact. Choosing excitation wavelengths so that excitation ratios were collected at 470 and 435 nm allowed a significant signal to quantitatively measure pH while the artifact was nearly abolished.