Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.     

Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition.

Seven-color analyses of immunofluorescence-stained tissue samples were accomplished using Fourier spectroscopy-based hyperspectral imaging and singular value decomposition. This system consists of a combination of seven fluorescent dyes, three filtersets, an epifluorescence microscope, a spectral imaging system, a computer for data acquisition, and data analysis software. The spectra of all pixels in a multicolor image were taken simultaneously using a Sagnac type interferometer. The spectra were deconvolved to estimate the contribution of each component dye, and individual dye images were constructed based on the intensities of assigned signals. To obtain mixed spectra, three filter sets, i.e., Bl, Gr, and Rd for Alexa488 and Alexa532, for Alexa546, Alexa568, and Alexa594, and for Cy5 and Cy5.5, respectively, were used for simultaneous excitation of two or three dyes. These fluorophores have considerable spectral overlap which precludes their separation by conventional analysis. We resolved their relative contributions to the fluorescent signal by a method involving linear unmixing based on singular value decomposition of the matrices consisting of dye spectra. Analyses of mouse thymic tissues stained with seven different fluorescent dyes provided clear independent images, and any combination of two or three individual dye images could be used for constructing multicolor images.     

Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry

BACKGROUND: Phycobiliproteins play an important role in fluorescent labeling, particularly for flow cytometry. The spectral properties of R-phycoerythrin (R-PE) and allophycocyanin (APC) have made them the dominant reagents in this class of fluorochromes. In this study, we evaluate a lesser-known but potentially important series of low-molecular weight cryptomonad-derived phycobiliproteins (commercially termed the CryptoFluortrade mark dyes) for their applicability to flow cytometry, both in extracellular and intracellular labeling applications. METHODS: Several cell lines were labeled with biotin-conjugated antibodies against expressed extracellular surface proteins, followed by streptavidin conjugates of three cryptomonad phycobiliproteins (CryptoFluor-2, CryptoFluor-4, and CryptoFluor-5). Cells were then analyzed by flow cytometry using a variety of laser lines and emission filters to establish the optimal excitation/emission characteristics for each fluorochrome. Some cells were permeabilized and labeled for intracellular antigens, also using the cryptomonad fluorochromes. Where appropriate, parallel samples were labeled with other fluorochromes (including R-PE, APC, the cyanin dyes Cy3 and Cy5, and others) to gauge the performance of the cryptomonad fluorochromes against fluorescent labels previously evaluated for flow cytometry. RESULTS: CryptoFluor-2 possessed excitation/emission maxima similar to those of APC and Cy5, with good excitation in the red (HeNe laser 632 nm) and strong emission in the far red (660 nm). CryptoFluor-4 possessed excitation/emission maxima similar to those of Cy3, with optimal excitation in the green (Kr 530 nm) and strong emission in the yellow/orange (585 nm). CryptoFluor-5 possessed excitation/emission maxima similar to those of lissamine rhodamine, with optimal excitation in the yellow (Kr 568 nm) and emission in the orange (610 nm). All cryptomonad fluorochromes gave satisfactory results for both intracellular and extracellular labeling, with detection sensitivities that were comparable or better than traditional phycobiliproteins and low- molecular weight synthetic fluorochromes such as the cyanin dyes. CONCLUSIONS: The CryptoFluor fluorochromes were applicable to flow cytometric immunodetection, with excitation and emission conditions commonly found on multilaser instruments. Performance of several of these dyes was at least comparable to existing fluorescent labels. The low molecular weights (30-60 kd) of phycobiliproteins may make them particularly useful in intracellular antigen detection. Cytometry 44:16-23, 2001. Published 2001 Wiley-Liss, Inc.     

Fluorescent rhodol derivatives: versatile, photostable labels and tracers.

A series of chemically reactive, fluorescent rhodol derivatives was prepared and evaluated. Reactive functional groups included activated esters, amines, haloacetamides, fixable hydrazide derivatives, acrylamides, and photoaffinity reagents. Depending on the choice of substituents, absorption maxima of the dyes varied from 490 to 550 nm with extinction coefficients that were generally greater than 50,000 M-1 cm-1 in aqueous solution and emission maxima from 520 to 580 nm. Most of the compounds investigated exhibited fluorescence lifetimes between 3 and 4 ns. Individual derivatives were suitable for excitation with the 488 and 514-nm lines of the argon ion laser and the 546-nm line of the mercury arc lamp and were compatible for use with standard fluorescein and rhodamine filter sets. The rhodol dyes were more photostable and less sensitive to pH changes in the physiological range than fluorescein derivatives. Some examples show absorption maxima at or near 514 nm, an excitation wavelength that is useful for multicolor fluorescence microscopy, flow cytometry, and DNA sequencing. Derivatives were also prepared that exhibit absorption and emission maxima similar to those of tetramethylrhodamine (TMR) analogs but with higher quantum yields in aqueous solution. A number of the dyes had higher solubilities in aqueous systems and were less quenched on conjugation to proteins than TMR derivatives. Appropriate substitution results in a wider range of solubilities in hydrophilic or lipophilic solvents than is easily accomplished with fluorescein or TMR derivatives. Conjugates of a number of the rhodol fluorophores were generally more photostable and less pH sensitive than fluorescein conjugates and more fluorescent than TMR conjugates.     

Improved double immunofluorescence for confocal laser scanning microscopy.

Reliable double immunofluorescence labeling for confocal laser scanning microscopy requires good separation of the signals generated by the fluorochromes. We have successfully overcome the limitation of a single argon ion laser in achieving effective excitation of dyes with well-separated emission spectra by employing the novel sulfonated rhodamine fluorochromes designated Alexa 488 and Alexa 568. The more abundant antigen was visualized using the red-emitting Alexa 568, with amplification of the signal by a biotinylated bridging antibody and labeled streptavidin. This was combined with the green-emitting Alexa 488, which yielded brighter images than fluorescein but exhibited comparable photodegradation. With appropriate controls to ensure the absence of crosstalk between fluorescence channels, these dyes permitted unequivocal demonstration of co-localization. This combination of fluorochromes may also offer advantages for users of instruments equipped with alternative laser systems.     

Engineered T4 viral nanoparticles for cellular imaging and flow cytometry

Viruses are of particular interest as scaffolds for biotechnology applications given their wide range of shapes and sizes and the possibility to modify them with a variety of functional moieties to produce useful virus-based nanoparticles (VNPs). In order to develop functional VNPs for cell imaging and flow cytometry applications, we used the head of the T4 bacteriophage as a scaffold for bioconjugation of fluorescent dyes. Bacteriophage T4 is a double-stranded DNA virus with an elongated icosahedron head and a contractile tail. The head is ～100 nm in length and ～90 nm in width. The large surface area of the T4 head is an important advantage for the development of functional materials since it can accommodate significantly larger numbers of functional groups, such as fluorescent dyes, in comparison with other VNPs. In this study, Cy3 and Alexa Fluor 546 were chemically incorporated into tail-less T4 heads (T4 nanoparticles) for the first time, and the fluorescent properties of the dye-conjugated nanoparticles were characterized. The T4 nanoparticles were labeled with up to 19?000 dyes, and in particular, the use of Cy3 led to fluorescent enhancements of up to 90% compared to free Cy3. We also demonstrate that the dye-conjugated T4 nanoparticles are structurally stable and that they can be used as molecular probes for cell imaging and flow cytometry applications.     

Development of a novel FRET immunosensor technique

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.     

Spectroscopic study and evaluation of red-absorbing fluorescent dyes

The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATTO655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATTO655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.     

Increased immunofluorescence sensitivity using 532 nm laser excitation.

OBJECTIVE: We evaluated the use of a high power, diode pulsed solid-state laser emitting 532 nm light for immunofluorescence applications. We compared the sensitivity and utility of this laser with the standard 488 nm excitation. METHODS: A flow cytometer was equipped with both a 488 nm and a 532 nm laser; fluorescence emissions from each laser were collected using the same filters and the same detector system. Cells or compensation beads (e.g. latex beads coated with anti-kappa antibodies) were stained with monoclonal antibodies conjugated to phycoerythrin (PE) as well as the PE tandem dyes TRPE, Cy5PE, Cy5.5PE, and Cy7PE. The sensitivity of detection of these reagents as well as those in heavily compensated channels was quantified by measuring the spreading error for a primary detector into a secondary detector. RESULTS: Measurement of the fluorescence emission of PE and PE-tandem dyes was considerably more sensitive when using 532 nm excitation (150 mW) as compared with 488 nm excitation (20 mW). In addition, as the absolute number of photoelectrons collected was greater, there was less measurement-error-induced spread into the compensated channels. As an example, when comparing the spreading error of PE labeled cells into the TRPE detector, the green laser was found to be 15-fold more sensitive as compared with the blue laser. In addition, the blue laser produced more autofluoresent signal from cells as compared with the green laser. Together, these advantages of the 532 nm excitation line provides for a significantly improved detection of immunofluorescence staining.     

Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability

Single and dual-labeled fluorescent oligodeoxynucleotides are used in many molecular biology applications. We investigated the effects of commonly used fluorescent dyes and quenchers on the thermodynamic stability of a model probe-target DNA duplex. We demonstrate that those effects can be significant. Fluorescent dyes and quenchers were attached to the probe ends. In certain combinations, these groups stabilized the duplex up to 1.8kcal/mol and increased T(m) up to 4.3 degrees C. None of the groups tested significantly destabilized the duplex. Rank order of potency was, starting with the most stabilizing group: Iowa Black RQ approximately Black Hole 2>Cy5 approximately Cy3>Black Hole 1>QSY7 approximately Iowa Black FQ>Texas Red approximately TAMRA>FAM approximately HEX approximately Dabcyl>TET. Longer linkers decreased stabilizing effects. Hybridizations to targets with various dangling ends were also studied and were found to have only minor effects on thermodynamic stability. Depending on the dye/quencher combination employed, it can be important to include thermodynamic contributions from fluorophore and quencher when designing oligonucleotide probe assays.     

Red‐shifted emission from 1,2‐dioxetane‐based chemiluminescent reactions

Commercial chemiluminescent reagents emit across a broad portion of the electromagnetic spectrum (400–500 nm). A challenge to the use of chemiluminescence to monitor biological processes is the presence of interfering substances in the biological optical window. In the present study, longer wavelength emitting fluorophores (the organic dyes Alexa 568 and Alexa 647), and a semiconductor nanoparticle (QDOT800) were used to red‐shift the emission from commercially available 1,2‐dioxetane‐based chemiluminescent substrate reactions. By adding non‐conjugated fluorescent emitters into chemiluminescent reaction mixtures, an emission peak occurred at the predicted wavelength of the fluorescent emitter. The excitation and emission from QDOT800 was preserved in the presence of a 100 µm‐thick glass barrier separating it from the chemiluminescent reaction components. The maximum tissue phantom penetration by QDOT800 emission was 8.5 mm; in comparison, the native chemiluminescent emission at 500 nm was unable to penetrate the thinnest tissue phantom of 2.5 mm. The described method for red‐shifted emissions from chemiluminescent reactions does not require direct interaction between the chemiluminescent reaction and the fluorescent emitters. This suggests that the mechanism of chemiluminescent excitation of fluorophores and QDOT800 is not exclusive to chemiluminescence resonance energy transfer or sensitized chemiluminescence, but rather by broad energization from the native chemiluminescent emission. Copyright © 2014 John Wiley & Sons, Ltd.     

Facile synthesis of symmetric, monofunctional cyanine dyes for imaging applications

Efficient syntheses of several members of a new class of symmetric, monocarboxylate-functionalized cyanine dyes have been developed. The synthesis is a simple two-step method, typically with greater than 60% yield and easy final product purification. The new monocarboxylate-functionalized cyanine dyes exhibit excellent water solubility and similar excitation and emission properties to those of Cy5 and Alexa Fluor 647. The application of the new dyes in cellular imaging has been demonstrated through direct conjugating of the dye with an antibody, then imaging of microtubules inside cells, visualized by near-infrared fluorescence microscopy.     

Immunofluorescence technique for 100-nm-thick semithin sections of Epon-embedded tissues

An immunofluorescence staining method for Epon-embedded materials is described. Rat kidney and liver were fixed by perfusion with 1% glutaraldehyde for 10 min. Tissue slices were embedded in Epon. Semithin sections with thicknesses ranging from 1,000 to 100 nm were cut and mounted on clean glass slides. Epoxy resin was removed by treatment with 10% sodium ethoxide. Sections were digested with 0.05% trypsin and then treated with sodium borohydride. Sections were immunostained for leucine aminopeptidase (plasma membrane), catalase (peroxisomes), 3-ketoacyl-CoA thiolase (mitochondria), cathepsin D (lysosomes), and LGP107 (lysosomal membrane) using Cy(2)- or Alexa 546-labeled secondary antibodies. In 1,000-nm-thick sections, non-specific fluorescence remained and such fluorescence decreased as the sections became thinner. Clear specific fluorescence was obtained in the sections with thicknesses ranging from 250 to 100 nm with Alexa 546-labeled antibody (red fluorescence) but was not specific enough with Cy(2)- or Alexa 430-labeled antibody (green fluorescence). Sodium borohydride greatly abolished autofluorescence of glutaraldehyde. The present method made it possible to obtain signals in cross-sections of biological materials with a thickness of 250-100 nm, which are difficult to obtain in optical section using a confocal laser microscope.     

Possible sources of dye-related signal correlation bias in two-color DNA microarray assays

DNA microarray analyses commonly use two spectrally distinct fluorescent labels to simultaneously compare different mRNA pools. Signal correlation bias currently limits accepted resolution to twofold changes in gene expression. This bias was investigated by (i) examining fluorescence and absorption spectra and changes in relative fluorescence of DNAs labeled with the Cy3, Cy5, Alexa Fluor 555, and Alexa Fluor 647 dyes and by (ii) using homotypic hybridization assays to compare the Cy dye pair with the Alexa Fluor dye pair. Cy3 or Cy5 dye-labeled DNA exhibited reduced fluorescence and absorption anomalies that were eliminated by nuclease treatment, consistent with fluorescence quenching that arises from dye-dye or dye-DNA-dye interactions. Alexa Fluor 555 and Alexa Fluor 647 dye-labeled DNA exhibited little or no such anomalies. In microarray hybridization, the Alexa Fluor dye pair provided higher signal correlation coefficients (R2) than did the Cy dye pair; at the 95% prediction level, a 1.3-fold change in gene expression was significant using the Alexa Fluor dye pair. Lowered signal correlation of the Cy dye pair was associated with high variance in Cy5 dye signals. These results indicate that fluorescence quenching may be a source of signal bias associated with the Cy dye pair.     

Boradiazaindacene dyes for technology of biological microchips

A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480-530 nm and fluorescence maxima at 500-550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled olgionucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction.     

Boradiazaindacene dyes for technology of biological microchips

A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480–530 nm and fluorescence maxima at 500–550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled oligonucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction.     

Cascade blue derivatives: water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers.

Fluorescent dyes based on the pyrenyloxytrisulfonic acid (Cascade Blue) structure were prepared and evaluated. The dyes contain functional groups that react with amines, thiols, acids, aldehydes, and ketones, forming covalently bonded, fluorescent derivatives of molecules with broad biological interest. Reactive groups in the Cascade Blue dyes include carboxylic acids and activated esters, amines, hydrazides, alcohols, photoaffinity reagents, acrylamides, and haloacetamides. The dyes exhibited absorption maxima at 374-378 nm and 399-403 nm, with extinction coefficients in the range of 1.9 x 10(4)-2.4 x 10(4) M-1cm-1 and 2.3 x 10(4)-3.0 x 10(4) M-1cm-1, respectively. Emission maxima ranged from 422-430 nm. The spectral properties of the fluorescent dyes are sufficiently different from fluorescein to permit simultaneous use of both dyes with minimum spectral interference. The Cascade Blue derivatives have narrower spectral bandwidths and smaller Stokes' shifts than other reactive dyes with similar spectral properties, do not show appreciable sensitivity to pH, have higher solubilities in aqueous solution, and have good to excellent quantum yields. Cascade Blue conjugates of a number of histochemically and biologically useful molecules were prepared, including dextrans, albumins, Fc receptor binding proteins, antibodies, lectins, membrane receptor binding proteins, and biotin binding proteins, as well as biological particles and bacteria. Cascade Blue conjugates of secondary and tertiary labels yielded specific fluorescence localization in the indirect immunofluorescent staining of human epithelial cell (HEp-2) nuclei.     

Which fluorophore is brightest? A comparison of the staining obtained using fluorescein,tetramethylrhodamine, lissamine rhodamine,texas red,and cyanine 3.18

Summary There are several red-emitting fluorophores available for immunofluorescence studies, including tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18; however, it is unclear which of these is best. The present study compared the brightness of these fluorophores to that of fluorescein. Staining was attempted using a primary antibody raised against serotonin and a secondary antibody that was conjugated with either fluorescein or one of the red fluorophores. The intensity of staining was determined densitometrically. It was found that a conjugate of cyanine 3.18 provided significantly brighter staining that conjugates of any of the other fluorophores, including fluorescein. It is concluded that cyanine 3.18 should be useful for multicolor fluorescence experiments and that it may be the brightest fluorophore available for single-color fluorescence immunocytochemistry.     

Fluorescent DNA nanotags featuring covalently attached intercalating dyes: synthesis, antibody conjugation, and intracellular imaging

We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, because the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal because of the large shift in emission wavelength allowed by energy transfer.     

Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red, and cyanine 3.18.

There are several red-emitting fluorophores available for immunofluorescence studies, including tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18; however, it is unclear which of these is best. The present study compared the brightness of these fluorophores to that of fluorescein. Staining was attempted using a primary antibody raised against serotonin and a secondary antibody that was conjugated with either fluorescein or one of the red fluorophores. The intensity of staining was determined densitometrically. It was found that a conjugate of cyanine 3.18 provided significantly brighter staining that conjugates of any of the other fluorophores, including fluorescein. It is concluded that cyanine 3.18 should be useful for multicolor fluorescence experiments and that it may be the brightest fluorophore available for single-color fluorescence immunocytochemistry.