Product Highlight: The Alexa Fluor Dye Series - Note 1.1

• Brightness — Alexa Fluor conjugates exhibit more intense fluorescence than other spectrally similar conjugates
• Photostability — Alexa Fluor conjugates are more photostable than most other fluorescent conjugates, allowing more time for image capture (, )
• Instrument compatibility — Absorption spectra of the Alexa Fluor conjugates are matched to the principal output wavelengths of common excitation sources
• Color selection — Alexa Fluor conjugates are available in several distinct fluorescent colors, ranging from blue to red
• pH insensitivity — Alexa Fluor dyes remain highly fluorescent over a broad pH range
• Water solubility — Alexa Fluor reactive dyes have good water solubility, so protein conjugations can be performed without organic solvents, and the conjugates are relatively resistant to precipitation during storage

The blue-fluorescent Alexa Fluor 350 dye produces conjugates that are typically greater than 50% more fluorescent than conjugates prepared from AMCA (Figure 7.31). Furthermore, because Alexa Fluor 350 conjugates have slightly shorter-wavelength emission maxima than AMCA conjugates (442 nm versus 448 nm), the fluorescence of Alexa Fluor 350 conjugates is better separated from that of commonly used green fluorophores (, ).

Figure 7.31 Comparison of the relative fluorescence of 7-amino-4-methylcoumarin-3-acetic acid (AMCA) streptavidin () and Alexa Fluor 350 streptavidin, a sulfonated AMCA derivative (S11249, ). Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of fluorophores per protein.

With excitation/emission maxima of 402/421 nm, our Alexa Fluor 405 dye () is a near-perfect match to the 405 nm spectral line of the blue diode laser recently developed for fluorescence microscopy and flow cytometry. The Alexa Fluor 405 succinimidyl ester is an amine-reactive derivative of our Cascade Blue dye, which was previously available in amine-reactive form only as an acetyl azide. Not only is it offered at higher purity than is Cascade Blue acetyl azide, but the Alexa Fluor 405 succinimidyl ester also contains a 4-piperidinecarboxylic acid spacer that separates the fluorophore from its reactive moiety, minimizing any interactions between the fluorophore and the biomolecule to which it is conjugated.

Few reactive dyes that absorb between 400 nm and 450 nm have appreciable fluorescence beyond 500 nm in aqueous solution. Our Alexa Fluor 430 dye fills this spectral gap. Excitation near its absorption maximum at ~430 nm is accompanied by strong emission near 540 nm (, ).

Protein conjugates prepared with the Alexa Fluor 488 dye are far superior to conjugates of fluorescein, and are indeed much better than conjugates of any other green fluorophore that we have tested, including those of the Cy2 dye (Figure 7.36). Not only are Alexa Fluor 488 conjugates significantly brighter than fluorescein conjugates (Figure 1.13), they are much more photostable (Figure 1.9, , , Figure 1.53). Furthermore, fluorescence of the Alexa Fluor 488 fluorophore is independent of pH from 4 to 10. This pH insensitivity is a major improvement over fluorescein, which emits fluorescence that is significantly affected by pH (Figure 1.12, , ).

Figure 7.36 Brightness comparison of Molecular Probes' Alexa Fluor 488 goat anti–mouse IgG antibody with Cy2 goat anti–mouse IgG antibody from Jackson ImmunoResearch. Human blood was blocked with normal goat serum and incubated with an anti-CD3 mouse monoclonal antibody; cells were washed, resuspended and incubated with either Alexa Fluor 488 or Cy2 goat anti–mouse IgG antibody at equal concentration. Red blood cells were lysed, and the samples were analyzed with a flow cytometer equipped with a 488 nm argon-ion laser and a 525 ± 10 nm bandpass emission filter.

Figure 1.9 Photobleaching resistance of the green-fluorescent Alexa Fluor 488, Oregon Green 488 and fluorescein dyes, as determined by laser-scanning cytometry. EL4 cells were labeled with biotin-conjugated anti-CD44 antibody and detected by Alexa Fluor 488 (S11223), Oregon Green 488 (S6368) or fluorescein (S869) streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6). The cells were then fixed in 1% paraformaldehyde, washed and wet-mounted. After mounting, cells were scanned 10 times on a laser-scanning cytometer; laser power levels were 25 mW for the 488 nm spectral line of the argon-ion laser. Scan durations were approximately five minutes apiece, and each repetition was started immediately after completion of the previous scan. Data are expressed as percentages derived from the mean fluorescence intensity (MFI) of each scan divided by the MFI of the first scan. Data contributed by Bill Telford, Experimental Transplantation and Immunology Branch, National Cancer Institute.

Figure 1.53 Photobleaching profiles of cells stained with Alexa Fluor 488 or fluorescein conjugates of goat anti–mouse IgG antibody F(ab')₂ fragment (A11017, F11021) were used to detect HEp-2 cells probed with human anti-nuclear antibodies. Samples were continuously illuminated and images were collected every 5 seconds with a cooled CCD camera. Normalized intensity data demonstrate the difference in photobleaching rates.

Figure 1.12 Comparison of pH-dependent fluorescence of the Oregon Green 488 (), carboxyfluorescein () and Alexa Fluor 488 () fluorophores. Fluorescence intensities were measured for equal concentrations of the three dyes using excitation/emission at 490/520 nm.

Figure 1.13 Comparison of the relative fluorescence of goat anti–mouse IgG antibody conjugates prepared from the Alexa Fluor 488 dye and from fluorescein isothiocyanate (FITC). Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of a reference dye and multiplying by the dye:protein labeling ratio.

The green-fluorescent Alexa Fluor 500 and Alexa Fluor 514 dyes are specifically designed to be detected simultaneously with other green fluorophores, despite their spectral overlap (, ). Though they appear similar in color by eye, the Alexa Fluor 500 dye can be optically separated from the Alexa Fluor 514 dye using the Zeiss META system or similar spectral imaging instruments with linear-unmixing software; these instruments have the capability of differentiating between fluorescence emission maxima <5 nm apart. Similarly, the fluorescent signal from the Alexa Fluor 514 dye can be resolved from both the Alexa Fluor 488 and the Alexa Fluor 500 fluorescence emissions. Furthermore, the photostable and pH-insensitive Alexa Fluor 514 dye is probably the best fluorophore available for the 514 nm spectral line of the argon-ion laser.

With excitation and emission spectra intermediate between those of the green-fluorescent Alexa Fluor 488 dye and orange-fluorescent Alexa Fluor 546 dye (), the Alexa Fluor 532 dye and its conjugates are ideal for use with 532 nm excitation sources, including the frequency-doubled Nd:YAG laser ().

Conjugates prepared with the Alexa Fluor 546 dye are perfect for applications that require fluorescent probes that emit in the orange region of the spectrum. These intensely fluorescent conjugates outperform conjugates of tetramethylrhodamine (TRITC and TAMRA) and Cy3 (Figure 1.23, ) and are readily excited by the strong 546 nm emission of mercury-arc lamps (, ).

Figure 1.23 Fluorescence output from an Alexa Fluor 546 goat anti–mouse IgG antibody (dye:protein ratio = 5.7) and a commercially available Cy3 goat anti–mouse IgG antibody (dye:protein ratio = 3.8). Antibody concentrations were adjusted to give equal absorbance at the excitation wavelength (535 nm). The relative fluorescence quantum yield of Alexa Fluor 546 conjugates is higher than that of Cy3 conjugates, even at high dye:protein ratios that would typically result in self-quenching effects with most other protein-labeling dyes.

Spectra of the Alexa Fluor 555 conjugates virtually match those of the Cy3 dye (Figure 1.18, ), resulting in an optimal match to filters designed for that dye. However, total fluorescence of Alexa Fluor 555 conjugates is higher (Figure 1.22, Figure 1.29). The Alexa Fluor 555 dye is also more photostable than Cy3 (Figure 1.20), providing researchers with additional time for image capture.

The red-orange–fluorescent Alexa Fluor 568 dye is optimally excited by the 568 nm spectral line of the Ar–Kr mixed-gas laser used in many confocal laser-scanning microscopes. Alexa Fluor 568 conjugates are considerably brighter than Lissamine Rhodamine B conjugates or even Rhodamine Red-X conjugates, which have similar excitation and emission maxima (, ).

Conjugates prepared from the Alexa Fluor 594 dye emit in the red region of the spectrum (), making them particularly useful for multilabeling experiments in combination with green-fluorescent probes. Alexa Fluor 594 conjugates are much more fluorescent than are Texas Red conjugates (Figure 1.21, ).

Figure 1.21 Comparison of the relative fluorescence of Alexa Fluor 594 and Texas Red-X goat anti–mouse IgG antibody F(ab')₂ fragment conjugates at different dye:protein ratios.

Our bright and photostable Alexa Fluor 610 dye emits an intense red fluorescence that can be visualized with the same optics used for the Texas Red and Alexa Fluor 594 dyes. With excitation/emission maxima of 612/628 nm (), the Alexa Fluor 610 dye can be easily differentiated from green fluorophores, making it an ideal candidate for multicolor labeling. Unlike the fluorescence of the Alexa Fluor 633 dye and longer-wavelength fluorophores, fluorescence of the Alexa Fluor 610 dye can still be seen with the human eye. We have principally utilized the Alexa Fluor 610 dye to prepare tandem conjugates of phycobiliproteins with improved spectral properties (Phycobiliproteins - Section 6.4).

Far-red–fluorescent dyes are among the most sought-after labels for fluorescence imaging because their spectra are well beyond the range of most sample autofluorescence. The growing popularity of the 633 nm spectral line of the He–Ne laser and the 635 nm spectral line of red diode lasers prompted us to create compatible dyes. Although their fluorescence is not visible to the human eye, Alexa Fluor 633 conjugates are bright and photostable, with peak absorption centered at 632 nm and a peak emission at 650 nm ().

Spectra of the Alexa Fluor 647 conjugates virtually match those of the Cy5 dye (Figure 1.25), resulting in an optimal match to optical filters designed for that dye. However, total fluorescence of the secondary antibody conjugates of the Alexa Fluor 647 dye is significantly higher than that of Cy5 conjugates supplied by other companies (Figure 1.32, Figure 1.30, Figure 1.31). Also, unlike the Cy5 dye, the Alexa Fluor 647 dye has very little change in absorbance or fluorescence spectra when conjugated to most proteins, oligonucleotides and nucleic acids (Figure 1.33), thus yielding greater total fluorescence at the same degree of substitution.

Figure 1.25 Comparison of the fluorescence spectra of the Alexa Fluor 647 and Cy5 dyes. Spectra have been normalized to the same intensity for comparison purposes.

Figure 1.32 Brightness comparison of Molecular Probes' Alexa Fluor 647 goat anti–mouse IgG antibody with Cy5 goat anti–mouse IgG antibody conjugates commercially available from other companies. Human blood was blocked with normal goat serum and incubated with an anti-CD3 mouse monoclonal antibody; cells were washed, resuspended and incubated with either Alexa Fluor 647 or Cy5 goat anti–mouse IgG antibody at an equal concentration. Red blood cells were lysed and the samples were analyzed with a flow cytometer equipped with a 633 nm He–Ne laser and a longpass emission filter (>650 nm).

Figure 1.30 Comparison of the brightness of Alexa Fluor 647 and Cy5 dye antibody conjugates (prepared by Molecular Probes, Inc.). More Alexa Fluor 647 dye molecules can be attached to proteins and nucleic acids without significant quenching, thus yielding conjugates that are much brighter than those possible using the Cy5 dye.

Figure 1.31 Flow cytometry was used to compare the brightness of Molecular Probes' Alexa Fluor 647 goat anti–mouse IgG antibody (red, A21235) with commercially available Cy5 goat anti–mouse IgG antibody from Jackson ImmunoResearch Laboratories (green) and Amersham-Pharmacia Biotech (blue). Human blood was blocked with normal goat serum and incubated with an anti-CD3 mouse monoclonal antibody; cells were washed, resuspended and incubated with either an Alexa Fluor 647 or Cy5 goat anti–mouse IgG secondary antibody at equal concentration. Red blood cells were lysed and the samples were analyzed on a flow cytometer equipped with a 633 nm He–Ne laser and a longpass emission filter (>650 nm).

Figure 1.33 The absorption spectra of the Cy5 dye conjugates of both proteins and nucleic acids show an additional peak at about 600 nm when compared to the spectrum of the free dye. However, light absorbed by the Cy5 dye conjugates at this wavelength does not result in fluorescence. Alexa Fluor 647 dye conjugates of proteins do not exhibit this spectral anomaly. Spectra have been normalized to the same peak intensity for comparison purposes.

The Alexa Fluor 660 dye is optimally excited by the 647 nm spectral line of the krypton-ion laser and well excited by the 633 nm spectral line of the He–Ne laser. Protein conjugates of the Alexa Fluor 660 dye produce bright far-red–fluorescence emission, with a peak at 690 nm. The wide separation of its emission from that of other fluorophores allows use of the Alexa Fluor 660 dye with other fluorescent labels, including the Alexa Fluor 546 and Cy3 dyes and phycoerythrin conjugates (). The Alexa Fluor 660 dye is the dye of choice as a "second label" with allophycocyanin (APC) conjugates in flow cytometry applications.

With a peak excitation at 679 nm and maximum emission at 702 nm, the Alexa Fluor 680 dye is spectrally similar to the Cy5.5 dye (Figure 1.26). Fluorescence emission of the Alexa Fluor 680 dye is well separated from that of other commonly used red fluorophores, such as the tetramethylrhodamine, Texas Red, R-phycoerythrin, Alexa Fluor 594 and Alexa Fluor 647 dyes, making it ideal for three- and four-color labeling ().

Figure 1.26 Comparison of the fluorescence spectra of the unconjugated Alexa Fluor 680 and Cy5.5 dyes. Spectra have been normalized to the same intensity for comparison purposes.

With an absorption maximum at 696 nm, the Alexa Fluor 700 dye can be excited with a xenon-arc lamp, far-red diode lasers or dye-pumped lasers operating in the 675–700 nm range. The Alexa Fluor 700 dye provides infrared fluorescence emission, with a peak at 719 nm ().

Spectrally similar to the Cy7 dye (Figure 1.27), the Alexa Fluor 750 dye is the longest-wavelength Alexa Fluor dye currently available. Its fluorescence emission maximum at 779 nm is well separated from commonly used far-red fluorophores such as Alexa Fluor 647, Alexa Fluor 660 or allophycocyanin (APC), facilitating multicolor analysis. With a peak excitation at ~752 nm, conjugates of the Alexa Fluor 700 dye are well excited by a xenon-arc lamp or dye-pumped lasers operating in the 720–750 nm range ().

 

Figure 1.27 Comparison of the fluorescence emission spectra of the Alexa Fluor 750 and Cy7 dyes. Spectra have been normalized to the same intensity for comparison purposes.