Atto Dyes for Superior Fluorescent Imaging

• Alternatives to common Fluorophores
• Reactive Atto Dyes
• Atto Dye Conjugates

Activated fluorescent dyes are routinely used to tag proteins, nucleic acids, and other biomolecules for use in life science applications including fluorescence microscopy, flow cytometry, fluorescence in situ hybridization (FISH), fluorescence resonance energy transfer (FRET) techniques, receptor binding assays, and enzyme assays. The Atto dyes are a series of fluorescent dyes that meet the critical needs of modern fluorescent technologies:

• Enhanced Photostability and Ozone Resistance
• Long Signal Lifetimes
• Reduced Background for Greater Sensitivity
• Extensive Selection of Alternatives to Common Dyes
• Recommended for Multiplex Applications

Atto 655 and Atto 647N – Photostable and Ozone Resistant for Microarray Applications

In contrast to fluorescein and cyanine-based dyes, Atto 655 and Atto 647N have a more rigid molecular structure and are more photostable under prolonged light exposure (see Figure 1). Excitation and emission wavelengths and the emission signal decay time are relatively insensitive to pH and other environmental changes such as temperature and atmospheric humidity.

Analyzing Properties of Fluorescent Dyes Used for Labeling DNA in Microarray Experiments.

Figure 1. Photostability of Atto 655 compared with Cy 5. Excitation by focused light of a 250 W hallogen lamp.

Long Signal Lifetimes

Atto dyes exhibit longer fluorescence signal lifetimes (0.6-3.8 ns) than either carbocyanine dyes or most of the autofluorescence inherent in cells and biomolecules.

Longer Excitation Wavelengths for Reduced Background

Diode laser excitation at 635 nm and red-absorbing fluorescent dyes were shown to reduce autofluorescence of biological samples sufficiently so that individual antigen and antibody molecules could be detected in human serum samples.^1,2 Excitation in the red spectral region also reduces cell damage when working with live cells.³

• Many of Atto dyes (Atto 590 and above) can be excited using wavelengths greater than 600 nm.
• Using long-wavelength activated Atto dyes with the appropriate excitation wavelength reduces autofluorescence due to sample, solvent, glass, or polymer support.
• Background due to Rayleigh and Raman scattering can be dramatically reduced.
• Improved overall sensitivity in biological analysis and imaging techniques can be obtained since Atto dyes have less interference from fluorophores with shorter lifetimes.

Atto 655 and Atto 680 — Less Molecular Inactivation for Greater Signal

Non-fluorescent triplet states and cis-conformations result in fluctuations that interfere with fluorescent signal yield.⁴ Dyes such as Cy5, Cy5.5, or Alexa Fluor^® 647 may form cis-isomers and triplet states, which precludes their usage in demanding techniques including fluorescence correlation spectroscopy (FCS), single molecule detection (SMD), and as acceptors in fluorescence resonance energy transfer (FRET).⁵ Atto 655 and Atto 680 have low intersystem crossing and lack an isomeric bond so they cannot undergo configuration isomerization.

• Fluorescent signals are stronger with the same molar amount of Atto 655 and Atto 680 since less dye is lost to inactivation. Other Atto dyes also have low triplet formation.

Recommended for Fluorescent Multiplex Detection

Atto dyes can be used to conjugate probes and biomolecules for multiplex applications. Selection of two Atto dyes with separated emission signals supports multiple excitation and measurement results from a single experiment.

Fluorescent Multiplex Detection using Antibody Atto Dye Conjugates.

Immunoblot detection of Protein 1 and Protein 2 using two primary antibodies and two anti-IgG-Atto dye conjugates. Imaging was done sequentially using a FLA-3000 Fuji® laser scanner, first at an excitation wavelength of 532 nm with a 580 nm emission filter, then at an excitation wavelength of 633 nm with a 675 nm emission filter. The image overlay was done using a software tool.

Alternatives to Common Fluorophores

With the extensive selection available, Atto dyes can replace commonly used fluorescent dyes. There are Atto dyes suitable for use with any common excitation light source.

Reactive Atto Dyes

Atto dyes produce intense fluorescent signals due to strong absorbance and high quantum yields.

• Strong Signal intensity – Most Atto dyes have σ_max values >100,000.
• Ideal for multiplex techniques using visible and near-IR emission wavelengths – Low excitation/emission overlap and good Stokes’ shift separation.
• Selection and Versatility – There is an Atto dye suitable to use with any common excitation light source.

Atto dyes are available as:

• Free acid dyes for all routine staining applications.
• NHS-esters for use in common conjugation protocols.
• Maleimides for use in coupling to thiol-containing groups such as cysteine residues and thiol (-SH) tags added during automated synthesis.

View the complete Atto Dye offering.

Convenient Atto Dye Conjugates

An extensive selection of Atto Dye conjugates and kits are available, including:

• Protein Labeling Kits
  • Atto 488 is a superior alternative to fluorescein and Alexa Fluor 488, producing conjugates with more photostability and brighter fluorescence.
  • Atto 550 is an alternative to rhodamine dyes, Cy3, and Alexa Fluor 550, offering more intense brightness and increased photostability.
  • Atto 594 is an alternative to Alexa Fluor 594 and Texas Red.
  • Atto 647N is an extraordinary highly fluorescent dye, and Atto 655 are alternatives to Cy5 and Alexa Fluor 647.
  • Atto 633 is an alternative to Alexa Fluor 633.

• Lectins for carbohydrate binding studies.

• Primary and Secondary Antibodies for direct and indirect ELISA, Immunoblotting, Immunohistochemistry, and other protein identification applications.

• Biotin and Streptavidin for avidin / streptavidin / biotin conjugation in applications including ELISA, immunohistochemistry, in situ hybridization, and flow cytometry.

• NTA Nickel conjugates for direct detection of polyhistidine-tagged recombinant proteins.

Fluorescent microscopy of human skin tissue section (paraffin fixation) with fungal infection. The target carbohydrate chitotriose of the pathogenic fungi are specifically bound to lectin from Phytolacca americana Atto 488 conjugate (green). The nuclei are counterstained with DAPI (blue). Image by J. Zbären, Inselspital, Bern.

His-tagged p38 MAPK protein (500 ng – 25 ng) was separated on a 4-20% Tris-glycine SDS-PAGE gel. After fixing and washing, the gel was incubated with Ni-NTA-Atto 647N (1:1000) in the dark. The gel was washed and then imaged using a FLA-3000 Fuji® laser scanner with 633 nm excitation and a 675 nm emission filter for Ni-NTA-Atto 647N (λ_ex 647 nm, λem 669 nm). The 50 ng band of His-tagged p38-MAPK is observed using fluorescence imaging.

References

1. Neuweiler, H. et al. Detection of individual p53-autoantibodies by using quenched peptide-based molecular probes. Angew. Chemie, 41, 4769-73 (2002).
2. Sauer, M. et al., Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers. Appl. Phys. B, 65, 427-31 (1997).
3. Terasaki, M., and Dailey, M. E. , Confocal microscopy on living cells. In Handbook of biological confocal microscopy. Pawley, J. B., Ed. 2nd ed., pp 327-346, Plenum Press, New York (1995).
4. Widengren, J., and Schwille, P., Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy. J. Phys. Chem. A, 104, 6416-28 (2000).
5. Widengren, J. et.al., Two new concepts to measure fluorescence resonance energy transfer via fluorescence correlation spectroscopy: theory and experimental realizations. J. Phys. Chem. A, 105, 6851-66 (2001).
6. Buschmann, V., Weston, K.D., and Sauer, M., Spectroscopic study and evaluation of red-absorbing fluorescent dyes. Bioconjugate Chem., 14, 195-204 (2003).

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