Atto Dyes for Superior Fluorescent Imaging

BioFiles 2011, 6.3, 5.

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), receptor binding assays, and enzyme assays. The Atto dyes are a series of fluorescent dyes that meet the critical needs of modern fluorescent technologies:

  • Stability - Atto 655 and Atto 647N are photostable and highly resistant to ozone degradation, making them ideal for microarray applications. See the related article "Analyzing Properties of Fluorescent Dyes used for Labeling DNA in Microarray Experiments".
  • Long Signal Lifetimes - Signal decay times of 0.6–4.1 nanoseconds allow timegate studies to reduce autofluorescence background and scattering.
  • Reduced Background - Several Atto dyes employ excitation wavelengths greater than 600 nm, reducing background fluorescence from samples, Rayleigh and Raman scattering.
  • Selection - Atto dyes have strong fluorescent signals that cover visible and near-IR emission wavelengths.

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Long Signal Lifetimes

Atto dyes exhibit longer fluorescence signal lifetimes (0.6–4.1 ns) in aqueous solution than either carbocyanine dyes or most of the autofluorescence inherent in cells and biomolecules. The signal from Atto dyes can be measured using pulsed laser excitation with a time-gated detection system to reduce interference from fluorophores with shorter lifetimes, background autofluorescence, and Rayleigh and Ramen light scattering, improving overall sensitivity.

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Longer Excitation Wavelengths for Reduced Background

Diode laser excitation at 635 nm and redabsorbing 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 in conjunction with the appropriate excitation wavelength reduces autofluorescence due to sample, solvent, glass, or polymer support, and improves overall sensitivity in biological analysis and imaging techniques. The background fluorescence due to Rayleigh and Raman scattering are also dramatically reduced by use of longer wavelength excitation.

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λEm Range from 479 to 764 nm for Fluorescent Multiplex Detection

Atto dyes have strong fluorescent signals with most having molar absorptivity values >100,000 and low excitation/emission overlap, making Atto dyes ideal for multiplex techniques using visible and near-IR emission wavelengths.

With excitation signal maxima ranging from 390 to 740 nm and good Stokes shift separation, there are Atto dyes suitable for use with any common excitation light source.

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Alternatives to Common Fluorophores

With the extensive selection of Atto dyes available, any common excitation light source can be used, and Atto dyes can replace other fluorescent dyes commonly used in life science.

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Reactive Atto Dyes and Conjugates

Atto dyes produce intense fluorescent signals due to strong absorbance and high quantum yields. 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
  • Atto Dyes conjugated to biotin, streptavidin, and antibodies are also available

Atto 655, Atto 680, and Atto 700 are quenched by guanosine, tryptophan and related compounds through direct contact between the dye and the quenching agent and using an electron transfer process. Fluorescent quenching of dyes by tryptophan residues in proteins has been used to differentiate unbound (nonfluorescent) protein from protein-antibody (fluorescent) interactions.1

Fluorescent Signal Information for Atto Dyes

Fluorescent signal information for Atto dyes. λabs - longest-wavelength absorption maximum; εmax - molar extinction coefficient at the longest-wavelength absorption maximum; λem - fluorescence maximum; ηem - fluorescence quantum yield; τem - fluorescence decay time.

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  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. (1995) Confocal microscopy on living cells. In Handbook of Biological Confocal Microscopy. Pawley, J. B., Ed. 2nd ed., pp 327–346, Plenum Press, NewYork.
  4. 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).
  5. Buschmann, V., Weston, K.D., and Sauer, M., Spectroscopic study and evaluation of red-absorbing fluorescent dyes. Bioconjugate Chem., 14, 195–204 (2003).
  6. 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).

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