Detection

Atto Dyes for Superior Fluorescent Imaging

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

WavelengthsActivated 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.

Atto 655

Figure 1. Photostability of Atto 655 compared with Cy 5. Excitation by focused light of a 250 W halogen 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 Dyes - Superior Tool for Super-Resolution Microscopy Application

Recent developments in microscopy application, like e.g. STED, PALM, STORM enables resolutions down to 10 nm. These applications require fluorescent dyes, that fulfill superior photo-physical criteria. Some Atto-dyes. Atto 488, Atto 647N, and Atto 655, have proven a suitable for techniques such as PALM, dSTORM, STED, etc.. Find more information about super-resolution dyes here.


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

Rat Stomach StainNon-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.

Protein 1 and Protein 2
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.

 

Fluorophone Recommended Atto Dye Alternative
Alexa Fluor 488 Atto 488
FITC Atto 488
FAM™ Atto 488
JOE™ Atto 520
TET™ Atto 520
Alexa Fluor 532 Atto 532
HEX™ Atto 532, Atto Rho6G
TAMRA™ Atto 550
Cy3 Atto 550
Cy3.5 Atto 565
ROX™ Atto 565, Atto Rho11
Alexa Fluor 594 Atto 590, Atto 594
Texas Red® Atto 590
Alexa Fluor 633 Atto 633, Atto Rho14
Cy5 Atto 647, Atto 647N, Atto 655
Alexa Fluor 647 Atto 647, Atto 647N, Atto 655
Cy5.5 Atto 680, Atto 700

 

Light source Main lines (nm) Recommended Atto dyes
Mercury arc lamp 365, 405, 436, 546 Atto 390, Atto 425, Atto 465, Atto 550, Atto 565
Mercury arc lamp 577 Atto 590, Atto Rho101, Atto 594; Atto Rho13, Atto 610, Atto 611x
Xenon arc lamp Continuum and peaks >800 nm Atto 610, Atto 620, Atto 647, Atto 647N, Atto 655, Atto 680
Halogen lamp Little UV and violet emission; Higher intensity toward longer wavelengths Atto 610, Atto 620, Atto 647, Atto 647N, Atto 655, Atto 680
Argon ion laser 488, 514 Atto 488, Atto 520, Atto 532, Atto 550
Argon-krypton laser 488, 514, 647, 676 Atto 520, Atto 647, Atto 647N, Atto 655, Atto 680
Krypton laser 647,676 Atto 647, Atto 647N , Atto 655, Atto Oxa12, Atto 665, Atto 680, Atto 700, Atto 725, Atto 740
He-Ne laser 633 Atto Rho14, Atto 633, Atto 647, Atto 647N
Nd-NAG laser 532 Atto 532, Atto Rho6G, Atto 550, Atto 565, Atto Rho11, Atto Rho 12
Common diode laser 635, 650, 670 Atto 633, Atto 647, Atto 647N, Atto 655, Atto 680

 

Reactive Atto Dyes

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

  • Strong Signal intensity – Most Atto dyes have σmax 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.

Fluorescent Microscopy of Human Skin Tissue
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.

 

p38 MAPK
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 (λex 647 nm, λem 669 nm). The 50 ng band of His-tagged p38-MAPK is observed using fluorescence imaging.

 

Atto labeled Phospholipids

Phospholipids are the major building blocks of biological membranes. The investigation of biological membranes, e.g. intracellular membranes of live cells, plasma membranes etc., has become a major area of interest. Sigma offers now a series of fluorescent labeled Phospholipid. The optical properties of the selected series of dyes allow application with all commonly used excitation and emission filter settings. We offer a variety of phospholipids based on glycerol carrying one or two fatty acids (lipophilic groups) and a phosphate monoester residue (hydrophilic group) like 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoylsn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-hydroxy-snglycero-3-phosphoethanolamine (PPE), and 1,2-dimyristoyl-sn-glycero- 3-phosphoethanolamine (DMPE). The fluorophores are covalently linked at the hydrophilic head group of the phospholipids.

Product No. Description
44039 Atto 488 DPPE
93580 Atto 532 DPPE
51016 Atto 550 DPPE
40924 Atto 594 DPPE
52816 Atto 633 DPPE
05152 Atto 647N DPPE
67335 Atto 488DOPE
40692 Atto 532 DOPE
42208 Atto 550 DOPE
05676 Atto594 DOPE
90077 Atto 633 DOPE
42247 Atto 647N DOPE
54368 Atto 488 DMPE
06713 Atto 532 DMPE
42971 Atto 550 DMPE
72567 Atto 594 DMPE
55284 Atto 633 DMPE
89522 Atto 647 DMPE
51028 Atto 488 PPE
94092 Atto 532 PPE
78998 Atto 550 PPE
19504 Atto 594 PPE
56530 Atto 633 PPE
91327 Atto 647N PPE

 

Förster Resonance Energy Transfer (FRET)

FRET is becoming more and more important as a method to determine distances at the molecular level and to study dynamic processes like binding of antibody/antigen pairs. If two dye molecules are located close to each other, their transition dipoles can interact, and energy can be transferred from one dye molecule (donor) to the other (acceptor). A practical measure of FRET efficiency is the distance (Förster-radius) at which the rate of energy transfer equals the rate of donor fluorescence. A table of Förster-radii for ATTO-dyes is presented on this PDF PDF icon (75 KB).

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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