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 have 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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Atto Dye's 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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Long Excitation Wavelengths Reduces 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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Emission Wavelength 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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Fluorescent Multiplex Detection using Atto Dyes

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 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.
 

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 and Conjugates

Atto dyes produce intense fluorescent signals due to strong absorbance and high quantum yields. Dyes are available in the following formats:

  • 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
  • Conjugated to biotin, streptavidin, and antibodies

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

 

  λabs ε max λem ηem τem Catalog Number
Dye nm m-1  cm-1 nm % ns Free Acid NHS Ester Maleimide Azide Amine Iodo- acetamide Biotin
Atto 390 390 24,000 479 90 3.8 89313 89204 89740 68321      
Atto 425 436 45,000 484 90 3.5 56749 16805 49349 78948     28616
Atto 430LS 433 32,000 547 65 4.0 06714 56387 41882        
Atto 465 453 75,000 508 55 2.2 50712 53404 55607        
Atto 488 501 90,000 523 80 3.2 41051 41698 28562 72709 74417 74402 30574
Atto 490LS 496 40,000 661 30 2.6 06715 78362 78363 93688      
Atto 495 495 80,000 527 45 2.4 16951 00379 41022        
Atto 514 511 115,000 533 85 3.0 52595 67455 81062 44311 93728 80027 00713
Atto 520 516 110,000 538 90 3.8 70706 77810 16590 61911     01632
Atto 532 532 115,000 553 90 3.8 06699 88793 68499        
Atto Rho6G 535 115,000 560 90 4.1 88821 93516 87785       94169
Atto 540Q 542 105,000       40592 61683 62453        
Atto 550 554 120,000 576 80 3.2 42424 92835 30730 41986 39271   28923
Atto 565 563 120,000 592 90 3.4 75784 72464 18507 43069     92637
Atto Rho3B 565 120,000 592 50 1.5 93685 28743 76531       07876
Atto Rho11 571 120,000 595 80 4.0 56611 89101 51431       19096
Atto Rho12 576 120,000 601 80 4.0 44432 68761 72763       91254
Atto Thio12 579 110,000 609 15 2.0 56963   94079       87784
Atto Rho101 586 120,000 610 80 4.2 77085 50492 73522       94379
Atto 580Q 586 110,000       03722 44756 68152        
Atto 590 594 120,000 624 80 3.7 70425 79636 39887       43208
Atto 594 601 120,000 627 85 3.5 08637 08741 08717 72998 61583   03927
Atto Rho13 600 120,000 625 80 3.9 16973 68538 44535       76141
Atto 610 615 150,000 634 70 3.3 78493 93259 41061       43292
Atto 612Q 615 115,000       04327 53988 50332        
Atto 620 619 120,000 643 50 2.9 92716 67351 49728       72978
Atto Rho14 625 140,000 646 80 3.7 59746 61909 14397       76140
Atto 633 629 130,000 657 64 3.2 18620 01464 n/a 68517 73245 68652  
Atto 647 645 120,000 669 20 2.3 97875 07376 41784        
Atto 647N 644 150,000 669 65 3.4 04507 18373 05316 91000 95349 73353 93606
Atto 655 663 125,000 684 30 1.9 93711 76245 80661 11774 14918 73948 06966
Atto Oxa12 663 125,000 684 30 1.8 92606 55785 01971       61938
Atto 665 663 160,000 684 60 2.9 16851 04022 01407       01376
Atto 680 680 125,000 700 30 1.8 94875 75999 04971       55819
Atto 700 700 120,000 719 25 1.5 30674 16986 50611 59825 19571   73911
Atto 725 729 120,000 752 10 0.5 47156 93725 90979       69616
Atto 740 740 120,000 764 10 0.6 91394 59808 77071       50842
Atto MB2 658 100,000       75118 73499 51797       02934

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

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

     

p38 MAPK

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

Atto Dye-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) has become a major area of interest. We offer a series of fluorescent-labeled phospholipids. 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) such as 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

Materials

     

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. (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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