Atto Dyes for Superior Fluorescent Imaging

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.

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 Raman light scattering, improving overall sensitivity.

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.

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.

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

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

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 (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

References

1. Neuweiler H, Schulz A, Vaiana AC, Smith JC, Kaul S, Wolfrum J, Sauer M. 2002. Detection of Individual p53-Autoantibodies by Using Quenched Peptide-Based Molecular Probes. Angew. Chem. Int. Ed.. 41(24):4769-4773. https://doi.org/10.1002/anie.200290044

2. Sauer M, Zander C, Müller R, Ullrich B, Drexhage K, Kaul S, Wolfrum J. 1997. Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers. Applied Physics B: Lasers and Optics. 65(3):427-431. https://doi.org/10.1007/s003400050292

3. Sauer M, Zander C, Müller R, Ullrich B, Drexhage K, Kaul S, Wolfrum J. 1997. Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers. Applied Physics B: Lasers and Optics. 65(3):427-431. https://doi.org/10.1007/s003400050292

4. Widengren J, Schweinberger E, Berger S, Seidel CAM. 2001. Two New Concepts to Measure Fluorescence Resonance Energy Transfer via Fluorescence Correlation Spectroscopy:  Theory and Experimental Realizations. J. Phys. Chem. A. 105(28):6851-6866. https://doi.org/10.1021/jp010301a

5. Buschmann V, Weston KD, Sauer M. 2003. Spectroscopic Study and Evaluation of Red-Absorbing Fluorescent Dyes. Bioconjugate Chem.. 14(1):195-204. https://doi.org/10.1021/bc025600x

6. Widengren J, Schwille P. 2000. Characterization of Photoinduced Isomerization and Back-Isomerization of the Cyanine Dye Cy5 by Fluorescence Correlation Spectroscopy. J. Phys. Chem. A. 104(27):6416-6428. https://doi.org/10.1021/jp000059s