Squaraine Rotaxanes

These dyes were developed by SETA BioMedicals to overcome some of the shortcomings of conventional dyes. They combine several desirable properties of fluorescent reporters: extremely high quantum yields in water (up to 65%) and high extinction coefficients up to 275,000 M-1cm-1, very high photostabilities and low sensitivity for oxygen species.

Polarization Labels
Structure of squaraine rotaxanes and relevant product types
Quantum yields of IgG-conjugates of SeTau-647 (K9-4149), Alexa 647 and Cy5 for different dye-to-protein ratios in phosphate buffer pH 7.4 
Chemical stability of SeTau-647 (K9-4149) vs. Cy5 in presence of ozone or H2O2
Fluorescence Lifetime of SeTau-647 vs. Alexa 647 or Cy5 in water
Squaraine rotaxanes exhibit high chemical stability in particular against oxidative reagents such as peroxides or ozone and therefore are excellent detection reagents for the use in microarrays where the fluorescence signals of cyanine dyes such as Cy5 and  Alexa 647 are strongly dependent on the concentration of ozone during post-hybridization array washing. Controlled exposures of microarrays to ozone confirmed the susceptibility of cyanine dyes (Cy5 or Alexa 647) to ozone levels as low as 5-10 ppb for periods as short as 10-30 s.

Squaraine rotaxanes are available in reactive form with carboxy, NHS ester and maleimide functionalities but also as hydrophobic probes for imaging.

Excitation Light Sources Characteristics
Product Number
(Specs Sheet)
Product Name
(Product Info)
Target Group 380 405 436 488 532 594 635 650 680 700 750 780 Medium λ abs
[M –1
λ em
K9-4119 new
NH2 PB 7.4 664 160000 712 53 3.1
NH2 PB 7.4 650 200000 694 65 3.2
CHCl3 634 88000 683 68
SH PB 7.4 648 200000 692 45 3.2
NH2 PB 7.4 649 200000 695 61 3.2
PB 7.4 647 211000 693 59 3.1
K9-4169 new
NH2 PB 7.4 673 275000 694 36 1.6

For a direct comparison of the imaging properties and characteristics of Alexa 647 and SeTau 647 we refer you to the following publication: D. Duggal et al. Degree of order and kinetics of phosphorylated (P) and de-P cross-bridges. Am J Physiol Regul Integr Comp Physiol (November 27, 2013): SeTau-647-maleimide (K9-4148) was chosen over Alexa 647 for the above imaging application mainly because it was more resistant to photobleaching and because it is brighter and has a twice as large Stokes' shift (44 nm) compared to Alexa 647 (20 nm).

Comparison of photobleaching of Alexa 647 (left) vs. SeTau 647 (right): the photobleaching rate for SeTau 647 was 4.2 times slower than for Alexa 647! Also the mean count rate of SeTau 647 was about 4 times higher than for Alexa 647.


SeTau 670-NHS  (Φ = 0.36,  ε = 275,000 and Φ. ε  = 99,000) with  the same absorption and emission as Cy5.5 is about 2 times brighter as Cy5.5 ((Φ. ε  = 44,850)  and also 2 times brighter than Alexa 680 (Φ = 0.36,  ε = 183,000 and Φ. ε  = 65,880), and exhibits a lower quenching tendency when labeled to biomolecules. Exc./Em. max of IgG-conjugates: 672/692 nm.

SeTau-665-NHS is a hydrophilic, mono-reactive squaraine rotaxane label for biomolecules. It is very bright (Φ = 0.53,  ε = 161,000 and Φ. ε  = 83,330) with a large Stokes' shift of 48 nm and a similar emission as Alexa 700 (Φ = 0.25,  ε = 205,000 and Φ. ε  = 51,250) but SeTau-665 is a factor of ~ 1.6 times brighter. It is optimally excited with a 665 nm diode laser. This dye also holds the record for 2PECSs of fluorescent labels with >8500 GM at 900 nm. Exc./Em. max of IgG-conjugates: 665/712 nm.

SeTau-647-NHS is a hydrophilic, mono-reactive squaraine rotaxane label for biomolecules. It is very bright (Φ = 0.53,  ε = 200,000 and Φ. ε  = 122,200) with a large Stokes' shift of 46 nm. It is a highly photostable dye that is optimally excited with the 647 nm spectral line of the Krypton-Argon laser. Exc./Em. max for conjugates: 649/695 nm. 

SeTau 647 was used by several research groups at highly reputed universities e.g. at  Harvard Medical School where the authors compared 3 imaging probes labelled with Alexa 488, Alexa 568 and SeTau 647 and concluded that SeTau-647 has near ideal in vivo imaging characteristics and at Weill Medical College of Cornell University where quantitative live-imaging with SeTau-647 (K9-4149) and mathematical modelling was used to outline the recycling pathway of the Megalin (LRP-2) receptor in MDCK cells (see below and relevant publication in Nature Comm. 2016).

Squaraine-Rotaxanes for fluorescence lifetime, fluorescence polarization and imaging applications 

The longer lifetimes in the order of 2 - 4 ns (see below), and larger Stokes' shifts (40 - 50 nm) of squaraine rotaxanes compared to conventional cyanine based NIR labels (τ = 0.5 – 1.5 ns) make them in particular suitable as labels for use in fluorescence polarization immunoassays.

Squaraine rotaxanes have unique properties for 2P imaging applications with two-photon action cross sections (2PACS) in the order of up to 10,000 GM: see (K. Podgorski et al. Ultra-bright and -stable red and NIR squaraine fluorophores for in vivo two-photon imaging. PLoS ONE 7(12): e51980).

Squaraine-Rotaxane imaging probes for live and fixed cells 

Our fluorescent rotaxane probes exhibit extremely high chemical and photostability and are therefore well suited for imaging applications. They also have extremly high 2-photon action cross sections in the order of several thousand GE.

Z-projection of a neuron filled
with a
squaraine rotaxane-labeled peptide

SeTau-633 is a carboxylic acid ethyl ester derivative that passively penetrates cell membranes. Once inside these residues are hydrolyzed by esterases thereby forming carboxyl groups that are cell-impermeable.

SeTau-647 is a highly water-soluble fluorescent probe with several negative charges and therefore will not passivele penetrate the cell-menbrane. This probe is very photostable and has a quantum yield of 60% in water.

Reactive versions (NHS-esters and maleimides) for covalent attachment to different biomolecules to target specific cellular locations for in vitro and in vivo optical imaging are also available. Images stained with these probes are stable for hundreds of hours:

[B.D. Smith et al. Squaraine Rotaxanes: Superior Substitutes for Cy-5 in Molecular Probes for Near-Infrared Fluorescence Cell Imaging. Angew. Chem. 2007, 119, 5624 –5627].

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