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Squaraine Rotaxanes

These dyes were developed by SETA BioMedicals to overcome some of the shortcomings of conventional dyes. Squaraine-rotaxanes combine many desirable properties of fluorescent reporters: extremely high quantum yields in water (up to 65%) and high extinction coefficients in the order of 200,000 M-1cm-1, high photostabilities and low sensitivity for oxygen species.

Polarization Labels

 

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 selected 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.

 

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
[nm]
ε
[M –1
cm–1]
λ em
[nm]
QY
[%]
FLT
[ns]
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K9-4119
K9-4119 new
SeTau-665-NHS
SeTau-665-NHS
NH2 PB 7.4 664 160000 712 53 3.1
Product Number
K9-4119
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K9-4142
K9-4142
SeTau-647-di-NHS
SeTau-647-di-NHS
NH2 PB 7.4 650 200000 694 65 3.2
Product Number
K9-4142
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K9-4145
K9-4145
SeTau-633-Ethyl-Ester
SeTau-633-Ethyl-Ester
CHCl3 634 88000 683 68
Product Number
K9-4145
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K9-4148
K9-4148
SeTau-647-Maleimide
SeTau-647-Maleimide
SH PB 7.4 648 200000 692 63 3.2
Product Number
K9-4148
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K9-4149
K9-4149
SeTau-647-NHS
SeTau-647-NHS
NH2 PB 7.4 649 200000 695 61 3.2
Product Number
K9-4149
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K9-4150
K9-4150
SeTau-647
SeTau-647
PB 7.4 647 211000 693 59 3.1
Product Number
K9-4150
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K9-4169
K9-4169 new
SeTau-670-NHS
SeTau-670-NHS
NH2 PB 7.4 673 275000 694 36 1.6
Product Number
K9-4169
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Squaraine-Rotaxane for fluorescence lifetime, fluorescence polarization and 2P-microscopy 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 also 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).

These lables were also mentioned in the recent publication by a group at the Harvard Medical Scchool  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!

 

 
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Quantum yields of IgG-conjugates of SeTau-647, Alexa 647 and Cy5 for different dye-to-protein ratios in phosphate buffer (pH 7.4)

             Multi-color two-photon image of a NOD FoxP3-GFP mouse                     (red: 2PEx SeTau 647, green: GFP, blue: collagen)

   
squ1  "SeTau-647,Cy5,Alexa-647.png"
Chemical stability plot of SeTau-647
vs. Cy5 in presence of ozone or H2O2
 Fluorescence Lifetime of SeTau-647
vs. Alexa 647 or Cy5 in water

 

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. 

2p-microscopy-neuron

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].

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

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