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Single Molecule Applications

A single molecule experiment investigates the properties of individual molecules. More recently, single-molecule fluorescence is the subject of intense interest for biological imaging, to study single, labelled biomolecules in-vivo in cells.

The photobleaching rates of 13 fluorescent dyes (see below), 4 of them conjugated to trolox, were investigated for single-molecule measurements at 37oC in living cells. For this purpose they were linked to a tag protein that was fused to CD47.

In this study SeTau-647 (K9-4149 and K9-4148), a squaraine rotaxane dye, exhibited the best photobleaching performance with an exponential lifetime of 105.8 sec and the best localization precision with 21.5 nm. As a membrane-impermeable dye it allowed for the observation of up to 12,000 frames, which is the longest single-fluorescent-molecule tracking ever reported (see images below and paper in Nature Chem. Biol. [32]).

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Photobleaching lifetimes of 13 dyes (including Atto-647N, Atto-594, DY-647, TMR, Cy3-Tx
and Cy5-Tx) under controlled conditions and conditions for slowest photobleaching [32].
Most dyes including SeTau-647 (arrow) exhibited the longest photobleaching time at 2%O2. 
SeTau-647 exhibited the best photobleaching performance with an exponential lifetime
of 105.8 sec and the best localization precision with 21.5 nm.

 

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TIRF microscopic images (top) of a time series for SeTau-647 linked to ACP-CD47
(fluorophore is located on the outer surface of a T24 epithelial cell).
Time-dependent reductions of the numbers of fluorescent spots found in each 33-ms
frame for TMR and SeTau-647 (K9-4148) on the extracellular surface (bottom) [32].

 

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]
Buy
                   
K8-1342
K8-1342
Seta-670-NHS
Seta-670-NHS
NH2 PB 7.4 667 180000 688 7 0.42
K8-3335
K8-3335
Seta-555-NHS
Seta-555-NHS
NH2 PB 7.4 555 155000 570 7
K8-3345
K8-3345
Seta-555-DBCO
Seta-555-DBCO
N3 PB 7.4 555 155000 570 7
K8-3346
K8-3346
Seta-555-Azide
Seta-555-Azide
triple-CC PB 7.4 555 155000 570 7
K8-5035
K8-5035
Seta-650-NHS
Seta-650-NHS
NH2 PB 7.4 651 200000 671 28
K8-5036
K8-5036
Seta-650-Maleimide
Seta-650-Maleimide
SH PB 7.4 652 200000 672 28
K8-5045
K8-5045
Seta-650-DBCO
Seta-650-DBCO
N3 PB 7.4 653 200000 674 28
K8-5046
K8-5046
Seta-650-Azide
Seta-650-Azide
triple-CC PB 7.4 651 200000 671 28
K9-4119
K9-4119 new
SeTau-665-NHS
SeTau-665-NHS
NH2 PB 7.4 664 160000 712 53 3.1
K9-4142
K9-4142
SeTau-647-di-NHS
SeTau-647-di-NHS
NH2 PB 7.4 650 200000 694 65 3.2
K9-4145
K9-4145
SeTau-633-Ethyl-Ester
SeTau-633-Ethyl-Ester
CHCl3 634 88000 683 68
K9-4148
K9-4148
SeTau-647-Maleimide
SeTau-647-Maleimide
SH PB 7.4 648 200000 692 45 3.2
K9-4149
K9-4149
SeTau-647-NHS
SeTau-647-NHS
NH2 PB 7.4 649 200000 695 61 3.2
K9-4150
K9-4150
SeTau-647
SeTau-647
PB 7.4 647 211000 693 59 3.1
K9-4169
K9-4169 new
SeTau-670-NHS
SeTau-670-NHS
NH2 PB 7.4 673 275000 694 36 1.6
K8-1341
K8-1341
Seta-670-Maleimide
Seta-670-Maleimide
SH PB 7.4 667 180000 688 7
K8-7522
K8-7522
Seta-750-NHS
Seta-750-NHS
NH2 PB 7.4 753 230000 780 14
K9-4179
K9-4179 new
SeTau-680-NHS
SeTau-680-NHS
NH2 PB 7.4 683 215000 705 58 2.9

Our cyanine-based dyes are more photostable compared to the conventional Cy dyes and Seta-555, Seta-650 and Seta-750 exhibit even higher photostability compared to Alexa dyes and are therefore are better suited for single molecule measurements.

Some of our Seta dyes e.g. Seta-670-NHS show extremely low blinking effects at the single molecule level and high photostability [16]. These labels exhibit easy conjugation chemistry and are available in both NHS and maleimide forms. Below is an example of a single molecule application that was done with Seta-670-NHS and a commercially available antibody.

 

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Relative photostability of Seta-555 and Seta-650 compared to Cy3, Cy5 and Alexa 647 upon irradiation with a 500 W halogen lamp
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Characteristic single-molecule time trajectories for Seta-670-labeled IgG coated on glass with a dye-to-protein ratio of 3. Insets to trajectories show values of fluorescence decay times for the specific label [17]

Single Molecule FRET

Fluorescence resonance energy transfer (FRET) is used to obtain distance information from 10 to 100 Ǻ, a range suitable for studying the global structure and interactions of biomolecules. Nevertheless, some conformational changes are difficult to detect using ensemble FRET. The development of single-molecule spectroscopy makes it possible to probe the conformational dynamics and interactions of biological systems at single-molecule level.

To date the most popular fluorescent reporter molecules for single molecule studies are still small size organic molecules. Among them SeTau-647 is currently the dye with the longest exponential lifetime of 105.8 sec. Besides small size and brightness an ideal fluorescent reporter for single-molecule studies should exhibit good photostability and a low tendency for aggregation including easy conjugation chemistry. FRET pairs should also have an adequate shift between the donor and acceptor emissions and similar brightness [16].

Seta-670-NHS has a significant overlap integral between its absorption and emission spectra. Its Förster distance (R0) for homo-FRET was calculated to be about 50 A, a distance comparable to the size of the antibody. The homo-FRET is expected to occur already when an antibody is labeled with only two fluorophores and increases quickly with the number of labels. Analysis of single molecule traces shows an interesting behavior where efficient non-radiative excitation energy transfer and self-quenching already manifests itself with only 2 or 3 labels. Detailed analyses of the overall and average residence times reveal that multiple labeling with fluorophores, such as Seta-670-NHS, could be a good approach for increasing the number of available photons and extending the overall observation time to study/observe binding at the single antibody level. Intrinsically, the signal and lifetime of the individual, intermediate fluorescently labeled species strongly depend on the number of labels, but the average residence time for each single species is similar. Apparently due to energy transfer the change in fluorescence intensity is compensated by the change in average fluorescence lifetime. Contrary to ensemble measurements where over-labeling is commonly recognized as a problem, this approach appears to have significant advantages for single antibody (protein) studies [17].

For additional FRET pairs we refer you to FRET applications, where we provide a more comprehensive list of donors and acceptors including the calculated Förster distances.

 

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