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Superresolution Microscopy

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
                   
K7-547
K7-547
SeTau-405-NHS
SeTau-405-NHS
NH2 PB 7.4 405 13800 518 80 9.3
K8-1342
K8-1342
Seta-670-NHS
Seta-670-NHS
NH2 PB 7.4 667 180000 688 7 0.42
K8-1357
K8-1357
Square-680-NHS
Square-680-NHS
NH2 MeOH 668 208000 690 10
K8-1384
K8-1384
Seta-700-NHS
Seta-700-NHS
NH2 PB 7.4 687 177000 703 6
K8-3002
K8-3002 new
Seta-470-NHS
Seta-470-NHS
NH2 water 469 50000 521 1.9
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
K8-1388
K8-1388
Seta-700-di-NHS
Seta-700-di-NHS
NH2 PB 7.4 688 180000 704 11
K4-205
K4-205 new
Seta-375-NHS
Seta-375-NHS
NH2 PB 7.4 372 24500 476 86 3.1
K4-225
K4-225 new
Seta-385-NHS
Seta-385-NHS
NH2 PB 7.4 384 25000 512 78
K4-215
K4-215 new
Seta-400-NHS
Seta-400-NHS
NH2 PB 7.4 399 24000 576 34

Novel Techniques:

Conventional microscopy is generally limited by the diffraction limit. In recent years a number of novel approaches have been developed to overcome the diffraction limit:

Near-field scanning optical microscopy (NSOM), stimulated emission depletion microscopy (STED), stochastic optical reconstruction microscopy (STORM) and structured illumination microscopy (SIM) are just a few of these new  techniques with improved lateral resolution down to tens of nanometers, more than an order of magnitude beneath that imposed by the diffraction limit, but each method has its limitations.

 

STED Dyes:

Until recently STED utilized mostly bright and photostable dyes with small Stokes shifts between 20–50 nm. Nevertheless, the need for multiplexing makes organic fluorophores having large Stokes shifts also a valuable asset in this technique. Two-color STED microscopy can be achieved by combining 2 fluorescent dyes, one with a small (10 − 30 nm) and the other having a large (>80 nm) Stokes shift. Provided the emission spectra of these dyes overlap, two different excitation wavelengths can be used to distinguish them and due to the overlapping emission spectra, the same  STED wavelength can be utilized for the depletion of both dyes.

Two-color STED microscopy: Combination of small (red) and large (green) Stokes' shift dyes, which can be depleted by one STED wavelength

 

Large Stokes' shift STED Dyes:

SETA BioMedicals produces a number of large Stokes' shift dyes that can be combined with small Stokes' shift dyes in two-color STED microscopy experiments:

Seta-375-NHS (K4-205) is an extremely bright, reactive label for use with the UV 375 - 380 nm diode lasers and has a  large Stokes' shift of 108 nm.  It is good alternative for Atto 390 or Alexa 350. Exc./Em. max: 372/480 nm.

Seta-385-NHS (K4-225) is a hydrophilic, positively charged, amine-reactive label containing one NHS-ester group. It has a large Stokes' shift of over 120 nm and a high extinction coefficient and a high quantum yield of 78% (Exc./Em. max: 384/512 nm).

Seta-400-NHS (K4-215) is a reactive label for use with the 405 nm diode laser. It has a very large Stokes' shift of 175 nm (Exc./Em. max: 400/576 nm) and can be combined with Seta-555-NHS (K8-3335) in dual-color STED microscopy. STED: 660 nm.

 

SeTau-405-NHS (K7-547) is a reactive dye that is well suited for excitation with the blue 404 nm diode laser. Because of its large Stokes’ shift of over 110 nm, the quenching tendency upon labeling to proteins, in particular IgG, is very low. Another important feature of this dye is that its fluorescence lifetime in the order of 9 ns, making it also a valuable label in lifetime based applications. Compatible with Fluorescein emission filter. Exc./Em. max: 405/518 nm.

Seta-670-NHS (K8-1342) is highly hydrophilic, amine-reactive fluorescent label containing one reactive NHS-ester group. It has a uniquely large Stokes' shift of 300 nm due to its short wavelength absorption around 390 nm (ε = 30,000 M–1 cm–1) and an emission maximum at 690 nm. It is excitable with the 405 nm diode laser (ε [M–1 cm–1]: 25,000).

Seta-700-NHS (K8-1389) is an amine-reactive fluorescent label containing one reactive NHS-ester group. It has a uniquely large Stokes' shift of > 300 nm due to its 2nd short wavelength absorption around 395 nm (ε [M–1 cm–1]: 31,000) and its emission maximum at 713 nm. It is excitable with the 405 nm diode laser (ε [M–1 cm–1]: 27,000).

 

Squaraine-Rotaxane STED Dye:

SeTau-647-NHS was used for STED measurements of a HeLa-M cell nucleus.  Squaraine-rotaxanes are a new class of extremely bright and photostable dyes for fluorescence imaging and they are also well suited for STED. The images below show HeLa-M cell nuclei labeled with primary antibody mAB414. This antibody is well characterized, and labels the FG-repeat nucleoporins of mature nuclear pore complexes. The overall size of the individual NPC puncta on the nucleus is 100–120 nm. The excitation laser was 635 nm, and the STED depletion wavelength for this dye is 770 nm. The emission filter was 685/40.

SeTau-647-Conventional-Image-249x249.gif

Conventional Image

SeTau-647-STED-Image-249x249.gif

STED Image

(Courtesy of J. Bewersdorf & J. Meyers, Yale University)

 

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