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t1   2-Photon Microscopy t1
 
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2-Photon Microscopy

Nonlinear 2 photon excitation is based on the simultaneous absorption of two photons. Since the energy of a photon is inversely proportional to its wavelength, the two absorbed photons must have a wavelength which is about twice that for one-photon excitation. In 2-photon microscopy, two excitation photons from a pulsed laser (Ti:sapphire laser) are combined to excite a fluorescent molecule. The molecule then emits a photon in the visible wavelength. 2-photon microscopy allows for out-of-focus background rejection similar to a confocal microscopy. The advantage of 2-photon microscopy over confocal microscopy is that it can penetrate deeper into tissue due to absence of out-of-focus absorption, the longer excitation wavelength and less scattered light. Nevertheless the achieved optical resolution is the same for both techniques.
photon

SETA BioMedicals offers several green, red and NIR emitting dyes with excellent 2-photon absorption cross sections. It is known that squaraine dyes have several times higher 2-photon absorption (2PA) efficiencies in comparison to other dyes [22-24]. Importantly the stronger, two-photon absorption bands can be identified by measurement of the excitation polarization spectra as shown in the Figure below. Excitation polarization spectra for squaraine- and cyanine-type molecules show large alternations of their maxima thereby revealing the positions of 1PA and 2PA transitions (see below).
2pl1
Absorption (solid black line), excitation polarization (blue), and corresponding 2PA spectrum measured
by two-photon fluorescence (red) and Z-scan (green) for a representative squaraine dye [23].

The 2PA cross section of common fluorescent labels are in the order of 100 GM (2PA of the di-anioic form of fluorescein in water at pH 13 is 36 GM and for Rhodamine B 150 GM for excitation at 800 nm). Even some of the best 2P labels published have 2PA cross sections of only several hundred GM [23]. Seta and SeTau dyes on the other hand have 2PA cross sections in the order of several thousand GM as can be seen from the above figure. Our SeTau and Seta labels are the best 2P labels currently available on the market combining high sensitivity and extremely high 2PA cross sections.

The relevant 2PA wavelengths for serveral Seta and Setau dyes are provided in the table below. For more information please contact us at info@setabiomedicals.com or call us at 217 417 2160.



2-Photon Labels:

SETA BioMedicals has developed several 2P labels with extremely high 2PA cross sections and emission wavelengths between 500 and 600 nm. Seta 646 (K8-1672), Seta 680 (K8-1682), and in particular the new squaraine rotaxanes are currently the most efficient 2P dyes available for the far red spectral range with an emission wavelengths between 660 and 710 nm. SeTau 405 (K7-547) with good 2P excitation properties is a dye with fluorescein emission characteristics. The 2PA wavelengths for these dyes are provided in the table below.
 
Product2PA wavelength [nm]Emission Wavelength [nm]Pack
Size		Product Code
750 - 850 518 0.5 mg K7-547-0.5mg
750 - 900 660 0.5 mg K8-1672-0.5mg
750 - 950 695 0.5 mg K9-4149-0.5mg
775 - 900 675 0.5 mg K8-1682-0.5mg

Pricing and Availability
Ordering

2-Photon FRET:

2P-FRET microscopy has significant advantages over laser confocal FRET microscopy: while in confocal FRET microscopy the actual FRET signal in the acceptor channel is always contaminated due to direct excitation of the acceptor molecule and the cross-talk between donor and acceptor emissions in the acceptor channel, in 2P-microscopy it is possible to prevent the direct excitation of the acceptor molecule. 2P-microscopy also avoids out-of-focus bleaching which allows repeated scanning in multiple focal planes and to obtain a more detailed picture of intracellular events. 2P-FRET microscopy is also a powerful tool to assay intracellular protein-protein co-localization/interaction events.

Below is some information of the Förster radii, 2P-excitation wavelengths and emission wavelengths of 2P- FRET pairs that are based on a combination of our most efficient 2P-donors with several of our acceptor dyes:
 
DonorAcceptorFörster Radii
(Å)		2P -Excitation Wavelength
[nm]		Emission Wavelengths [nm]
DonorAcceptor
71 - 76* 750 - 900 660 695
60 - 67* 750 - 900 660 695
60 - 68* 750 - 900 660 693
56 - 67* 750 - 900 660 780
58 - 70* 750 - 900 660 -
56 - 68* 750 - 900 660 712
78 - 85* 750 - 850 520 573
50 - 53* 750 - 850 520 571
41 - 45* 750 - 850 520 -
72 - 78* 740 - 900 675 695
65 - 70* 740 - 900 675 695
55 - 60* 740 - 900 675 -
72 - 76* 740 - 900 675 780
70 - 76* 740 - 900 675 -
62 - 70* 740 - 900 675 712
  * depending on Q.Y. of donor
 
 
 
Product Specifications:
Excitation Light Sources Free Dye Dye Conjugate
Product Number Product NameTarget
Group		 380405 436488532594635670SolventλAb max. [nm]ε [M–1
cm–1]		 λEm max. [nm]QY [%] Carrier
PB 7.4		λAb max. [nm]λEm max. [nm]QY [%]
                         
K7-545 SeTau-425-NHS NH2 • 425 4200 545 39 0.0 427 528 16
K7-547 SeTau 405 NHS NH2 • • PB 7.4 405 13800 518 80 BSA, D/P=8 406 518 20
K7-549 SeTau-404-NHS NH2 • • PB 7.4 402 10600 515 52 BSA, D/P=2 402 503
K8-1262 Seta-635-di-NHS NH2 • • PB 7.4 640 120000 653 5 IgG, D/P=1 646 658 13
K8-1641 Seta-632-Maleimide SH • PB 7.4 633 270000 642 5
K8-1642 Seta-632-NHS NH2 • PB 7.4 632 280000 641 6 IgG, D/P=1 637 647 18
K8-1663 Seta-633-NHS NH2 • PB 7.4 633 250000 644 7 IgG, D/P=1 637 647 26
K8-1669 Seta-635-NH-NHS NH2 • PB 7.4 638 136000 655 31
K8-1672 Seta-646-NHS NH2 • PB 7.4 646 207000 656 10 IgG, D/P=1 650 661 33
K8-1682 Seta-660-di-NHS NH2 • PB 7.4 661 151000 672 11 IgG, D/P=1 665 675 49
K8-3002 Seta-470-NHS NH2 • • • PB 7.4 469 50000 521 2 BSA, D/P=1 469 515 28
K9-4112 SeTau-655-di-NHS NH2 • PB 7.4 665 200000 673 25 IgG, D/P=1 657 675 31
K9-4113 SeTau-665-di-NHS NH2 • • PB 7.4 665 140000 715 62 IgG, D/P=1 664 709 43
K9-4142 SeTau-647-di-NHS NH2 • PB 7.4 650 368000 694 65 IgG, D/P=0.5 649 691 58
K1-204 SeTau-380-NHS NH2 • Water 270 23800 480 56 BSA, 383 468 44
K8-1261 Square-650-NHS NH2 • • • CHCl3 649 138000 664 17
K8-1377 Seta-690-NH-di-NHS NH2 • • PB 7.4 693 113000 714 14 BSA, D/P=1 711 726 14
K9-4149 Seta-647-NHS NH2 • PB 7.4 649 200000 695 61 IgG, D/P=1 648 694 59
K9-4119 SeTau-665-NHS NH2 • • • PB 7.4 664 160000 712 53 IgG, D/P=1.2 653 716 50
 
 
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