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2-Photon Microscopy

2-Photon excitation

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.

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
K7-567
K7-567
SeTau-405-Azide
SeTau-405-Azide
triple-CC 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
K7-545
K7-545
SeTau-425-NHS
SeTau-425-NHS
NH2 PB 7.4 425 4200 545 39 26.2
K8-1346
K8-1346
Seta-670-Azide
Seta-670-Azide
triple-CC PB 7.4 667 180000 686 7
K8-1351
K8-1351
Square-660-Carboxy
Square-660-Carboxy
PB 7.4 657 182000 676 3 0.27
K8-1352
K8-1352
Square-660-NHS
Square-660-NHS
NH2 PB 7.4 657 182000 676 3 0.27
K8-1355
K8-1355
Square-680-Carboxy
Square-680-Carboxy
PB 7.4 654 138000 670 1
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
K8-1407
K8-1407
Square-650-pH-NHS
Square-650-pH-NHS
NH2 PB 5.6 653 135000 671 16 1.17
K8-1626
K8-1626
Seta-633-di-NHS
Seta-633-di-NHS
NH2 PB 7.4 635 192000 644 12 0.7
K8-1641
K8-1641
Seta-632-Maleimide
Seta-632-Maleimide
SH PB 7.4 633 270000 642 5
K8-1642
K8-1642
Seta-632-NHS
Seta-632-NHS
NH2 PB 7.4 632 280000 641 6
K8-1663
K8-1663
Seta-633-NHS
Seta-633-NHS
NH2 PB 7.4 633 250000 644 7 0.25
K8-1672
K8-1672
Seta-646-NHS
Seta-646-NHS
NH2 PB 7.4 646 207000 656 10 0.38
K8-1682
K8-1682
Seta-660-di-NHS
Seta-660-di-NHS
NH2 PB 7.4 661 220000 672 11 0.9
K8-1696
K8-1696
Seta-633-Azide
Seta-633-Azide
triple-CC PB 7.4 633 250000 644 7
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
K8-1388
K8-1388
Seta-700-di-NHS
Seta-700-di-NHS
NH2 PB 7.4 688 180000 704 11
K8-1341
K8-1341
Seta-670-Maleimide
Seta-670-Maleimide
SH PB 7.4 667 180000 688 7
K8-1671
K8-1671
Seta-646-Maleimide
Seta-646-Maleimide
SH PB 7.4 647 210000 657 8
K8-1405
K8-1405
Square-650-pH-Carboxy
Square-650-pH-Carboxy
NH2 PB 5.6 653 135000 671 16 1.17
K8-1389
K8-1389 new
Seta-700-NHS
Seta-700-NHS
NH2 PB 7.4 688 162000 703 14
K9-4179
K9-4179 new
SeTau-680-NHS
SeTau-680-NHS
NH2 PB 7.4 683 215000 705 58 2.9

SETA BioMedicals has developed several green, red and NIR emitting dyes (probes and labels) with excellent 2-photon action cross sections. Due to the donor-acceptor-donor structure of squaraine dyes they exhibit much higher 2-photon absorption (2PA) efficiencies in comparison with other dyes [22-24] and in particular the new class of squaraine-rotaxanes show very high two-photon action cross-sections (2PACS) of up to 10,000 GM at near-infrared wavelengths critical for in vivo imaging. Dyes and labels that undergo two-photon absorption (2PA) in the NIR and fluoresce in the far-red to NIR region with high quantum yields are very desirable to achieve deep tissue imaging.

 

2-photon action cross sections for several squaraines and squaraine rotaxanes
2-photon action cross sections for several squaraines
and squaraine rotaxanes [24]
2-photon image of neuron loaded with squaraine-rotaxane SeTau-647
2-photon image of neuron loaded with SeTau-647

While the 2PACSs of common fluorescent labels are in the order of 10 - 200 GM (2PACS of the di-anioic form of fluorescein in water at pH 13 is 37 GM and for Rhodamine B it is 204 GM for excitation at 830 nm in MeOH - see table below) and even some of the best 2P labels published have 2PACSs of only several hundred GM [23], Seta and SeTau dyes have 2PACSs in the order of thousand to several thousand GM in aquous solution. SeTau and Seta labels are the best 2P probes and labels currently available on the market combining high sensitivity and extremely high 2PACS: (K. Podgorski et al. Ultra-bright and -stable red and NIR squaraine fluorophores for in vivo two-photon imaging. PLoS ONE 7(12): e51980)

The outstanding imaging properties of SeTau 647 are mentioned in the publication by the group of Prof. Weissleder at the Harvard Medical School: "Here we present the first generation of two-photon beta cell specific in vivo imaging probes based on GLP1R targeting peptides. Among the three compounds of potential interest, we found quite unexpectedly that a squaraine-rotaxane conjugate (2PEx-647, K9-4148) had near ideal in vivo imaging characteristics.

The relevant 2PA wavelengths for several Seta and SeTau dyes are provided in the table below.

 

Probes and Labels for in-vivo 2P-microscopy:

SETA BioMedicals has developed several 2P probes and labels with extremely high 2PACSs and emission wavelengths between 500 and 700 nm. Seta-660, Seta-670 and in particular the squaraine rotaxanes (SeTau-665 and SeTau-647) are currently the most efficient 2P dyes available for the far red and NIR spectral emission range. SeTau-405 with good 2P excitation properties is a dye with fluorescein emission characteristics. The 2P absorption wavelengths and cross-sections for these dyes are provided in the table below.

Seta and Squaraine rotaxane dyes offer unprecedented properties ideal for in vivo two-photon imaging. These dyes have two-photon cross-sections and photostabilities approaching those of quantum dots, but with molecular weights similar to other organic dyes, allowing labeling of subcellular structures, such as synapses, at low concentrations and with reduced functional impact. Importantly they are non-toxic and allow long-term neuronal imaging. The demonstrated brightness and stability of these dyes promise to extend the limits of fluorophore concentration, imaging rate, illumination depth, and imaging duration for in vivo two-photon microscopy [24].

Below is a comparison of the 2PACSs for some popular dyes and our Seta and SeTau dyes:

 

Product Number
(Specs Sheet)
Product Name
(Product Info)
Maximum 2PA
Wavelength [nm]
Emission
Wavelength [nm]
2P-Excitation
Cross-Section [GM]
-
Alexa 594
780 617 100
-
Alexa 647
1238 665 44
-
Alexa 700
1320 723 52
-
Cy5
1219 665 40
-
Fluorescein
770 - 790 515 37
-
Rhodamine B
830 620 204
-
Lucifer Yellow
840 528 1.3
-
Fura-2
700 512 11
-
Indo-1
700 475 1
-
DDTC
1550 785 105
750–850 518 >100
K8-1672 Seta-646-NHS 840 660 480
K8-1384 800 705 1050
K8-1682 Seta-660-NHS 900 675 1625
K8-1342 Seta-670-NHS 840 690 1925
K9-4150 SeTau-647 920 693 >3000
K9-4149 920 695 3700
K9-4119 SeTau-665-NHS 900 716 >8500

 

 

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 on the Forster 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:

 

2P-Donor Acceptor Förster Radii
(Å)
2P-Excitation
Wavelength
[nm]
Emission Wavelengths [nm]
Donor Acceptor
71–76* 840 660 695
60–67* 840 660 695
60–68* 840 660 693
56–67* 840 660 780
78–85* 750–850 520 573
50–53* 750–850 520 571
41–45* 750–850 520
55–60* 900 675
72–76* 900 675 780

* Varies with Q.Y. of the donor

 

 

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