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 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	13,800	518	80	9.3
K7-567 K7-567	SeTau-405-Azide SeTau-405-Azide	triple-CC	•	•											PB 7.4	405	13,800	518	80	9.3
K8-1342 K8-1342	Seta-670-NHS Seta-670-NHS	NH2	•	•					•	•					PB 7.4	667	180,000	688	7	0.42
K7-545 K7-545	SeTau-425-NHS SeTau-425-NHS	NH2	•	•	•										PB 7.4	425	4,200	545	39	26.2
K8-1346 K8-1346	Seta-670-Azide Seta-670-Azide	triple-CC	•	•					•	•					PB 7.4	667	180,000	686	7
K8-1351 K8-1351	Square-660-Carboxy Square-660-Carboxy		•	•					•	•					PB 7.4	657	182,000	676	3	0.27
K8-1352 K8-1352	Square-660-NHS Square-660-NHS	NH2	•	•					•	•					PB 7.4	657	182,000	676	3	0.27
K8-1355 K8-1355	Square-680-Carboxy Square-680-Carboxy		•	•					•	•	•				PB 7.4	654	138,000	670	1
K8-1357 K8-1357	Square-680-NHS Square-680-NHS	NH2	•	•					•	•					MeOH	668	208,000	690	10
K8-1384 K8-1384	Seta-700-NHS Seta-700-NHS	NH2	•	•						•	•	•			PB 7.4	687	177,000	703	6
K8-1407 K8-1407	Square-650-pH-NHS Square-650-pH-NHS	NH2						•	•	•					PB 5.6	653	135,000	671	16	1.17
K8-1641 K8-1641	Seta-632-Maleimide Seta-632-Maleimide	SH							•						PB 7.4	633	270,000	642	5
K8-1642 K8-1642	Seta-632-NHS Seta-632-NHS	NH2							•						PB 7.4	632	280,000	641	6
K8-1663 K8-1663	Seta-633-NHS Seta-633-NHS	NH2						•	•						PB 7.4	633	250,000	644	7	0.25
K8-1672 K8-1672	Seta-646-NHS Seta-646-NHS	NH2							•						PB 7.4	646	207,000	656	10	0.38
K8-1682 K8-1682	Seta-660-di-NHS Seta-660-di-NHS	NH2							•	•					PB 7.4	661	220,000	672	11	0.9
K8-1696 K8-1696	Seta-633-Azide Seta-633-Azide	triple-CC						•	•						PB 7.4	633	250,000	644	7
K9-4119 K9-4119	SeTau-665-NHS SeTau-665-NHS	NH2								•	•	•			PB 7.4	664	160,000	712	53	3.1
K9-4142 K9-4142	SeTau-647-di-NHS SeTau-647-di-NHS	NH2							•	•					PB 7.4	650	200,000	694	65	3.2
K9-4145 K9-4145	SeTau-633-Ethyl-Ester SeTau-633-Ethyl-Ester								•	•					CHCl3	634	105,000	683	68
K9-4148 K9-4148	SeTau-647-Maleimide SeTau-647-Maleimide	SH							•	•					PB 7.4	648	200,000	692	45	3.2
K9-4149 K9-4149	SeTau-647-NHS SeTau-647-NHS	NH2							•	•					PB 7.4	649	200,000	695	61	3.2
K9-4150 K9-4150	SeTau-647 SeTau-647								•	•					PB 7.4	647	211,000	693	59	3.1
K9-4169 K9-4169	SeTau-670-NHS SeTau-670-NHS	NH2							•	•	•				PB 7.4	673	275,000	694	36	1.6
K8-1388 K8-1388	Seta-700-di-NHS Seta-700-di-NHS	NH2	•	•						•	•	•			PB 7.4	688	180,000	704	11
K8-1341 K8-1341	Seta-670-Maleimide Seta-670-Maleimide	SH	•	•					•	•					PB 7.4	667	180,000	688	7
K8-1671 K8-1671	Seta-646-Maleimide Seta-646-Maleimide	SH							•						PB 7.4	647	210,000	657	8
K8-1405 K8-1405	Square-650-pH-Carboxy Square-650-pH-Carboxy	NH2						•	•	•					PB 5.6	653	135,000	671	16	1.17
K8-1389 K8-1389	Seta-700-NHS Seta-700-NHS	NH2		•						•	•	•			PB 7.4	688	162,000	703	14
K9-4179 K9-4179	SeTau-680-NHS SeTau-680-NHS	NH2							•	•	•				PB 7.4	683	295,000	705	58	2.9
K9-4154 K9-4154	SeTau-647-DBCO SeTau-647-DBCO	N3							•	•					PB 7.4	649	200,000	690	60	3.2
K9-3152 K9-3152	SeTau-488-NHS SeTau-488-NHS	NH2				•									PBS 7.4	486	59,000	532	27
K9-4159 K9-4159 new	SeTau-660-NHS SeTau-660-NHS	NH2							•	•					PB 7.4	663	240,000	694	50	3.36

SETA BioMedicals has developed several green, red and NIR emitting dyes (probes and labels) with very high 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 extremely 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.

 

While the 2PACSs of common fluorephores 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 the 2PACS 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. 2012; 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 [24,42,43,44,45].

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
K7-547	SeTau-405-NHS	750–850	518	>100
K8-1672	Seta-646-NHS	840	660	480
K8-1384	Seta-700-NHS	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	SeTau-647-NHS	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
Seta-646 (K8-1672)	SeTau-647 (K9-4142)	71–76*	840	660	695
Seta-646 (K8-1672)	SeTau-647 (K9-4149)	60–67*	840	660	695
Seta-646 (K8-1672)	Seta-670 (K8-1342)	60–68*	840	660	693
Seta-646 (K8-1672)	Seta-750 (K8-7522)	56–67*	840	660	780
SeTau-405 (K7-547)	R-PE (PE-5mg)	78–85*	750–850	520	573
SeTau-405 (K7-547)	Seta-555 (K8-3335)	50–53*	750–850	520	571
SeTau-405 (K7-547)	SQ-565 (K8-1902)	41–45*	750–850	520	–
Seta-660 (K8-1682)	SQ-740 (K8-1649)	55–60*	900	675	–
Seta-660 (K8-1682)	Seta-750 (K8-7522)	72–76*	900	675	780

* Varies with Q.Y. of the donor