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	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
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
K8-3002 K8-3002 ^new	Seta-470-NHS Seta-470-NHS	NH₂		•	•	•									water	469	50,000	521	1.9
K8-3335 K8-3335	Seta-555-NHS Seta-555-NHS	NH₂					•								PB 7.4	555	155,000	570	7
K8-3345 K8-3345	Seta-555-DBCO Seta-555-DBCO	N₃					•								PB 7.4	555	155,000	570	7
K8-3346 K8-3346	Seta-555-Azide Seta-555-Azide	triple-CC					•								PB 7.4	555	155,000	570	7
K8-5035 K8-5035	Seta-650-NHS Seta-650-NHS	NH₂							•	•					PB 7.4	651	200,000	671	28
K8-5036 K8-5036	Seta-650-Maleimide Seta-650-Maleimide	SH							•	•					PB 7.4	652	200,000	672	28
K8-5045 K8-5045	Seta-650-DBCO Seta-650-DBCO	N₃							•	•					PB 7.4	653	200,000	674	28
K8-5046 K8-5046	Seta-650-Azide Seta-650-Azide	triple-CC							•	•					PB 7.4	651	200,000	671	28
K9-4119 K9-4119 ^new	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
K8-1388 K8-1388	Seta-700-di-NHS Seta-700-di-NHS	NH₂	•	•						•	•	•			PB 7.4	688	180,000	704	11
K4-205 K4-205 ^new	Seta-375-NHS Seta-375-NHS	NH₂	•												PB 7.4	372	24,500	476	86	3.1
K4-225 K4-225 ^new	Seta-385-NHS Seta-385-NHS	NH₂	•												PB 7.4	384	25,000	512	78
K4-215 K4-215 ^new	Seta-400-NHS Seta-400-NHS	NH₂	•	•											PB 7.4	399	24,000	576	34
K9-4179 K9-4179 ^new	SeTau-680-NHS SeTau-680-NHS	NH₂							•	•	•				PB 7.4	683	295,000	705	58	2.9
K9-4154 K9-4154 ^new	SeTau-647-DBCO SeTau-647-DBCO	N₃							•	•					PB 7.4	649	200,000	690	60	3.2

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^–1cm^–1) and an emission maximum at 690 nm. It is excitable with the 405 nm diode laser (ε [M^–1cm^–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 2^nd short wavelength absorption around 395 nm (ε [M^–1cm^–1]: 31,000) and its emission maximum at 713 nm. It is excitable with the 405 nm diode laser (ε [M^–1cm^–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.

Also check out our other extremely bright and photostable squaraine rotaxane products.

Conventional Image

STED Image

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

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