Abberior^® Dyes for Super-Resolution Microscopy Applications

Microscopic methods in life sciences are of tremendous importance for visualization cellular and tissue structures. The development of new microscopy concepts has overcome the resolution barrier given by the diffraction limit, enabling a resolution limit down to about 10 nm. The visualization of cellular structures and molecular interactions can reveal new understanding in biological processes. Due to tremendous efforts in the development of super-resolved fluorescence microscopy, The Nobel Prize in Chemistry for 2014 was awarded to Eric Betzig, Stefan W. Hell, and to William E. Moerner.

These super-resolution microscopy principles are based on several technological approaches. Conventional light microscopy enables a resolution limit of about 250 nm in the x- and y- direction and 450 – 700 nm in the z –direction. Super-resolution techniques have overcome the resolution-limit (point-spread function) at least by a factor of 2. The resolution of super-resolution microscopy depends on the number of points that can be resolved on the structure of interest. Crucial for a successful super-resolution imaging is the choice of fluorescent probe. Brightness and high-contrast ratio between the states are important. In most super-resolution methods, the states of the probe must be controllable, reversible or irreversible, and switchable between a light or a dark state. Depending on the super-resolution method, further photo-physical criteria the probe must be fulfilled. Established techniques include the following:

• STED (Stimulated emission depletion)
• GSDIM (Ground State Depletion)
• PALM (Photoactivated localization microscopy)
• STORM (Stochastic optical reconstruction microscopy)
• RESOLFT (reversible saturable optical (flurorescence) transitions)

Figure 1. Three-color STED image of primary hippocampal neurons imaged with the Abberior^® Instruments Expert Line STED microscope. Note: The characteristic ~190 nm beta II spectrin periodicity along distal axons (green), which is only visible in the STED image. Labeled structures and dyes: beta II Spectrin (green, Abberior^® STAR580), Bassoon (red, Abberior^® STAR635P), Actin cytoskeleton (blue, Phalloidin, Oregon Green 488). Sample was prepared by Elisa D’Este at MPIBPC, Göttingen.

We offer the superior series of Abberior^® dyes that are especially designed and tested for super-resolution microscopy such as STED, RESOLFT, PALM, STORM, GSDIM and others. Abberior^® STAR, Abberior^® CAGE, Abberior^® FLIP, Abberior^® RSFP – the specific requirements of the super-resolution techniques are served with dedicated dye series. These Dyes are developed and produced by Abberior^® GmbH. Stefan Hell is it’s Co-founder.

Benefits

• Optimized for brightness and low background
• Optimized switching behavior being the key for super-resolution
• All markers are tested for different super-resolution methods
  • Abberior^® STAR for STED, confocal and epifluorescence imaging
  • Abberior^® CAGE & FLIP for PALM, STORM and GSDIM
• Abberior^® dyes are recommended by microscope vendors
• Proprietary, IP protected products
• Detailed characteristics of the dyes provided, e.g. optimal STED wavelength

Super-resolution microscopy is dependent on fluorescent labels. Manufactured by Abberior^®, the STAR, CAGE and FLIP dyes as well as RSFPs are exceptionally bright and photostable and provide optimized photoswitching for RESOLFT and PALM/STORM imaging. They are the only commercially available dyes that are tailored specifically to the needs of super-resolution microscopy.

Abberior^® dyes are also well suited for confocal microscopy, epifluorescence imaging and single molecule applications. Fluorescence applications that depend on a good signal-to-noise ratios and low background cam benefit from Abberior^® dyes.

Confocal and STED images. Two subunits of the nuclear pore complex were immunolabeled using antibodies against gp210 and antibodies with multiple specificities (PAN4/5) and secondary antibodies coupled to Abberior^® STAR580 and Abberior^® STAR635P. Note that gp210 is localized in an eight-fold symmetric structure at the rim of the nuclear pore complex. Imaged with the Abberior^® Instruments STEDYCON (compact line).

Dyes	Description	Absorption Maximum, λabs	Extinction Coefficient, ε	Fluorescence Maximum, λfl	Recommended STED	Cat No. NHS	Cat No. Maleimide
Abberior® CAGE 500	for single-molecule switching microscopy (e.g. PALM, STORM, GSDIM)	230, 299, 340 nm (non-activated, PBS, pH 7.4) 511 nm (photoactivated, PBS, pH 7.4)	50,000 M-1cm-1 (photoactivated, PBS, pH 7.4)	525 nm (PBS, pH 7.4)	595-615 nm	44254	92546
Abberior® CAGE 532	for single-molecule switching microscopy (e.g. PALM, STORM, GSDIM)	237, 302, 350 nm (non-activated, PBS, pH 7.4) 533 nm (photoactivated, PBS, pH 7.4)	82,000 M-1cm-1 (photoactivated, PBS, pH 7.4)	541 nm (PBS, pH 7.4)	610-640 nm		95705
Abberior® CAGE 552	for single-molecule switching microscopy (e.g. PALM, STORM, GSDIM)	231, 308, 350 nm (non-activated, PBS, pH 7.4) 552 nm (photoactivated, PBS, pH 7.4)	66,000 M-1cm-1 (photoactivated, PBS, pH 7.4)	574 nm (PBS, pH 7.4)	650-670 nm	94822	92545
Abberior® CAGE 590	for single-molecule switching microscopy (e.g. PALM, STORM, GSDIM)	262, 325, 351 nm (non-activated, PBS, pH 7.4) 595 nm (photoactivated, PBS, pH 7.4)	75,000 M-1cm-1 (photoactivated, PBS, pH 7.4)	615 nm (PBS, pH 7.4)	685-715 nm	77958	no
Abberior® FLIP 565	for single-molecule switching microscopy (e.g. PALM, STORM, GSDIM)	314 nm (closed form, PBS, pH 7.4) 566 nm (open form, PBS, pH 7.4)	47,000 M-1cm-1 (open form, PBS pH, 7.4)	580 nm (open form, PBS, pH 7.4)	-	79189	92544
Abberior® STAR 440SXP	for long Stokes STED and 2-color STED application	432 nm (PBS, pH 7.4)	33,000 M-1cm-1 (PBS, pH 7.4)	511 nm (PBS, pH 7.4)	590-620 nm	68221	38361
Abberior® STAR 470SXP	for long Stokes STED and 2-color STED application	467 nm (PBS, pH 7.4)	29,000 M-1cm-1 (PBS, pH 7.4)	598 nm (PBS, pH 7.4)	740-770 nm	94716	no
Abberior® STAR 488	for STED application	503 nm (PBS, pH 7.4)	65,000 M-1cm-1 (PBS, pH 7.4)	524 nm (PBS, pH 7.4)	585-605 nm	61048	no
Abberior® STAR 512	for STED application	511 nm (PBS, pH 7.4)	85,000 M-1cm-1 (PBS, pH 7.4)	530 nm (PBS, pH 7.4)	590-620 nm	38922	03004
Abberior® STAR 580	for STED application	587 nm (PBS, pH 7.4)	85,000 M-1cm-1 (PBS, pH 7.4)	607 nm (PBS, pH 7.4)	690-720 nm	38377	no
Abberior® STAR 635	for STED application	635 nm (PBS, pH 7.4)	110,000 M-1cm-1 (PBS, pH 7.4)	655 nm (PBS, pH 7.4)	740-770 nm	30558	96013
Abberior® STAR 635P	for STED application	638 nm (PBS, pH 7.4)	120,000 M-1cm-1 (PBS, pH 7.4)	651 nm (PBS, pH 7.4)	740-770 nm	07679	no

For North America, we also provide a series of antibody labeled Abberior^® conjugates.

Cat No.	Description	Availability	Pack size
30483	Anti-Mouse IgG-Abberior® CAGE 635 antibody produced in goat	US only	500 µg
54287	Anti-Rabbit IgG-Abberior® CAGE 590 antibody produced in goat	US only	500 µg
41155	Anti-Rabbit IgG-Abberior® CAGE 500 antibody produced in goat	US only	500 µg
52283	Anti-Mouse IgG-Abberior® STAR  RED antibody produced in goat	US only	500 µg
53399	Anti-Rabbit IgG-Abberior® STAR 635P antibody produced in goat	US only	500 µg
41699	Anti-Rabbit IgG-Abberior® STAR RED antibody produced in goat	US only	500 µg
41348	Anti-Rabbit IgG-Abberior® STAR 635 antibody produced in goat	US only	500 µg
53654	Anti-Rabbit IgG-Abberior® STAR 600 antibody produced in goat	US only	500 µg
41367	Anti-Rabbit IgG-Abberior® STAR 580 antibody produced in goat	US only	500 µg
52403	Anti-Mouse IgG-Abberior® STAR 580 antibody produced in goat	US only	500 µg
00289	Anti-Rabbit IgG-Abberior® STAR 512 antibody produced in goat	US only	500 µg
52944	Anti-Rabbit IgG-Abberior® STAR 488 antibody produced in goat	US only	500 µg
53366	Anti-Mouse IgG-Abberior® STAR 488 antibody produced in goat	US only	500 µg

References

1. Leica Microsystems recommendations for 2-color applications.

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15. K. Kolmakov, et al. Red-Emitting Rhodamines with Hydroxylated, Sulfonated, and Phosphorylated Dye Residues and Their Use in Fluorescence Nanoscopy" Chem. Eur. J. 2012, 18, 12986 –12998 (2013).

16. C. A. Wurm, et al. Novel red fluorophores with superior performance in STED microscopy, Optical Nanoscopy 2012; DOI: 10.1186/ 2192-2853-1-7 (2013).

17. S. Rocha, H. et al. Photoswitchable Fluorescent Proteins for Superresolution Fluorescence Microscopy Circumventing the Diffraction Limit of Light in Fluorescence Spectroscopy and Microscopy (Eds.: Y. Engelborghs, A. J. W. G. Visser), Humana Press, Vol. pp. 793-812 (2013).

18. J. V. Chacko, et al. Probing Cytoskeletal Structures by Coupling Optical Superresolution and AFM Techniques for a Correlative Approach. Cytoskeleton, 70(11), pp. 729–740, DOI: 10.1002/cm.21139 (2013).

19. I.-H. Wang, et al. Tracking Viral Genomes in Host Cells and Single Molecule Resolution. Cell Host Microbe, 14(4), pp. 468–480, DOI: 10.1016/j.chom.2013.09.004 (2013).

20. H. Schill, et al. 4-Trifluoromethyl-Substituted Coumarins with Large Stokes Shifts: Synthesis, Bioconjugates, and Their Use in Super-Resolution Fluorescence Microscopy. Chem. Eur. J., 19(49), pp. 16556–16565, DOI: 10.1002/chem.201302037 (2013).

21. Y. Wang, et al. Time-Gated Stimulated Emission Depletion Nanoscopy. Opt. Eng., 52(9), 093107, DOI: 10.1117/1.OE.52.9.093107 (2013).

22. M. V. Sednev, et al. Carborhodol: A New Hybrid Fluorophore Obtained by Combination of Fluorescein and Carbopyronine Dye Cores. Bioconjugate Chem., 24(4), pp 690–700, DOI: 10.1021/bc3006732 (2013).

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