Cover Picture: Masked Rhodamine Dyes of Five Principal Colors Revealed by Photolysis of a 2-Diazo-1-Indanone Caging Group: Synthesis, Photophysics, and Light Microscopy Applications (Chem. Eur. J. 41/2014)

• Published in: Chemistry : a European journal Vol. 20; no. 41; p. 13037
• Main Authors: Belov, Vladimir N., Mitronova, Gyuzel Yu, Bossi, Mariano L., Boyarskiy, Vadim P., Hebisch, Elke, Geisler, Claudia, Kolmakov, Kirill, Wurm, Christian A., Willig, Katrin I., Hell, Stefan W.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 06.10.2014; Wiley Subscription Services, Inc
• Subjects: bioconjugation; diazo compounds; fluorescence; photolysis; rhodamines
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.201490170

Caged rhodamine dyes (RhodaminesNN) of five basic colors were synthesized and used as "hidden" markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, either by irradiation with UV- or violet light (one-photon process), or by exposure to intense red light (λ750nm; two-photon mode). All dyes possess a very small 2-diazoketone caging group incorporated into the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a spiro-9H-xanthene fragment. Initially they are non-colored (pale yellow), non-fluorescent, and absorb at λ=330-350nm (molar extinction coefficient (ε)[asymptotically =]10(4) M(-1) cm(-1)) with a band edge that extends to about λ=440nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (λ=511-633 and 525-653nm, respectively). The unmasked dyes are highly colored and fluorescent (ε=3-8×10(4) M(-1) cm(-1) and fluorescence quantum yields ()=40-85% in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water-soluble caged red-emitting dye with two sulfonic acid residues was prepared. RhodaminesNN were decorated with amino-reactive N-hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photoactivated (uncaged) with λ=375-420nm light or intense red light (λ=775nm). Protein conjugates with optimal degrees of labeling (3-6) were prepared and uncaged with λ=405nm light in aqueous buffer solutions (=20-38%). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10-40% of the non-fluorescent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged dyes. Photoactivation of the individual molecules of RhodaminesNN (e.g., due to reversible or irreversible transition to a "dark" non-emitting state or photobleaching) provides multicolor images with subdiffractional optical resolution. The applicability of these novel caged fluorophores in super-resolution optical microscopy is exemplified.