Captivated: in the cage, in the cell

Max Planck scientists invent and apply novel class of masked fluorescent dyes

12. April 2010

The novel caged rhodamines may be used in microscopy either as single labels, or in combination with conventional fluorescent dyes and photoswitchable rhodamine spiroamides. For example, a set consisting of Rhodamine NN, photochromic spiroamide of "Rhodamine S" and conventional (uncaged) N,N,N',N'-Tetramethylrhodamine enabled the stepwise activation and detection of several fluorescent markers using only one excitation source and one detection channel. Based on these three fluorophores with very similar absorption and emission spectra, a monochromatic multi-label imaging scheme with low cross-talk was realized (Figure 2a). Moreover, the new caged dyes may be used in optical nanoscopy, e. g. for sub-diffraction imaging by single molecule switching (Figure 2b).

Figure 2. Application of the new caged compounds. (a) Three fluorophores with very similar absorption and emission spectra but different switching characteristics allow new multi-label and monochromatic imaging schemes. For this purpose, the actin cytoskeleton was labeled with conventional TMR fluorescent dye; the peroyxsomes were immunolabeled with caged TMR-NN and the tubulin cytoskeleton was immunostained with switchable Rhodamine S. The different structures can be distinguished and imaged by stepwise activation and bleaching of the individual fluorophores. (b) The new caged fluorescent dyes may be used in optical nanoscopy, e. g. in sub-diffraction imaging with single molecule switching. A conventional microscopy image of the tubulin cytoskeleton and its sub-diffraction representation are shown.

In summary, the novel caging procedure can be applied to a wide variety of dyes (rhodamines, carbopyronines and probably other cationic dyes, e. g. triarylmethyl derivatives). Thus, the present approach can be extended to a larger set of (fluorescent) dyes and excitation sources.

Moreover, the compact structure and the very small size of the caging group facilitate the ability of Rhodamines NN to cross the membranes of living cells (see Figure 1b). In combination with the site specific labeling protocols, as realized for the Halo- and SNAP-tags, the availability of the new cell-permeable and caged Rhodamines NN may extend the set of live cell labelling strategies based on binding the genetically encoded protein tags with organic fluorophores. The spatially restricted photoactivation of Rhodamines NN followed by tracking of the uncaged molecules will enable the measurements of molecular dynamics (e. g. diffusion parameters or flow velocities). The combination of Rhodamine NN with nanoscopic techniques (e.g. STED or single molecule switching followed by localization) is expected to provide additional information on subcellular structures. Moreover, measurements of the fluorescence resonance energy transfer (FRET) signal of the newly uncaged fluorescent areas located in the close proximity to the "permanent" colored regions are likely to reveal additional structural details for the relatively short distances of 1-10 nm. (vb/cw)

Original publications

[1] V. N. Belov, C. Wurm, V. P. Boyarskiy, S. Jakobs, and S. W. Hell (2010): Rhodamines NN: a novel class of caged fluorescent dyes. Angew. Chem. Int. Ed. 122 | DOI: 10.1002/ange.201000150

[2] S. W. Hell, V. N. Belov, V. P. Boyarskiy, C. A. Wurm, S. Jakobs, and C. Geisler (Max-Planck-Innovation GmbH): PCT/EP/2009/006578 (10.09.2009).

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