Photoactivation of silicon rhodamines via a light-induced protonation

Abstract

We present a new type of photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent isomerization followed by protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired sideproducts released. Using this photoactivatable fluorophore, we created probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore's outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

“…Furthermore, fixed U-2 OS cells which stably express HaloTag fused to the microtubule (MT) binding protein Cep41 were imaged with 26. 51 The resulting images showed fine structures of tubulin with a full-width-half-maximum (FWHM) of 40.7 nm ± 5.7 nm, which is in agreement with previous reports on MT diameter measurements (Figures 5b, c and Supplementary Figure 23). [51][52] These observations corroborate the applicability of 26 in SMLM and its spontaneous blinking in standard phosphate-buffered saline (pH 7.4) without the need for low wavelength irradiation and additives (Supplementary Movies 1 and 2).…”

“…However, both results lie within the range of previously reported values. [51][52] Thus, our synthetic approach enabled us to develop 26 and 33, two spectrally distinct and spontaneously blinking probes for SMLM.…”

Targeted Photoactivatable Green-Emitting BODIPY Based on Directed Photooxidation Induced Activation and its Application to Live Dynamic Super-Resolution Microscopy

“…The usefulness of fluorescent probes in bioimaging is no longer to be proven and has considerably contributed to the advancement of knowledge in biology. Among fluorescent probes, those having the ability to irreversibly turn on their fluorescence upon photoirradiation, also called photoactivatable fluorescent probes, [1], [2], [3] mainly found applications in single molecule tracking, [4], [5] and super resolution microscopy based on Photoactivated Localization Microscopy (PALM). [6], [7], [8], [9] In the field, photoactivatable fluorescent proteins are the most used in the bioimaging community, as they can be fused to proteins of interest.…”

“…In the light of these disadvantages, small molecular probes have emerged as appealing complementary alternatives. Consequently, several photoactivation approaches have been proposed, including: photoelimination of nitrogen (N2), [3],, [12] photoinduced disconnection of a spiro-oxazine, [8] photo-uncaging of a fluorescence quencher, [13], [14], [15], [16] nitrosamine caging groups, [17] diazoketone-caging, [18] light-induced protonation of rhodamine, [4] as well as radical reaction on xanthone dyes. [19], [20] Interestingly, photoactivatable dyes based on photooxidation mediated by 1 O2 led to general and efficient approaches to develop visible-light-activated probes.…”