# Photoactivatable Carborhodol and Carborhodamine Dyes with One Cleavable Group: Synthesis, Spectra, and Fluorescence Nanoscopy Applications

## TL;DR

Scientists developed new fluorescent dyes with a single caging group for better solubility and faster photoactivation in super-resolution microscopy.

## Contribution

The introduction of monocaged photoactivatable dyes with one cleavable group improves biocompatibility and simplifies activation.

## Key findings

- Monocaged dyes have lower molecular mass and improved solubility compared to traditional dyes with two caging groups.
- Probes with COOH groups allow for irreversible single-step photoactivation emitting yellow or orange light.
- Carborhodol showed reversible emission changes at pH > 7, useful for orthogonal activation.

## Abstract

Photoactivatable
(PA) dyes with symmetric structures
and two caging
groups, rhodamines and carbo- and silicon-rhodamines, have been widely
applied in super-resolution microscopy of subcellular structures with
optical resolution far below the diffraction limit. The presence of
two “heavy” caging groups reduces the solubility of
a probe and, eventually, makes it less biocompatible. The photocleavage
in two steps prolongs the photolysis time required for complete PA;
it may cause excessive bleaching and secondary photoreactions. Here,
we introduce “monocaged” PA fluorescent dyes based on
xanthene cores with two heteroatoms (N, O, or N, N). In contrast to
standard approaches, we protected only one heteroatom (either N or
O) with a photocleavable (4,5-dimethoxy-2-nitrobenzyl) group and demonstrate
that it is sufficient to mask the fluorescence of carbo-rhodol and
carbo-rhodamine dyes. The monocaged PA probes have significantly lower
molecular masses than their analogs with two bulky caging groups.
The probes with a reactive group (COOH) provide facile labeling and
undergo irreversible single-step photoactivation toward products emitting
yellow or orange light. For carborhodol with a free hydroxyl and the
protected N-methyl group, the reversible increase
in emission was found at pH > 7 (as an activation tool, orthogonal
to photolysis). Carboxamides incorporating the HaloTag amine (O2)
ligand were applied for targeting and imaging of the HaloTag self-labeling
enzyme fused with a protein of interest (vimentin). The utility and
imaging performance of the probes with two heteroatoms belonging to
a fluorophore, but only one caging group, were demonstrated in live
cell labeling, conventional (confocal) microscopy, Minimal Photon
Fluxes (MINFLUX) nanoscopy, and single-molecule localization methods
(SMLM).