Far-red switching DNA probes for live cell nanoscopy

Herein we present DNA probes composed of Hoechst 33258 and spontaneously blinking far-red hydroxymethyl silicon-rhodamine (HMSiR). The best performing probe, 5-HMSiR-Hoechst, contains the 5′-regioisomer, shows ∼400-fold fluorescence increase upon DNA binding and is compatible with wash-free single molecule localization and 3D stimulated emission depletion microscopy of chromatin nanostructures in living cells

Enhancing the biocompatibility of rhodamine fluorescent probes by a neighbouring group effect

Fluorescence microscopy is an essential tool for understanding dynamic processes in living cells and organisms. However, many fluorescent probes for labelling cellular structures suffer from unspecific interactions and low cell permeability. Herein, we demonstrate that the neighbouring group effect which results from positioning an amide group next to a carboxyl group in the benzene ring of rhodamines dramatically increases cell permeability of the rhodamine-based probes through stabilizing a fluorophore in a hydrophobic spirolactone state. Based on this principle, we create probes targeting tubulin, actin and DNA. Their superb staining intensity, tuned toxicity and specificity allows long-term 3D confocal and STED nanoscopy with sub-30 nm resolution. Due to their unrestricted cell permeability and efficient accumulation on the target, the new probes produce high contrast images at low nanomolar concentrations. Superior performance is exemplified by resolving the real microtubule diameter of 23 nm and selective staining of the centrosome inside living cells for the first time

Synthesis, structure–property relationships and absorbance modulation of highly asymmetric photochromes with variable oxidation and substitution patterns

Asymmetric diarylethenes with benzo[b]thiophen-3-yl and 2-thienyl residues having variable oxidation degrees (S and/or SO2) remained unexplored. These photochromes provide reversibly photoswitchable absorbance and multicolor emission modulation. Here we report 18 photochromic 1,2-diarylperfluorocyclopentenes with oxidized and non-oxidized 2-methylbenzo[b]thiophen-3-yl, as well as 5-aryl-3-methylthiophen-2-yl groups. The structure–property relationships were studied for three groups of compounds: non-oxidized, mono-oxidized (to SO2 in the benzothiophene part), and fully-oxidized (to 2 × SO2). The quantum chemistry calculations helped to interpret the substituents’ effects in each group and predict the photophysical properties of yet unavailable photochromes. The photochromic systems with absorbance modulation introduced in this work were designed for the use in diffraction-unlimited writing and reading with light, nanopattering and optical lithography

Blinking fluorescent probes for tubulin nanoscopy in living and fixed cells

Here we report a small molecule tubulin probe for single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy and MINFLUX nanoscopy, which can be used in living and fixed cells. We explored a series of taxane derivatives containing spontaneously blinking far-red dye hydroxymethyl silicon–rhodamine (HMSiR) and found that the linker length profoundly affects the probe permeability and off-targeting in living cells. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and the 6′-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameter of ≤50 nm was routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin allows a complementary use of different nanoscopy techniques for investigating microtubule functions and developing imaging methods. For the first time, we resolved the inner microtubule diameter of 16 ± 5 nm by optical nanoscopy and thereby demonstrated the utility of a self-blinking dye for MINFLUX imaging

The use of Hoechst dyes for DNA staining and beyond.

Hoechst dyes are among the most popular fluorophores used to stain DNA in living and fixed cells. Moreover, their high affinity and specificity towards DNA make Hoechst dyes excellent targeting moieties, which can be conjugated to various other molecules in order to tether them to DNA. The recent developments in the fields of microscopy and flow cytometry have sparked interest in such composite molecules, whose applications range from investigating nucleus microenvironment to drug delivery into tumours. Here we provide an overview of the properties of Hoechst dyes and discuss recent developments in Hoechst-based composite probes

Application of STED imaging for chromatin studies

Chromatin is the information center of a cell. It comprises proteins and nucleic acids that form a highly complex and dynamic structure within the nucleus. Its multiple organization levels span from micrometre to nanometre scale. For many years, the lower levels of chromatin organization have been beyond the resolution limit of fluorescent microscopy, thus impeding research on nucleus architecture, transcription, translation and DNA repair. Recent development in super-resolution fluorescence microscopy enables us to more easily observe objects at the nanometre scale and allows the study of complex cellular structures at unprecedented detail. This review focuses on the application of stimulated emission depletion microscopy for imaging two main components of the chromatin-DNA and the proteins interacting with it

Rhodamine-Hoechst positional isomers for highly efficient staining of heterochromatin.

Hoechst conjugates to fluorescent dyes are popular DNA stains for live-cell imaging, but the relationship between their structure and performance remains elusive. This study of carboxyrhodamine–Hoechst 33258 conjugates reveals that a minimal change in the attachment point of the dye has dramatic effects on the properties of the final probe. All tested 6′-carboxyl dye-containing probes exhibited dual-mode binding to DNA and formed a dimmer complex at high DNA concentrations. The 5′-carboxyl dye-containing probes exhibited single-mode binding to DNA which translated into increased brightness and lower cytotoxicity. Up to 10-fold brighter nuclear staining by the newly developed probes allowed acquisition of stimulated emission depletion (STED) nanoscopy images of outstanding quality in living and fixed cells. Therefore we were able to estimate a diameter of ∼155 nm of the heterochromatin exclusion zones in the nuclear pore region in living cells and intact chicken erythrocytes and to localize telomeres relative to heterochromatin in living U-2 OS cells. Employing the highly efficient probes for two-color STED allowed visualization of DNA and tubulin structures in intact nucleated erythrocytes – a system where imaging is greatly hampered by high haemoglobin absorbance