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.

Excellent live-cell staining and nanoscopy imaging with rhodamine 4′-isomer probes boosted by a neighboring group effect.

The development of live-cell fluorescence nanoscopy is powered by the availability of suitable fluorescent probes. Rhodamines are among the best fluorophores for labeling intracellular structures. Isomeric tuning is a powerful method for optimizing the biocompatibility of rhodamine-containing probes without affecting their spectral properties. An efficient synthesis pathway for 4-carboxyrhodamines is still lacking. We present a facile protecting-group-free 4-carboxyrhodamines’ synthesis based on the nucleophilic addition of lithium dicarboxybenzenide to the corresponding xanthone. This approach drastically reduces the number of synthesis steps, expands the achievable structural diversity, increases overall yields and permits gram-scale synthesis of the dyes. We synthesize a wide range of symmetrical and unsymmetrical 4-carboxyrhodamines covering the whole visible spectrum and target them to multiple structures in living cells – microtubules, DNA, actin, mitochondria, lysosomes, Halo-tagged and SNAP-tagged proteins. The enhanced permeability fluorescent probes operate at submicromolar concentrations, allowing high-contrast STED and confocal microscopy of living cells and tissues.

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.

Fluorescent actin probes made of 6′-carbopyronines and jasplakinolide are insensitive to efflux pumps and stain F- and G-actin efficiently in living cells, allowing high quality 2D and 3D nanoscopy of dynamic actin structures.

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.