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.

Efflux pump insensitive rhodamine-jasplakinolide conjugates for G- and F-actin imaging in living cells.

The actin cytoskeleton is crucial for endocytosis, intracellular trafficking, cell shape maintenance and a wide range of other cellular functions. Recently introduced cell-permeable fluorescent actin probes, such as SiR-actin, suffer from poor membrane permeability and stain some cell populations inhomogeneously due to the active efflux by the plasma membrane pumps. We analyzed a series of new probes composed of jasplakinolide and modified rhodamine fluorophores and found that rhodamine positional isomerism has a profound effect on probe performance. The probes based on the 6'-carboxy-carbopyronine scaffold are considerably less susceptible to efflux and allow efficient staining without efflux pump inhibitors. They can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity. We show that jasplakinolide-based fluorescent probes bind not only to actin filaments, but also to G-actin, which enables imaging highly dynamic actin structures. We demonstrate an excellent performance of the new probes in multiple organisms and cell types: human cell lines, frog erythrocytes, fruit fly tissues and primary neurons.

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.

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system.

Bioluminescence-based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has, so far, not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterial luxCDABE and frp genes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells.

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.