Biology subjects
Publications and source records attributed to Koenen, T..
The bacterial bioluminescence system can be applied to produce autonomous bioluminescence in mammalian cells. Until now, the system has only been stably inserted into cells cultured in vitro. Here we report the generation of an autobioluminescent transgenic mouse line constitutively expressing the genes of the bacterial bioluminescence system, enabling substrate-free in vivo luminescence imaging of a living mammal.
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 biocompatibility of the rhodamine-containing probes without affecting their spectral properties. However, the efficient synthesis pathway for rhodamine 4-isomers is still lacking. Herein, we present a facile protecting-group-free 4-carboxyrhodamines synthesis based on nucleophilic addition of lithium dicarboxybenzenide to the corresponding xanthone. This approach drastically reduces the number of synthesis steps and expands the achievable structural diversity, increases overall yields and permits a cheap gram-scale synthesis of the dyes. We prepared a wide range of symmetric and asymmetric 4-carboxyrhodamines covering the whole visible spectrum and targeted 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 probe for single molecule localisation microscopy (SMLM) of tubulin in living and fixed cells. We explored a series of constructs composed of taxanes and spontaneously blinking far-red dye hydroxymethyl silicon-rhodamine (HMSiR). We found that the linker length profoundly affects the probe permeability and off-targeting. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and 6-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameters of [≤]50 nm can be routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin also performs well in 3D stimulated emission depletion (STED) microscopy, allowing a complementary use of both nanoscopy methods for investigating microtubule functions in living cells.