Biology subjects

Kiszka, K. A.

Publications and source records attributed to Kiszka, K. A..

4 recordsLinked to original sources

bioRxiv2025

Regioisomer-controlled red-shifted DNA probes for imaging of living tissues

Fluorescence imaging of chromatin DNA with high resolution and specificity is key to understanding cellular processes and enabling molecular diagnostics. However, choosing the best DNA probe for in vivo imaging is often a challenging task, as systematic studies investigating the biocompatibility of these molecules are lacking. Red-shifted fluorescent probes are particularly advantageous for imaging thick tissues, as light scattering decreases at red and far-red wavelengths. We synthesized and characterized the 4', 5', and 6' carboxyrhodamine regioisomers of SiR-Hoechst and 610CP-Hoechst DNA probes and assessed their performance in mice following intravenous injection. All variants entered tissues and produced stable staining that persisted for several hours ex vivo. In particular, 5' regioisomers show the highest performance, yielding bright and specific nuclear labeling. Renal clearance of the probes was nearly complete within 24 h, as indicated by fluorescence analysis of urine samples. Owing to their low cytotoxicity, high specificity, and favourable photophysical properties, these probes enabled high-quality confocal, two-photon, and STED microscopy in liver, kidney, lung, and heart tissue. Our findings extend the application of rhodamine-based DNA probes to in vivo imaging highlight their potential for deep-tissue imaging in live animals.

biochemistry↗

bioRxiv2024

MINFLUX fluorescence nanoscopy in biological tissue

Optical imaging access to nanometer-level protein distributions in intact tissue is a highly sought-after goal, as it would provide visualization in physiologically relevant contexts. Under the unfavorable signal-to-background conditions of increased absorption and scattering of the excitation and fluorescence light in the complex tissue medium, super-resolution fluorescence microscopy methods are severely challenged in attaining precise localization of molecules. We reasoned that the typical use of a confocal detection pinhole in MINFLUX nanoscopy, suppressing background and providing optical sectioning, should facilitate the detection and resolution of single fluorophores even amid scattering and optically challenging tissue environments. Here, we investigated the performance of MINFLUX imaging for different synaptic targets and fluorescent labels in tissue sections of the mouse brain. Single fluorophores were localized with a precision of < 5 nm at up to 80 {micro}m sample depth. MINFLUX imaging in two color channels allowed to probe PSD95 localization relative to the spine head morphology, while also visualizing presynaptic VGlut clustering and AMPA receptor clustering at the post-synapse. Our two-dimensional (2D) and three-dimensional (3D) two-color MINFLUX results in tissue, with < 10 nm 3D fluorophore localization, open up new avenues to investigate protein distributions on the single-synapse level in fixed and living brain slices.

biophysics↗

bioRxiv2024

Autonomous bioluminescence emission from transgenic mice

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.

bioengineering↗

bioRxiv2021

Blinking fluorescent probes for tubulin nanoscopy in living and fixed cells

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.

cell biology↗