Long-Chain Fluorescent Probe for Straightforward and Nondestructive Staining Mitochondria in Fixed Cells and Tissues.

Normally, small-molecule fluorescent probes dependent on the mitochondrial membrane potential (MMP) are invalid for fixed cells and tissues, which limits their clinical applications when the fixation of pathological specimens is imperative. Given that mitochondrial morphology is closely associated with disease, we developed a long-chain mitochondrial probe for fixed cells and tissues, DMPQ-12, by installing a C12-alkyl chain into the quinoline moiety. In fixed cells stained with DMPQ-12, filament mitochondria and folded cristae were observed with confocal and structural illumination microscopy, respectively. In titration test with three major phospholipids, DMPQ-12 exhibited a stronger binding force to mitochondria-exclusive cardiolipin, revealing its targeting mechanism. Moreover, mitochondrial morphological changes in the three lesion models were clearly visualized in fixed cells. Finally, by DMPQ-12, three kinds of mitochondria with different morphologies were observed in situ in fixed muscle tissues. This work breaks the conventional concept that organic fluorescent probes only stain mitochondria with normal membrane potentials and opens new avenues for comprehensive mitochondrial investigations in research and clinical settings.

Ratiometric and Discriminative Visualization of Autophagy and Apoptosis with a Single Fluorescent Probe Based on the Aggregation/Monomer Principle.

Autophagy and apoptosis play a central role in maintaining homeostasis in mammals. Therefore, discriminative visualization of the two cellular processes is an important and challenging task. However, fluorescent probes enabling ratiometric visualization of both autophagy and apoptosis with different sets of fluorescence signals have not been developed yet. In this work, we constructed a versatile single fluorescent probe (NKLR) based on the aggregation/monomer principle for the ratiometric and discriminative visualization of autophagy and apoptosis. NKLR can simultaneously perform two-color imaging of RNA (deep red channel) and lysosomes (yellow channel) in aggregation and monomer states, respectively. During autophagy, NKLR migrated from cytoplasmic RNA and nuclear RNA to lysosomes, showing enhanced yellow emission and sharply decreased deep red fluorescence. Moreover, this migration process was reversible upon the recovery of autophagy. Comparatively, during apoptosis, NKLR immigrated from lysosomes to RNA, and the yellow emission decreased and even disappeared, while the fluorescence of the deep red channel slightly increased. Overall, autophagy and apoptosis could be discriminatively visualized via the fluorescence intensity ratios of the two channels. Meanwhile, the cells in three different states (healthy, autophagic, apoptotic) could be distinguished by three point-to-point fluorescence images via the localization and emission color of NKLR. Therefore, the probe NKLR can serve as a desirable molecular tool to reveal the in-depth relation between autophagy and apoptosis and facilitate the study on the two cellular processes.

Construction of fluorescent rotors with multiple intramolecular rotation sites for visualization of cellular viscous compartments with elevated fidelity.

Viscosity-sensitive fluorescent dyes are widely utilized to image viscous intracellular compartments with high fidelity. However, the sensitivity of many fluorescent rotors needs improvement for bioimaging applications. Herein, we proposed to construct a fluorescent rotor with multiple intramolecular rotation sites to elevate its sensitivity to environmental viscosity. The fabricated fluorescent rotor showed evidently increased sensitivity to viscosity and had the potential to image intracellular viscous compartments with improved fidelity. By decorating the rotor with sidechains of different lengths, we successfully fabricated two fluorescent probes, TAPI-6 and TAPI-16, to visualize the mitochondria and plasma membrane, respectively, with high fidelity. The two probes were also successfully utilized to clearly visualize the mitochondria and plasma membranes in skeletal muscle tissue, cardiac muscle tissue, and liver tissue, demonstrating the potential of the fluorescent rotor for bioimaging applications. We believe that the strategy of increasing the sensitivity to viscosity using multiple rotation sites is valuable for the construction of fluorescent rotors, and the presented fluorescent probes in this work can serve as powerful tools for biological research.

Fabrication of small-structure red-emissive fluorescent probes for plasma membrane enables quantification of nuclear to cytoplasmic ratio in live cells and tissues.

Nuclear to cytoplasmic ratio is one of the vital parameters in diagnosis of cancer by means of hematoxylin-eosin (HE) stained histopathology. However, HE histopathology dependent on mechanical tissue slice damages biosamples and exhibits insufficient accuracy. Herein, we rationally prepared two small-molecule plasma membrane fluorescent probes with red-emitting fluorescence for visualizing plasma membrane in living cells and tissues. Their fluorescence intensities are strongly affected by environmental viscosity, which enables the exclusive imaging of plasma membrane in high fidelity. The probes can visualize plasma membrane in SiHa and rat blood red cells. Particularly, the probes are able to visualize T-tubule (transverse tubule) in skeletal muscle tissues successfully, suggesting their ability to image plasma membrane in tissues. In cooperation with Hoechst 33342, the nuclear to cytoplasmic ratio was successfully qualified in live cells and tissues. We believe these probes may have potential applications in facilitating the study on histopathology and the related areas.