Bioisostere-conjugated fluorescent probes for live-cell protein imaging without non-specific organelle accumulation.

Specific labeling of proteins using membrane-permeable fluorescent probes is a powerful technique for bioimaging. Cationic fluorescent dyes with high fluorescence quantum yield, photostability, and water solubility provide highly useful scaffolds for protein-labeling probes. However, cationic probes generally show undesired accumulation in organelles, which causes a false-positive signal in localization analysis.

No-wash fluorogenic labeling of proteins for reversible photoswitching in live cells.

Photoswitchable fluorescent molecules (PSFMs) are positioned as valuable tools for biomolecule localization tracking and super-resolution imaging technologies due to their unique ability to reversibly control fluorescence intensity upon light irradiation. Despite the high demand for PSFMs that are suitable for live-cell imaging, no general method has been reported that enables reversible fluorescence control on proteins of interest in living cells. Herein, we have established a platform to realize reversible fluorescence switching in living cells by adapting a protein labeling system.

Visualization of multiple localizations of GLUT4 by fluorescent probes of PYP-tag with designed unnatural warhead.

Within a cell, multiple copies of the same protein coexist in different pathways and behave differently. Being able to individually analyze the constant actions of proteins in a cell is crucial to know the pathways through which they pass and which physiological functions they are deeply involved in. However, until now, it has been difficult to distinguish protein copies with distinct translocation properties by fluorescence labeling with different colors in living cells.