# Cell wash-free fluorescent probes based on phenothiazine and phenoxazine with high photostability and large stokes shifts for targeted imaging of subcellular organelles

## TL;DR

This paper introduces new fluorescent probes that allow real-time imaging of cell organelles without washing, reducing cellular damage and enabling continuous observation.

## Contribution

The development of wash-free fluorescent probes based on phenothiazine/phenoxazine with high photostability and large Stokes shifts for multi-organelle imaging.

## Key findings

- Nitro-modified probes show strong polarity sensitivity and large Stokes shifts (up to 191 nm).
- Probes enable real-time, wash-free imaging of multiple organelles in living cells.
- PXZ-Lipid probe successfully monitors dynamic lipid droplet changes in real time.

## Abstract

Most organelle-targeting probes require the removal of excess dye to enhance the signal-to-noise ratio before microscopic imaging experiments. However, this washing step may cause cellular damage and interfere with the continuous observation of cellular activities. Here, we report a series of wash-free probes based on small molecule phenothiazine/phenoxazine. Simple modification of phenothiazine/phenoxazine by nitro group significantly enhances the polarity-sensitive characteristic, which are well-suited for wash-free cellular imaging. These probes feature low molecular weight, excellent photostability, and large Stokes shifts (up to 191 nm). By conjugation with targeting groups, a series of probes are developed for specific imaging in different organelles such as lysosomes, mitochondria, the endoplasmic reticulum, lipid droplets, and the plasma membrane, without any washing step. Moreover, by simply replacing S atom in the central core with O atom, the emission of probes shifts from red to green-yellow. Using these two probes, high-contrast, dual-color wash-free imaging can be achieved. Among them, the PXZ-Lipid probe was successfully applied for real-time monitoring of dynamic changes in lipid droplets within living cells. This work establishes a general strategy for designing small-molecule, wash-free fluorescent probes based on phenothiazine/phenoxazine scaffolds, enabling real-time, multi-organelle imaging with minimal cellular disturbance.

Nitro-modified phenothiazine/phenoxazine small-molecule fluorescent probes exhibit strong polarity sensitivity, high photostability, large Stokes shifts (up to 191 nm), and low molecular weight. Conjugation with targeting groups enables wash-free, real-time imaging of multiple organelles (lysosomes, mitochondria, ER, lipid droplets, plasma membrane) in living cells. The PXZ-Lipid probe allows dynamic monitoring of lipid droplet changes, providing a general strategy for minimally invasive multi-organelle visualization.Image 1

•The probes achieve a pronounced environment-sensitive response through simple modification with the nitro group•Large Stokes shifts (up to 191 nm) and excellent photostability.•Specific targeting of lysosomes, mitochondria, ER, lipid droplets, and membrane.•Real-time imaging of lipid droplet dynamics in living cells without washing.

The probes achieve a pronounced environment-sensitive response through simple modification with the nitro group

Large Stokes shifts (up to 191 nm) and excellent photostability.

Specific targeting of lysosomes, mitochondria, ER, lipid droplets, and membrane.

Real-time imaging of lipid droplet dynamics in living cells without washing.