Live-cell fluorescence nanoscopy is a powerful tool to study cellular biology on a molecular scale, yet its use is held back by the paucity of suitable fluorescent probes. Fluorescent probes based on regular fluorophores usually suffer from low cell permeability and unspecific background signal. We report a general strategy to transform regular fluorophores into fluorogenic probes with excellent cell permeability and low unspecific background signal. The strategy is based on the conversion of a carboxyl group found in rhodamines and related fluorophores into an electron-deficient amide. This conversion does not affect the spectroscopic properties of the fluorophore but permits it to exist in a dynamic equilibrium between two different forms: a fluorescent zwitterion and a non-fluorescent, cell permeable spirolactam. Probes based on such fluorophores generally are fluorogenic as the equilibrium shifts towards the fluorescent form when the probe binds to its cellular targets. The resulting increase in fluorescence can be up to 1000-fold. Using this simple design principle we created fluorogenic probes in various colours for different cellular targets for wash-free, multicolour, live-cell nanoscopy. The work establishes a general strategy to develop fluorogenic probes for live-cell bioimaging.

Super-resolution fluorescence microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to super-resolution microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this Perspective, we discuss the role of small-molecule fluorescent probes for live-cell super-resolution microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell super-resolution microscopy are given.

Hypochlorous acid (HOCl), as a highly potent oxidant, is well-known as a key "killer" for pathogens in the innate immune system. Recently, mounting evidence indicates that intracellular HOCl plays additional important roles in regulating inflammation and cellular apoptosis. However, the organelle(s) involved in the distribution of HOCl remain unknown, causing difficulty to fully exploit its biological functions in cellular signaling pathways and various diseases. One of the main reasons lies in the lack of effective chemical tools to directly detect HOCl at subcellular levels due to low concentration, strong oxidization, and short lifetime of HOCl. Herein, the first two-photon fluorescent HOCl probe (TP-HOCl 1) and its mitochondria- (MITO-TP) and lysosome- (LYSO-TP) targetable derivatives for imaging mitochondrial and lysosomal HOCl were reported. These probes exhibit fast response (within seconds), good selectivity, and high sensitivity (<20 nM) toward HOCl. In live cell experiments, both probes MITO-TP and LYSO-TP were successfully applied to detect intracellular HOCl in corresponding organelles. In particular, the two-photon imaging of MITO-TP and LYSO-TP in murine model shows that higher amount of HOCl can be detected in both lysosome and mitochondria of macrophage cells during inflammation condition. Thus, these probes could not only help clarify the distribution of subcellular HOCl, but also serve as excellent tools to exploit and elucidate functions of HOCl at subcellular levels.

Peroxynitrite (ONOO) is a kind of reactive oxygen species (ROS) with super activity of oxidization and nitration, and overproduction of ONOO is associated with pathogenesis of many diseases. Thus, accurate detection of ONOO with high sensitivity and selectivity is imperative for elucidating its functions in health or disease states. Herein we for the first time present a new two-photon ratiometric fluorescent ONOO probe (MITO-CC) based on FRET mechanism by combining rational design strategy and dye-screening approach. MITO-CC, with fast response rate (within 20 s), excellent sensitivity (detection limit = 11.30 nM) and outstanding selectivity toward ONOO, was successfully applied to ratiometric detection of endogenous ONOO produced by HepG2/RAW264.7 cells and further employed for imaging oxidative stress in an inflamed mouse model. Therefore, probe MITO-CC could be a potential biological tool to explore the roles of ONOO under different physiological and pathological settings.

Intracellular thermometry at the microscopic level is currently a hot topic. Herein we describe a small molecule fluorescent thermometer targeting mitochondria (Mito thermo yellow). Mito thermo yellow successfully demonstrates the ability to monitor the intracellular temperature gradient, generated by exogenous heating, in various cells.