Novel fluorescent probes for highly selective two-photon imaging of mitochondria in living cells

• Two novel two-photon mitochondrial probes were synthesized successfully.
• They possess larger two-photon action cross-sections than MTG and MTR.
• High-brightness two-photon fluorescence images can be obtained by them.
• High co-localization coefficient with MTR demonstrated their selectivity.
• Many excellent properties, such as photostability, were revealed.

The synthesis and characterization of a pair of novel pyridine cation derivatives possessing two-photon excitation fluorescence (TPEF) properties and selectively staining mitochondria in living SiHa cells within 30 min, CAI and CAEI, were reported. And the green-emitting CAI displayed a much larger two-photon excitation fluorescence action absorption cross-section (δ×Φ) of 328 g at 860 nm in comparison with commercial MitoTracker Green (MTG) with maximum δ×Φ value of 2.18 g at 850 nm. As is known to all, δ×Φ is a crucial parameter to obtain a high-quality microscopic photo in living cells in two-photon microscopy (TPM). Moreover, the fact that the co-localization coefficient between CAI and conventional MitoTracker Red (MTR) was 0.95 in SiHa cells demonstrated specific staining performance of CAI to mitochondria. As biosensors, both CAI and CAEI possessed a number of beneficial properties such as large δ×Φ and Stokes shifts, good membrane permeability, long retention time, high photostability and excellent counterstain compatibility with different biosensors for instance Hoechst 33342 and D 307, which ranked them as one of the best TPEF mitochondrial probes. Furthermore, related mechanism research suggested that their localization properties were dependent on the mitochondrial membrane potential in living cells. And their remarkable properties can extend the investigation on mitochondria in a biological context.