An imaging method comprising bringing a biological specimen into association with a compound of Formula I； irradiating the specimen with light； and observing two-photon excited fluorescence images emitted from the compound of Formula I； Wherein X and Y is as defined herein； B is an optionally substituted aromatic or heteroaromatic ring； M is 1-3； p is 1-3； Z is an anion having a negative charge of n； n is 1-3； q is 1-3； provided that m x p = n x q. B may be selected from phenyl, pyridyl, thienyl, furanyl, naphthyl, quinolyl and isoquinolyl, particularly 4-pyridyl. m, p, n and q are preferably 1. Z may be a halide anion, particularly iodide. Compounds of Formula 1 wherein B is phenyl substituted with 1-3 groups independently selected from fluoro and C1-C3, particularly trifluoromethyl are disclosed. The disclosed compounds are indicated to have greater imaging depth and photostability than known agents, whilst having lower toxicity. The methods are disclosed to have selectivity for mitochondria. The methods are disclosed to have utility in both in vitro and in vivo imaging, in particular, imaging of tumours.
