Abstract

Optical microscopy is an indispensable tool that is driving progress in cell biology, and is still the only practical means of obtaining spatial and temporal resolution within living cells and tissues. Much effort is being devoted recently to achieve intrinsic three-dimensional (3D) spatial resolution by exploiting optical nonlinear effects which can only take place in the small focal volume where high photon densities are reached. One of the most utilised multiphoton (ie nonlinear) microscopy techniques is two-photon fluorescence where the biomolecules of interest are labelled with fluorophores, which are optically excited via simultaneous absorption of two photons. However, these modified biomolecules raise questions if their behaviour is real or artefactual. Furthermore, all organic fluorophores are prone to photo-bleaching which severely limits time-course observations and is accompanied by toxicity effects and consequent cell damage.

© 2011 Optical Society of America