Two-photon-like microscopy with orders-of-magnitude lower illumination intensity via two-step fluorescence.

ABSTRACT: We describe two-step fluorescence microscopy, a new approach to non-linear imaging based on positive reversible photoswitchable fluorescent probes. The protein Padron approximates ideal two-step fluorescent behaviour: it equilibrates to an inactive state, converts to an active state under blue light, and blue light also excites this active state to fluoresce. Both activation and excitation are linear processes, but the total fluorescent signal is quadratic, proportional to the square of the illumination dose. Here, we use Padron's quadratic non-linearity to demonstrate the principle of two-step microscopy, similar in principle to two-photon microscopy but with orders-of-magnitude better cross-section. As with two-photon, quadratic non-linearity from two-step fluorescence improves resolutio

SUBMITTER: Ingaramo M 

PROVIDER: S-EPMC4559865 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature