Simultaneous imaging of multiple cellular events using high-accuracy fluorescence polarization microscopy.

Förster resonance energy transfer (FRET) has been widely used to design indicators for biomolecules. Conventional FRET-based indicators enable quantitative measurements of analyzes by calculating the ratio between donor and acceptor fluorophores. However, such 'hetero-FRET'-based indicators, which use multiple differently colored fluorophores, restrict the simultaneous use of other colors of fluorescent molecules. To overcome this problem, we developed a 'homo-FRET'-based Ca2+ indicator, W-Cameleon, composed of two identical yellow fluorescent proteins. The binding of Ca2+ to the indicator induces a change in FRET efficiency, which in turn transforms into changes in fluorescence anisotropy. Given that the fluorescence polarization is depolarized by light passing through a high numerical aperture lens and reflecting on a dichroic mirror, we also developed a microscopy technique that reliably detects fluorescence anisotropy with high precision. Our design is aided by photonic-crystal technology, to compensate for the fluorescence depolarization. We thereby succeeded in the simultaneous visualization of three individual intracellular events by using three different fluorescent indicators. Our system may contribute to an expansion of the number of events that can be observed, which will enable a more quantitative understanding of biological phenomena.