A novel method for spatially complex diffraction‐limited photoactivation and photobleaching in living cells

Abstract

Photoactivated probes have gained interest as experimental tools to study intracellular signalling pathways all the way to the molecular level. However technical limitations of the means to activate such compounds have put constraints on their use in spatially highly restricted subcellular areas. The Mosaic digital illumination system uses a high-speed array of individually addressable, tiltable micromirrors to direct continuous-wave laser light onto a specimen with diffraction-limited precision. The system, integrated into a Nikon A1R confocal microscope, was used to uncage Ca²⁺ or IP3 and conduct photobleaching experiments from multiple geometrically complex subcellular regions while simultaneously measuring [Ca²⁺]i with high-speed confocal imaging.