Two-color nanoscopy of organelles for extended times with HIDE probes

Performing multi-color nanoscopy for extended times is challenging due to the rapid photobleaching rate of most fluorophores. Here we describe a new fluorophore (Yale-595) and a bio-orthogonal labeling strategy that enables two-color super-resolution (STED) and 3D confocal imaging of two organelles simultaneously for extended times using high-density environmentally sensitive (HIDE) probes. Because HIDE probes are small, cell-permeant molecules, they can visualize dual organelle dynamics in hard-to-transfect cell lines by super-resolution for over an order of magnitude longer than with tagged proteins. The extended time domain possible using these tools reveals dynamic nanoscale targeting between different organelles.