Optics

Physics

Biophysics

Actin

Membrane protein

Microscopy

Cell

Live cell imaging

Endoplasmic reticulum

Integral membrane protein

Organelle

Nanotechnology

Chemistry

Materials science

Membrane

Biochemistry

Biology

Membrane contact site

Filopodia

Cell biology

Nature Biotechnology

Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.

Long time-lapse nanoscopy with spontaneously blinking membrane probes