Nanoscopy with Multiple Off-States

Coordinate-targeted super-resolution techniques distinguish fluorophores within a diffraction-limited zone by differing molecular states (typically on vs. off) that are imprinted with light. In STED microscopy, state contrast is generated via stimulated emission. For strong spatial confinement, this requires high STED light intensities. Here we show that enhanced state contrast can be achieved by combining multiple off-state transitions, specifically by combining stimulated emission with reversible photoswitching in "protected STED".

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