Macromolecular-scale resolution in biological fluorescence microscopy

TLDR

Triplet‑state relaxation is achieved by lowering the pulsed‑laser repetition rate or speeding the scan to prevent triplet accumulation, thereby enabling the high‑resolution imaging. The technique delivers 15–20 nm far‑field resolution, a 10–12‑fold boost over diffraction, up to 30‑fold higher fluorescence signal, and a ≈140‑fold smaller focal spot, revealing nanoscale protein structures and opening new avenues in life‑science research.

Abstract

We demonstrate far-field fluorescence microscopy with a focal-plane resolution of 15–20 nm in biological samples. The 10- to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitation–depletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to ≈140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffraction-unlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences.