Breaking the Diffraction Barrier in Fluorescence Microscopy by Optical Shelving

We report the breaking of the diffraction resolution barrier in far-field fluorescence microscopy by transiently shelving the fluorophore in a metastable dark state. Using a relatively modest light intensity of several $\mathrm{kW}/{\mathrm{cm}}^2$ in a focal distribution featuring a local zero, we confine the fluorescence emission to a spot whose diameter is a fraction of the wavelength of light. Nanoscale far-field optical resolution down to 50 nm is demonstrated by imaging microtubules in a mammalian cell and proteins on the plasma membrane of a neuron. The presence of dark states in virtually any fluorescent molecule opens up a new venue for far-field microscopy with resolution that is no longer limited by diffraction.

Figure 1

Fluorescence depletion of the organic dye Atto532 by triplet state ($T_1$) buildup. (a) Fluorescence (black) and fraction of bleached molecules after 25 depletion-probe cycles (gray) as a function of depletion intensity $I_D$ or power $P_D$; error bars as relative standard deviation from 5 consecutive measurements. Inset: Fluorescence depletion is induced by $T_1$ buildup. (b) Depletion-probe cycle with strong depletion illumination ($500\mu \mathrm{s}$, $I_D=85\mathrm{kW}/{\mathrm{cm}}^2$), depleting the fluorescence due to $T_1$ buildup; subsequent low-power illumination ($100\mu \mathrm{s}$, $I_P=7\mathrm{kW}/{\mathrm{cm}}^2$) probing the residual fluorescence signal. After depletion and complete relaxation of the $T_1$ ($\sim 60\mathrm{ms}$), fluorescence emission is completely recovered, as observed by comparison with the signal gained just with the probe beam (Control).

Figure 2

Creating effective PSFs of subdiffraction extent in a single point scanning GSD microscope. (a) Squeezing the spot just along the x axis and (b) along all directions in the focal plane. The depletion spot (Depletion) overlaid with the regularly focused probe spot (Probe) produces the effective PSF (Eff. PSF). The probe spot probes the fluorescence right after the depletion spot has pumped the dye into the $T_1$. Focal plane cross section of the PSF (upper panel) and profiles along the x axis [dashed line in upper panels] showing FWHM values (lower panels). Wavelengths for depletion and probing: 532 nm; fluorescence wavelength: 560 nm.

Figure 3

GSD microscopy provides focal plane resolution $\le 80\mathrm{nm}$ in biological imaging, demonstrated with Atto532-stained microtubules: (a) confocal, (b) GSD, and (c) GSD plus linear deconvolution (LD). (d) Normalized line profiles through the data of (a) and (b) where indicated by the dashed line; images were smoothed using a Lagrangian interpolation of third order. Scale bar $=500\mathrm{nm}$.

Figure 4

GSD microscopy provides super-resolution in the focal plane. Orange fluorescent beads recorded confocally (a) and with GSD (b). SNAP-25 protein agglomerations stained with Atto532 on a cell membrane: confocal (c) and GSD (d). All images after linear deconvolution (LD). Scale bar $=500\mathrm{nm}$.