Fluorescence Fluctuation Spectroscopy in Subdiffraction Focal Volumes

We establish fluorescence fluctuation spectroscopy (FFS) with nanoscale detection volumes generated by stimulated emission depletion. Our method applies fluorescence correlation spectroscopy and fluorescence intensity distribution analysis to extract molecular information about mobilities and fluorescence emission in solution. The combination of correlation analysis with that of photon intensity distributions reveals a fivefold squeezing of the detection volume over current diffraction-limited systems, which is in full agreement with the simultaneously demonstrated 25-fold reduction in (axial) focal transit time. Our method significantly extends the potential of far-field FFS, including for the noninvasive investigation of molecular reactions at higher concentrations.

Figure 1

(a) Diffraction-limited excitation spot $H_0(\overrightarrow{r})$ along with the STED PSF $h_{\mathrm{STED}}(\overrightarrow{r})$ and the resulting fluorescence spot $H_{\mathrm{spot}}(\overrightarrow{r})$, calculated using vectorial diffraction theory. Conditions: 64° semiaperture angle, water immersion, $x$-polarized excitation, and $\sigma h_{\mathrm{STED}}^{\max }=15$. (b) Focal shape parameter $K$ of the spot size with increasing STED fluence.

Figure 2

(a) Measured FCS curves with and without a time-averaged STED power of ${\overline{P}}_{\mathrm{STED}}=22\mathrm{mW}$. (b) Deduced axial diffusion time ${\tau }_2$ (triangles) and apparent mean number of focal spot molecules $N$ (circles) as a function of ${\overline{P}}_{\mathrm{STED}}$.

Figure 3

(a) Number of molecules $N^{\prime }$. (b) Total signal $S$ and uncorrelated signal component $U$ as extracted by FIDA. (c) Experimental fluorescence suppression through STED. (d) Calculated axial fluorescence profile without (gray) and with STED at $\sigma h_{\mathrm{STED}}^{\max }=30$ (black and dashed lines corresponding to $\delta =0$ and 0.2, respectively).

Figure 4

Scaling of the detection volume as a function of ${\overline{P}}_{\mathrm{STED}}$ determined from (triangles) the axial diffusion time ${\tau }_2$ gained by FCS, (squares) the corrected number of molecules $\tilde{N}$ from the FCS amplitude, and (circles) the number of molecules $N^{\prime }$ from FIDA alone. Right axis shows the detection volume in attoliters (${10}^{-18}\mathrm{L}$) obtained according to $V=8{\pi }^{3/2}D{\tau }_1\sqrt{D{\tau }_2}$ and using $D=2.5\times {10}^{-6}{\mathrm{cm}}^2/\mathrm{s}$.