Quantitative fluorescence correlation spectroscopy in three-dimensional systems under stimulated emission depletion conditions

Background reduction in STED-FCS using coherent-hybrid STED

“…From these three quantities, we could estimate background levels as follows. Undepleted background increases the apparent number of molecules in the observation surface [5], [9], [10]:…”

“…As an alternative measure of the noise in experimental FCS curves, we calculated the root-meansquare values of the fitting residuals up to a lag time of 50 μs (smaller than transit times), normalised by the amplitude, as applied before [24]. Recent work showed that the relatively poor signal-to-background ratio (SBR) obtained with 2D STED-FCS when studying 3D diffusion was due to low-intensity contributions originating from undepleted areas of the excitation focus [5], [9]. To extend this study to different STED confinement modes, we first obtained better insight into spatial origins of signal and background by simulating the excitation and depletion foci of our system.…”

“…In the resulting effective intensity distributions in confocal and different STED confinement modes (Figure 3), low-and high-intensity areas, which yield respectively weakly and strongly correlating contributions, can be considered as regions giving rise to background and signal, respectively. In practice, we used a threshold defined as 1/e 2 of the maximum intensity (black dashed contours in Figure 3), as previously in STED-FCS [9]. Because intensity distributions are symmetric with respect to the optical axis, they are conveniently represented in cylindrical coordinates (Figure 3(a), top).…”

“…The most commonly used depletion pattern is a ring-shaped focus, which constrains the lateral resolution but leaves unchanged the axial resolution (2D STED). This depletion pattern has been extensively used to study two-dimensional systems like cellular membranes [4]- [8], but faces severe limitations when studying three-dimensional diffusion, due to varying axial cross-sections and high background originating from undepleted areas [5], [9] that leads to an increase in the apparent number of molecules in the observation volume [5], [10]. Alternatively, a bottle-shaped depletion beam can be used to essentially constrain the axial resolution (z-STED), but is more sensitive to optical aberrations [11], [12], which can be mitigated with adaptive optics [13].…”

Adaptive optics allows 3D STED-FCS measurements in the cytoplasm of living cells

“…2D STED-FCS in 3D environments constrains the lateral resolution and assumes uniform diffusion properties along the optical axis. In this configuration, the estimation of both average number of molecules in the observation volume and transit times is complicated by varying axial cross-section of the beam and biased by out-of-focus contributions (5,6), requiring either specific fitting models (6,7), background subtraction using separation of photons by lifetime tuning (SPLIT) (8), or stimulated emission double depletion (STEDD) (9). In principle, the 3D depletion pattern should greatly simplify the analysis and interpretation of STED-FCS measurements of 3D diffusion.…”