STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics

Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells. STED-FLCS allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme, as demonstrated by a comparison between STED-FLCS and previous conventional STED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells. The STED-FLCS data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds. Drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity. STED-FLCS specifically demonstrates a significant improvement over previous gated STED-FCS experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality.

We applied different concentrations of BSA-lipid complexes for incubation on ice depending on the lipid type: 5-50 nM for SM and 10-300 nM for PE. For details see 4,5 . An Atto488-labeled PE lipid analogue (head group, Atto-Tec) was used for the measurements on the commercial system using a 592 nm STED laser.

Supported lipid bilayers (SLBs).
A single-component supported lipid bilayer was used as a control for free Brownian diffusion. We created the supported lipid bilayer on cover glass following the procedure described in reference 6  lipid solution had become transparent. This solution was placed into a microscopy chamber with plasmacleaned (Femto-RF, Diener Electronic) standard microscope cover glass. Bilayers were the spin-coated on the cover glass. Thirty minutes later, the sample was thoroughly rinsed and kept under buffer solution. In figure S1, the bilayer was formed on freshly cleaved mica.
Cells. Cell culture, transfection and mounting of the cells. PtK2 cells were cultivated in DMEM medium with Glutamax and 4.5% (w/v) glucose (Invitrogen) supplemented with 50 μg ml −1 penicillin, 50 μg ml −1 streptomycin, 1 mM Na-pyruvate and 10% (v/v) FCS (Invitrogen) at 37 °C, 7% CO 2 . Cells were grown overnight on glass cover slides until they reached ~80% confluence. For FCS measurements, incorporation of the fluorescent lipid analogues into the plasma membrane of living PtK2 cells was accomplished via bovine serum albumin (BSA) complexes as described previously 4,5 . Briefly, cells were washed with Dulbecco's modified Eagle medium without Phenol Red buffered with 10 mM HEPES (HDMEM) and incubated with BSA-lipid complexes on ice for 30 min, washed in cold HDMEM and incubated at 37 °C for 4 min in HDMEM. The exact measurement conditions (room temperature (22 °C), S4 measurements performed on the plasma membrane facing the cover glass and measurement time 10-15 s) as well as controls for precluding potential influence by laser light or labeling are given in references 4,5 .
Drug treatments. Cholesterol Oxidase: The cells were treated with 1 U/ml Streptomyces spec. COase Nitrogen-vacancy color centers. Figures 1 and 2 show images of single luminescent negatively charged nitrogen-vacancy (NV) color centers 1, 2 , whose absorption and emission maxima are at around 560 nm and 700 nm, respectively. These experiments were performed on type IIa polycrystalline diamond grown by chemical vapor deposition (CVD) using the 532 nm excitation laser. The sample was prepared by mounting the bulk CVD diamond on a standard glass coverslip.

FCS data analysis
Correlation data on membranes were recorded for diffusing fluorescent lipid analogues either in supported lipid bilayers or in the plasma membrane of living cells. Correlation data G(t c ) were analyzed using a model of two-dimensional anomalous subdiffusion as outlined previously 4,5 : Here, t xy is the focal transit time given by the diameter of the Gaussian-like assumed observation spot, the

Theory
In this section we revisit the theory of the time t evolution of the effective observation spots in a STED microscope operating with a pulsed excitation and a continuous-wave (cw) STED laser 1, 2 . First, we quantify the emitted fluorescence in time t following the excitation laser pulse. The emitted fluorescence is proportional to the population of the first excited state. We assume that: (i) the fluorescent marker can be described by a simple two-level model with ground and first excited states S 0 and S 1 ; the population of additional dark state and vibrational sub-levels is neglected in the theory; (ii) the probability to excite the fluorescent marker from S 0 to S 1 with the STED beam is negligible as well; (iii) the fluorescent marker is initially in S 1 due to the short excitation pulse; (iv) spontaneous S 0 to S 1 de-excitation takes place with a rate constant k S1 =1/ (with denoting the excited state lifetime), and fluorescence photons are emitted with a quantum yield q fl , i.e., with a rate k fl = k S1 q fl ; (v) the time period T=1/f between two excitation pulses is longer than the excited-state lifetime τ of the markers, i.e., all markers have relaxed to S 0 before the arrival of the next excitation pulse; (vi) the rate of stimulated emission is given by k STED = σ STED~ISTED with σ S TED~ = σ S TED λ STED /(hc) being the stimulated emission cross-section σ S TED divided by the photon energy (hc)/λ STED (λ STED is the wavelength of the STED light, and hc = 1.99•10 -25 Jm the product of Planck's constant h and the velocity of light c); (vii) the saturation intensity I s is the intensity of the STED laser at which k STED = k S1 , i.e. I s = k S1 / σ STED~, revealing the so-called saturation factor ς = I STED /I s = k STED /k S1 . Under these assumptions, the relevant rate equation for the population P S1 of S 1 is . (1) If we normalize the population, i.e. P S1 (0) = 1, the excited state population is given by P(t) = exp(-(k S1k STED )t). Hence the fluorescence emission rate at a time t after the excitation event and under constant illumination by cw STED light is (Fig. S1) . (2) The first term is a normalization factor, the second term represents the fluorescence rate of the undisturbed molecule (ς = 0) and the third term is the reduction of the fluorescence emission due to the STED laser action. The most important implication out of this equation is that that the efficiency of fluorescence suppression improves by increasing either ς (i.e. the STED laser intensity) and/or the time t of STED laser action. At low excitation intensity the tE-PSF of a general STED microscope is given by the excitation probability times the probability of spontaneous decay times the detection efficiency .
The spatial r-dependence of ς(r) is given by the doughnut-like intensity distribution of the STED light, with ς m = I m STED /I s . The new FWHM is therefore (5) and the total intensity detected decreases with t as h CW-STED (t,0) = exp(-k s1 t).

Equation 5 clearly states that the observation spot (or E-PSF) changes dynamically in time (tE-PSF).
In particular, for a given STED intensity ς m, the observation spot is equivalent to a confocal system during or immediately after the excitation pulse and it decreases with the time t of the STED laser action.
Consequently, by sorting detected photons depending on their delay t relative to the excitation pulse (time gating) one can generate different observation spots from the same measurement. By using a timecorrelated-single-photon-counting approach and choosing a-posteriori specific time gates it is possible to generate observation spots with different size.

S7
For gSTED-FCS and STED-FLCS the E-PSF (or observation spot) reads , (6) and , respectively. Figure S2c,d shows the characteristics of the E-PSFs for a series of time-gates with increasing time delay T g . As expected, for gSTED-FLCS the FWHM of the E-PSF decreases continuously from quasi confocal down to sub-diffraction size (~3, ~4 and ~6 fold reduction in comparison to confocal for ς m equals to 5, 10 and 20, respectively) for increasing time delay.
One has to keep in mind that time-gated detection reduces also the overall signal (inset Figure S2b   Inset: unnormalized observation spots. The excitation intensity profile h exc (r), the detection efficiency profile h det (r) and the STED intensity profile I STED (r) are computed using Fourier theory 3 . Given I STED (r), h exc (r) and h det (r) the observation spots are calculated using Equations (4, 6, 7). We assumed an oil immersion objective of 1.4 numerical aperture, λ exc = 635 nm, λ S TED = 760 nm and λ det = 670 nm and a detection pinhole with a projected diameter of 500 nm in the sample space.