Confocal fluorescence correlation spectroscopy (FCS) allows for the determination of lateral diffusion coefficients and surface densities in planar phospholipid systems. The determination of the vertical (z-) position of the laser focus relative to the phospholipid surface plane is of crucial importance for the accuracy of the confocal FCS experiment. In this work we determine for the first time this vertical (z-) position of the laser focus by a so-called “Z-scan”, which is based on the determination of diffusion times and particle numbers in 0.2 μm steps along the vertical (z-) axis. Experiments on supported phospholipid bilayers composed of dioleoylphosphatidylcholine (DOPC) and small amounts of Rhodamine Red-X 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Rhodamine Red-X DHPE) adsorbed onto atomically flat mica and borosilicate glass demonstrate that results obtained by the Z-scan approach are significantly more precise than those results obtained when the fluorescence intensity maximum is used as an indicator in the determination of the vertical (z-) position of the sample. In addition to this basic contribution for the investigation of planar bilayer systems by confocal FCS, the lateral diffusion coefficients of Rhodamine Red-X DHPE in supported phospholipid bilayers composed of DOPC and cholesterol as well as in DOPC or dipalmitoylphosphatidylcholine (DPPC) monolayers adsorbed at a liquid−liquid interface were determined.

The lateral diffusion coefficients of a BODIPY tail-labeled lipid in two model systems, namely, free-standing giant unilamellar vesicles (GUVs) and supported phospholipid bilayers (SPBs), were determined by fluorescence correlation spectroscopy (FCS) using the Z-scan approach. For the first time, the performed measurements on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers maintain exactly the same experimental conditions for both systems, which allows for a quantitative comparison of lipid diffusion in these two commonly used model membranes. The results obtained revealed that the lipid mobility in free-standing bilayers (D=7.8+/-0.8 microm2 s-1) is significantly higher than in the bilayer created on the solid support (mica) (D=3.1+/-0.3 microm2 s-1).

Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.

This article explains the basic principles of FLCS, a genuine fusion of Time-Correlated Single Photon Counting (TCSPC) and Fluorescence Correlation Spectroscopy (FCS), using common terms and minimum mathematics. The usefulness of the method is demonstrated on simple FCS experiments. The method makes possible to separate the autocorrelation function of individual components of a mixture of fluorophores, as well as purging the result from parasitic contributions like scattered light or detector afterpulsing.