The distribution and dynamics of phosphatidylserine are studied in the plasma membrane and in organellar membranes of live cells using two novel fluorescent probes in combination with various biophysical techniques, including fluorescence correlation spectroscopy and single-particle tracking.

Fluorescence cross-correlation spectroscopy (FCCS) is a single-molecule sensitive technique to quantitatively study interactions among fluorescently tagged biomolecules. Besides the initial implementation as dual-color FCCS (DC-FCCS), FCCS has several powerful derivatives, including single-wavelength FCCS (SW-FCCS), two-photon FCCS (TP-FCCS), and pulsed interleaved excitation FCCS (PIE-FCCS). However, to apply FCCS successfully, one needs to be familiar with procedures ranging from fluorescent labeling, instrumentation setup and alignment, sample preparation, and data analysis. Here, we describe the procedures to apply FCCS in various biological samples ranging from live cells to in vivo measurements, with the focus on DC-FCCS and SW-FCCS.

This study shows that low-dose curcumin stimulates the proliferation of NSCs, which is probably by inhibiting the mRNA and protein expressions of GR and directly or indirectly regulating the STAT3 via the synergistic effect of GR and STAT3 pathways and its related signal pathways.

Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS) has been applied to the quantitative determination of molecular interactions at equilibrium for different molecular systems in vitro and in vivo, including living cells and organisms. Here we report for the first time the measurement of an activation and time dependent interaction between a cytosolic and a membrane bound protein by SW-FCCS in live cells. On the example of the epidermal growth factor (EGF) receptor (EGFR) we confirm the existence of pre-formed dimers in the absence of stimulation and demonstrate that the activation of the receptor can be detected by the phosphorylation dependent binding of a phosphotyrosine binding (PTB) domain. SW-FCCS results indicate that in CHO cells there is low specific interaction between PTB and EGFR, possibly indicating a low level of EGFR phosphorylation even in the absence of EGF stimulation. After EGF stimulation the interaction between PTB and EGFR increases significantly in a time dependent manner.

fluorophore via photoinduced electron transfer (PeT) [3]. By carefully tuning the redox potential of the BODIPY fluorophores, [4] we can have one redox state fluorescent, and the other quenched through PeT (e.g. reduced state off and oxidized state on). Furthermore we are able to tailor the BODIPY dyes to enhance lipid solubility [5] and target organelles. In this presentation I will discuss the general concepts behind the new probes and will focus on the reactivity and imaging opportunities in live cells arising from a novel a-tocopherol based fluorogenic probe that readily targets mitochondria [6]. I will also discuss the opportunities that a related probe based on a fluorogenic ubiquinone provides towards monitoring key metabolic processes within mitochondria.

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