Co-imaging extrinsic, intrinsic and effector caspase activity by fluorescence anisotropy microscopy.

In order to overcome intercellular variability and thereby effectively assess signal propagation in biological networks it is imperative to simultaneously quantify multiple biological observables in single living cells. While fluorescent biosensors have been the tool of choice to monitor the dynamics of protein interaction and enzymatic activity, co-measuring more than two of them has proven challenging. In this work, we designed three spectrally separated anisotropy-based Förster Resonant Energy Transfer (FRET) biosensors to overcome this difficulty. We demonstrate this principle by monitoring the activation of extrinsic, intrinsic and effector caspases upon apoptotic stimulus. Together with modelling and simulations we show that time of maximum activity for each caspase can be derived from the anisotropy of the corresponding biosensor. Such measurements correlate relative activation times and refine existing models of biological signalling networks, providing valuable insight into signal propagation.

Interdependence between EGFR and Phosphatases Spatially Established by Vesicular Dynamics Generates a Growth Factor Sensing and Responding Network.

The proto-oncogenic epidermal growth factor receptor (EGFR) is a tyrosine kinase whose sensitivity to growth factors and signal duration determines cellular behavior. We resolve how EGFR's response to epidermal growth factor (EGF) originates from dynamically established recursive interactions with spatially organized protein tyrosine phosphatases (PTPs). Reciprocal genetic PTP perturbations enabled identification of receptor-like PTPRG/J at the plasma membrane and ER-associated PTPN2 as the major EGFR dephosphorylating activities. Imaging spatial-temporal PTP reactivity revealed that vesicular trafficking establishes a spatially distributed negative feedback with PTPN2 that determines signal duration. On the other hand, single-cell dose-response analysis uncovered a reactive oxygen species-mediated toggle switch between autocatalytically activated monomeric EGFR and the tumor suppressor PTPRG that governs EGFR's sensitivity to EGF. Vesicular recycling of monomeric EGFR unifies the interactions with these PTPs on distinct membrane systems, dynamically generating a network architecture that can sense and respond to time-varying growth factor signals.

Photofunctional surfaces for quantitative fluorescence microscopy: monitoring the effects of photogenerated reactive oxygen species at single cell level with spatiotemporal resolution.

Herein, we report on the implementation of photofunctional surfaces for the investigation of cellular responses by means of quantitative fluorescence microscopy. The developed substrates are able to produce reactive oxygen species under the fluorescence microscope upon irradiation with visible light, and the behavior of cells grown on these surfaces can be consequently investigated in situ and in real time. Moreover, a suitable methodology is presented to simultaneously monitor phototriggered morphological changes and the associated molecular pathways with spatiotemporal resolution employing time-resolved fluorescence anisotropy at the single cell level. The results showed that morphological changes can be complemented with a quantitative evaluation of the associated molecular signaling cascades for the unambiguous assignment of reactive oxygen species-related photoinduced apoptosis. Indeed, similar phenotypes are associated with different cellular processes. Our methodology facilitates the in vitro design and evaluation of photosensitizers for the treatment of cancer and infectious diseases with the aid of functional fluorescence microscopy.

flatFLIM: enhancing the dynamic range of frequency domain FLIM.

Fluorescence Lifetime Imaging Microscopy (FLIM) is a quantitative technique to probe the nanoenvironment of fluorescent molecules. It is the most robust way to quantify Förster Resonance Energy Transfer (FRET) as it allows reliable differentiation between concentration changes and quenching. In this way, molecular interactions can be imaged in single living cells. The most common wide-field implementation is homodyne Frequency Domain (FD) FLIM, which determines the fluorescence lifetime by measuring the phase and modulation changes of the fluorescence in each pixel upon excitation with a light source modulated at a high frequency. The fluorescence lifetimes are derived from a stack of images acquired at different phase shifts between excitation and detection. In this work we describe a simple method to enhance the dynamic range of FD-FLIM based on precompensating the expected fluorescence modulation by varying the laser power through the phase stack. We show theoretically and experimentally that most of the dynamic range of the camera can be recovered to quantify cells with different intensities. This improvement can be added to any FD-FLIM setup with minimal modifications, enhancing the throughput of information content.

Luminescent ruthenium tripod complexes: properties in solution and on conductive surfaces.

Two luminescent ruthenium complexes containing tripod-type end groups linked through a rigid spacer to a phenanthroline derivative, able to confer an axial geometry to the complexes, are described. One of the compounds is functionalized with thioacetate groups in order to link the metal complex to metallic surfaces. The photophysical and electrochemical behavior of the complexes are studied in solution and on conductive substrates and, furthermore, self-assembled monolayers are investigated in a junction using gold and an indium gallium eutectic, as electrodes, and by time-resolved confocal microscopy. The results show that the complexes form very stable and well-ordered monolayers because of the tripod system, which can anchor the complex almost perpendicular to the surfaces.

Controllable growth and field-effect property of monolayer to multilayer microstripes of an organic semiconductor.

The controllable growth of partially aligned monolayer to multilayer micrometer stripes was demonstrated by adjusting the pulling speed in a dip-coating process. The number of molecular layers decreases with the increasing pulling speed. A lower pulling speed yields mixed multilayers (3-9 monolayers). It is noteworthy that pure monolayer and bilayer microstripes over large areas can be obtained at high pulling speeds. The stripe morphology strongly depends on the pulling speed or the number of molecular layers. XRD and confocal fluorescence measurements manifest that monolayer stripes are amorphous, while multilayer stripes (> or = 2) consist of crystalline states. FET devices were fabricated on these stripes. Monolayer stripes failed to reveal a field effect due to their amorphous state. In contrast, multilayer stripes exhibit good field-effect behavior. This study provides useful information for future molecular design in controlling molecular architectures. The controllable growth from monolayer to multilayer offers a powerful experimental system for fundamental research into the real charge accumulation and transporting layers for OFETs.