Quantitative Analysis of Protein-Protein Interactions: One- and Two-photon Excitation FRET-FLIM Microscopy

Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging.

The simultaneous observation of multiple fluorescent proteins (FPs) by optical microscopy is revealing mechanisms by which proteins and organelles control a variety of cellular functions. Here we show the use of visible-light based two-photon excitation for simultaneously imaging multiple FPs. We demonstrated that multiple fluorescent targets can be concurrently excited by the absorption of two...

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noi...

Corrigendum: Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

Molecular Imaging: FLIM-FRET Microscopy

Molecular interactions in living cells are dynamic, and techniques that rely on chemical fixation or disruption of cell structure can provide only limited information about these interactions. Technological advances in light microscopy imaging, combined with the availability of genetically encoded fluorescent proteins now provide the tools to obtain spatial and temporal distribution of protein ...

Two-photon excitation STED microscopy.

We report sub-diffraction resolution in two-photon excitation (TPE) fluorescence microscopy achieved by merging this technique with stimulated-emission depletion (STED). We demonstrate an easy-to-implement and promising laser combination based on a short-pulse laser source for two-photon excitation and a continuous-wave (CW) laser source for resolution enhancement. Images of fluorescent nanopar...