Dual-color metal-induced and Förster resonance energy transfer for cell nanoscopy.

We report a novel method, dual-color axial nanometric localization by metal--induced energy transfer, and combine it with Förster resonance energy transfer (FRET) for resolving structural details in cells on the molecular level. We demonstrate the capability of this method on cytoskeletal elements and adhesions in human mesenchymal stem cells. Our approach is based on fluorescence-lifetime-imaging microscopy and allows for precise determination of the three-dimensional architecture of stress fibers anchoring at focal adhesions, thus yielding crucial information to understand cell-matrix mechanics. In addition to resolving nanometric structural details along the z-axis, we use FRET to gain precise information on the distance between actin and vinculin at focal adhesions.

Cell-Substrate Dynamics of the Epithelial-to-Mesenchymal Transition.

The biological process of the epithelial-to-mesenchymal transition (EMT) allows epithelial cells to enhance their migratory and invasive behavior and plays a key role in embryogenesis, fibrosis, wound healing, and metastasis. Among the multiple biochemical changes from an epithelial to a mesenchymal phenotype, the alteration of cellular dynamics in cell-cell as well as cell-substrate contacts is crucial. To determine these variations over the whole time scale of the EMT, we measure the cell-substrate distance of epithelial NMuMG cells during EMT using our newly established metal-induced energy transfer (MIET) microscopy, which allows one to achieve nanometer axial resolution. We show that, in the very first hours of the transition, the cell-substrate distance increases substantially, but later in the process after reaching the mesenchymal state, this distance is reduced again to the level of untreated cells. These findings relate to a change in the number of adhesion points and will help to better understand remodeling processes associated with wound healing, embryonic development, cancer progression, or tissue regeneration.

Simultaneous Measurement of the Three-Dimensional Orientation of Excitation and Emission Dipoles.

The emission properties of most fluorescent emitters, such as dye molecules or solid-state color centers, can be well described by the model of an oscillating electric dipole. However, the orientations of their excitation and emission dipoles are, in most cases, not parallel. Although single molecule excitation and emission dipole orientation measurements have been performed in the past, no experimental method has so far looked at the three-dimensional excitation and emission dipole geometry of individual emitters simultaneously. We present the first experimental study, using defocused imaging in conjunction with radially polarized excitation scanning, to measure both the excitation as well as emission dipole orientations of single molecules, which allows us to sample the distribution of their mutual orientation. We find an unexpectedly broad distribution of the angle between both dipoles which we attribute to the interaction between the observed molecules and the substrate they are immobilized on.

Fourier interpolation stochastic optical fluctuation imaging.

Stochastic Optical Fluctuation Imaging (SOFI) is a super-resolution fluorescence microscopy technique which allows to enhance the spatial resolution of an image by evaluating the temporal fluctuations of blinking fluorescent emitters. SOFI is not based on the identification and localization of single molecules such as in the widely used Photoactivation Localization Microsopy (PALM) or Stochastic Optical Reconstruction Microscopy (STORM), but computes a superresolved image via temporal cumulants from a recorded movie. A technical challenge hereby is that, when directly applying the SOFI algorithm to a movie of raw images, the pixel size of the final SOFI image is the same as that of the original images, which becomes problematic when the final SOFI resolution is much smaller than this value. In the past, sophisticated cross-correlation schemes have been used for tackling this problem. Here, we present an alternative, exact, straightforward, and simple solution using an interpolation scheme based on Fourier transforms. We exemplify the method on simulated and experimental data.