Label-Free Pump–Probe Nanoscopy

In the last few decades fluorescence microscopy has been the most widely used microscopy technique and much effort has been put into the development of advanced super-resolution fluorescence microscopy techniques to circumvent the diffraction limit. Despite their well-established benefits, these techniques have to rely on the photo-physical properties of fluorescent molecules to obtain the desired contrast and spatial resolution. The labeling procedure may cause unwanted alterations in the sample. With the advent of ultrashort-pulsed laser sources, it became possible to better explore novel non-fluorescent-based contrast mechanisms that rely solely on intrinsic properties of the molecules of interest and which led to the development of label-free microscopy approaches. In this chapter, the imaging capabilities of absorption-based pump\u2013probe microscopy are presented. This technique explores the ultrafast dynamic properties of the sample with high spatial and temporal resolution, as well as high sensitivity and chemical specificity. Two pulses, a pump and a probe, with a proper spatial and temporal overlap are used. The pump is absorbed, inducing a measurable change in the sample carrier population, which is then monitored by a delayed probe pulse. The development of new label-free approaches also represents a key challenge for the exploration of super-resolution approaches in non-fluorescence-based methods

General RNA-binding proteins have a function in poly(A)-binding protein-dependent translation

The interaction between the poly(A)-binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA-binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB-1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB-1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP-dependent after the addition of YB-1. In this system, eIF4E binding to the cap structure is inhibited by YB-1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB-1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB-1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP