STED-SPIM: Stimulated Emission Depletion Improves Sheet Illumination Microscopy Resolution

We demonstrate the first, to our knowledge, integration of stimulated emission depletion (STED) with selective plane illumination microscopy (SPIM). Using this method, we were able to obtain up to 60% improvements in axial resolution with lateral resolution enhancements in control samples and zebrafish embryos. The integrated STED-SPIM method combines the advantages of SPIM with the resolution enhancement of STED, and thus provides a method for fast, high-resolution imaging with >100 μm deep penetration into biological tissue

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo

Life is driven by a set of biological events that are naturally dynamic and tightly orchestrated from the single molecule to entire organisms. Although biochemistry and molecular biology have been essential in deciphering signaling at a cellular and organismal level, biological imaging has been instrumental for unraveling life processes across multiple scales. Imaging methods have considerably improved over the past decades and now allow to grasp the inner workings of proteins, organelles, cells, organs and whole organisms. Not only do they allow us to visualize these events in their most-relevant context but also to accurately quantify underlying biomechanical features and, so, provide essential information for their understanding. In this Commentary, we review a palette of imaging (and biophysical) methods that are available to the scientific community for elucidating a wide array of biological events. We cover the most-recent developments in intravital imaging, light-sheet microscopy, super-resolution imaging, and correlative light and electron microscopy. In addition, we illustrate how these technologies have led to important insights in cell biology, from the molecular to the whole-organism resolution. Altogether, this review offers a snapshot of the current and state-of-the-art imaging methods that will contribute to the understanding of life and disease

Model Bio\u2010Membranes Investigated by AFM and AFS: A Suitable Tool to Unravel Lipid Organization and their Interaction with Proteins

The study of the biological membrane has largely benefitted from the exploitation of model bilayer systems. These simplified models of the complex biological membrane composed of thousands of different types of molecules allow both to understand basic physical principles underlying the membrane functioning and to test new techniques that will be subsequently applied to biological membranes. Here we concentrate on one of the most used model systems for this kind of investigations: the Supported Lipid Bilayer (SLB). In particular, we analyze the possibilities of investigation offered by Atomic Force Microscopy and Spectroscopy (AFM/AFS) on this model system. We discuss the information that this techniques is able to provide on the phase behavior of the lipid bilayers and on the partitioning of membrane proteins relative to the bilayer lateral heterogeneity. We discuss also the possibility to characterize the mechanical properties of lipid bilayers on the nanometer scale lateral resolution

STED super-resolved microscopy

Stimulated emission depletion (STED) microscopy provides subdiffraction resolution while preserving useful aspects of fluorescence microscopy, such as optical sectioning, and molecular specificity and sensitivity. However, sophisticated microscopy architectures and high illumination intensities have limited STED microscopy's widespread use in the past. Here we summarize the progress that is mitigating these problems and giving substantial momentum to STED microscopy applications. We discuss the future of this method in regard to spatiotemporal limits, live-cell imaging and combination with spectroscopy. Advances in these areas may elevate STED microscopy to a standard method for imaging in the life sciences