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

The nano-architecture of the axonal cytoskeleton

International audienceThe cytoskeleton is a cellular shapeshifter. Like these creatures of mythology and speculative fiction, the cytoskeleton can alter its physical form and shape to accommodate the immediate needs of the cell. Indeed, many a rapt student has watched the cytoskeleton of a dividing cell seemingly miraculously transform into an organized mitotic spindle and then dissolve into an indiscrete mass, right before their eyes. The extreme polarization of neurons (that is, their fundamental asymmetry, arising due to the presence of elongated processes), along with their lifelong plasticity, creates unique demands on the cytoskeleton. A remarkable example is the axon, which can grow to enormous lengths and must generate and maintain its form and function throughout life — a burden that rests largely on the cytoskeleton. The axonal cytoskeleton has three major constituents: microtubules, neurofilaments and actin (BOX 1). Each is unique, associating with its own set of binding proteins and performing specialized roles within the axon. Most of these cytoskeletal proteins are synthesized in the neu­ ronal soma and are transported along the axon. Such transport is a constitutive phenomenon that occurs throughout the life of the neuron. Thus, the axonal cytoskeleton is best understood by considering both its anatomical organization and its dynamics (including axonal transport). Given the complex morphology and physiology of neurons, the field of neurobiology has traditionally been at the forefront of adopting new optical techno­ logies as they arise. Advances in microscopy now allow us to observe cells with unprecedented spatial resolu­ tion and to follow dynamic processes with exquisite temporal detail 1. For example, super­resolution strat­ egies that circumvent the diffraction limit of optical microscopy appeared more than ten years ago (BOX 2) and have been used to reveal aspects of neuronal organ­ ization down to the scale of macromolecular com­ plexes 2. These techniques have provided key insights into the organization and function of the axonal cytoskeleton — and in particular the organization of actin and microtubules — revealing how it builds the axon and maintains its intricate architecture. Focusing on the cytoskeleton within the axon initial segment (AIS) and the more distal axon shaft, in this Review, we highlight these recent discoveries and place them in the context of earlier findings, giving the reader a tentative vision of the future. Overview of the axonal cytoskeleton The history of cytoskeletal research is essentially the pursuit of tools and techniques that allowed an ever­ closer view of this elaborate structure, a quest that continues to this day. Early studies by 17th­century microscopy pioneers highlighted a network of 'neu­ rofibrils' , which we now know were most likely neuro­ filaments 3. With the advent of electron microscopy, two types of fibrils were seen in axons: filaments measuring approximately 10 nm in diameter, corresponding to neurofilaments, and others measuring approximately 20–30 nm in diameter, corresponding to structures that eventually came to be known as microtubules 4,5. Abstract | The corporeal beauty of the neuronal cytoskeleton has captured the imagination of generations of scientists. One of the easiest cellular structures to visualize by light microscopy, its existence has been known for well over 100 years, yet we have only recently begun to fully appreciate its intricacy and diversity. Recent studies combining new probes with super-resolution microscopy and live imaging have revealed surprising details about the axonal cytoskeleton and, in particular, have discovered previously unknown actin-based structures. Along with traditional electron microscopy, these newer techniques offer a nanoscale view of the axonal cytoskeleton, which is important for our understanding of neuronal form and function, and lay the foundation for future studies. In this Review, we summarize existing concepts in the field and highlight contemporary discoveries that have fundamentally altered our perception of the axonal cytoskeleton

Fluorescence Microscopy with Nanometer Resolution

Throughout the twentieth century, it was widely accepted that a light microscope relying on propagating light waves and conventional optical lenses could not discern details that were much finer than about half the wavelength of light, or 200−400nm, due to diffraction. However, in the 1990s, the potential for overcoming the diffraction barrier was realized, and microscopy concepts were defined that now resolve fluorescent features down to molecular dimensions. This chapter discusses the simple yet powerful principles that make it possible to neutralize the resolution-limiting role of diffraction in far-field fluorescence nanoscopy methods such as STED, RESOLFT, PALM/"​"​STORM, or PAINT. In a nutshell, feature molecules residing closer than the diffraction barrier are transferred to different (quantum) states, usually a bright fluorescent state and a dark state, so that they become discernible for a brief period of detection. With nanoscopy, the interior of transparent samples, such as living cells and tissues, can be imaged at the nanoscale. A fresh look at the foundations shows that an in-depth description of the basic principles spawns powerful new concepts. Although they differ in some aspects, these concepts harness a local intensity minimum (of a doughnut-shaped or a standing wave pattern) for determining the coordinate of the fluorophore(s) to be registered. Most strikingly, by using an intensity minimum of the excitation light to establish the fluorophore position, MINFLUX nanoscopy has obtained the ultimate (super)resolution: the size of a molecule (≈1nm)