Research Article: From single molecules to life: microscopy at the nanoscale

Date Published: September 9, 2016

Publisher: Springer Berlin Heidelberg

Author(s): Bartosz Turkowyd, David Virant, Ulrike Endesfelder.

http://doi.org/10.1007/s00216-016-9781-8

Abstract

Super-resolution microscopy is the term commonly given to fluorescence microscopy techniques with resolutions that are not limited by the diffraction of light. Since their conception a little over a decade ago, these techniques have quickly become the method of choice for many biologists studying structures and processes of single cells at the nanoscale. In this review, we present the three main approaches used to tackle the diffraction barrier of ∼200 nm: stimulated-emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM). We first present a theoretical overview of the techniques and underlying physics, followed by a practical guide to all of the facets involved in designing a super-resolution experiment, including an approachable explanation of the photochemistry involved, labeling methods available, and sample preparation procedures. Finally, we highlight some of the most exciting recent applications of and developments in these techniques, and discuss the outlook for this field.

Partial Text

The timescales and spatial scales of the processes and molecules associated with life span extremely broad ranges, covering many orders of magnitude (Fig. 1). For instance, intracellular regulation (e.g., conformational changes or biochemical reactions within molecules) takes place at submillisecond timescales, nanosized molecules such as ATP (which serves the energy demands of cells) diffuse in milliseconds through cell volumes ranging from several micrometers up to millimeters, while (clustered) membrane receptors move at speeds that are about a magnitude slower. Large multicomponent machineries realize and control complex multilayered cellular functions that occur in seconds to hours. The ribosome, a large macromolecule which consists of two functional subunits of several dozen proteins on nucleic acid chain scaffolds, takes a matter of seconds to synthesize new peptide chains comprising hundreds of amino acids, which then quickly fold up into functional proteins. On the other hand, the replication of a full genome requires at least about 40 min for the 4.6 million nucleic acid base pairs of the bacterium Escherichia coli, and the cellular division cycle ranges from tens of minutes for E. coli to several hours for mammalian cells.Fig. 1a–bSpatial and temporal scales in the life sciences and microscopy. a Selected characteristic submicrometer objects are separated on the basis of biological (above the axis, green) and technical (below the axis, blue) significance. The IgG antibody structure (15 nm) contains two other notable structures: the antigen-binding region, called the Fab fragment (10 nm, blue) and the single-variable domain (3 nm, red), from which so-called nanobodies from cameloids are derived. Structures are taken from the PDB [GFP 1KYS, IgG 1IGT, SNAP 3KZZ, DNA 4LEY] and PubChem [ATP CID 5957, Alexa Fluor 647 CID 102227060]. b Timescales of various important biological processes (above the axis, green) and physical events, as well as typical timescales associated with microscopy procedures (below the axis, blue)

The resolution of light microscopy is often introduced via the Rayleigh criterion. Light from point-like sources is convolved by the so-called point-spread function (PSF) of an optical system when transmitted through a diffraction-limited microscope (Fig. 2a). In 1896, Lord Rayleigh defined the maximum resolution of an optical system as the minimum distance between two point-like objects which can be separated as individual sources. He regarded two point sources of equal strength as just discernible when the main diffraction maximum of one image coincides with the first minimum of the other. For an epifluorescence microscope with a circular aperture where the light is collected with the same objective, this yieldsdocumentclass[12pt]{minimal}
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Direct observations of the molecular processes that take place in cells can help to advance our understanding of life and how the complex interdependencies of single molecules enable it. Using super-resolution microscopy, we can follow these molecules, measure diffusion and progress in assembly processes, and quantify the molecules in subcellular structures at unrivaled spatiotemporal resolution. Over the past decade, rapid developments in these techniques have created a wide spectrum of advanced experimental settings that have already unraveled several mysteries associated with cells, some of which are depicted in Fig. 4 and are briefly summarized below.Fig. 4a–bQuantitative super-resolution microscopy. a SMLM allows the stoichiometry of a molecule to be determined, with several over- or undercounting effects taken into account. (i) The photochemical properties of fluorescent proteins lead to specific blinking and bleaching behaviors. The high-blinking and fast-bleaching behaviors shown by mEos2 (left) and Dendra2 (right), respectively, are largely determined by the orientation of the single residue arginine 66. Reprinted with permission from [191]. (ii) Fluorophore blinking behavior can be corrected for using kinetic fluorophore schemes. In this strategy, the number of FliM proteins per flagellar motor is counted in vivo. Reprinted from [192]. (iii) Spatial organization of E. coli RNA polymerases under minimal as well as rich growth conditions. Reprinted with permission from [193]. (iv) Maturation of endocytic vesicles into late endosomes. Reprinted from [194]. (b) Structural super-resolution microscopy reveals the molecular architecture of cellular multicomponent complexes. (i) Mutual organization of various pre- and postsynaptic proteins in relation to the proteins Bassoon and Homer1. Reprinted with permission from [139]. (ii) Combining data from identical particles yields a high-resolution average. Systematic SMLM imaging of the Y-shaped subunit of the nuclear pore complex allows it to be aligned onto the electron density of the nuclear pore (bottom). Reprinted with permission from [104]. (iii) Aligning different pairs of synaptonemal proteins onto a helical template yields the three-dimensional model of the synaptonemal complex with isotropic resolution. Reprinted from [105]. Scale bars: aii and aiii 500 nm; aiv 100 nm; bi 200 nm; biii 2 μm

Over the last few decades, super-resolution microscopy has proven its value in the life sciences, and a myriad of biological applications of super-resolution microscopy have emerged. The super-resolution toolbox currently consists of many diverse methods and application strategies that complement traditional cell biology studies as well as techniques from molecular biology or biochemistry. Super-resolution microscopy is on its way to becoming a standard research tool, which is leading to a huge demand for computer-based data processing and openly available analysis software for (advanced) data evaluation, visualization, and comparison, as well as accessible, affordable, and simple-to-use hardware implementations. Also, super-resolution microscopy traditionally yields large volumes of microscopic data, which would ideally be stored and handled in open-access public platforms. Whether this vision comes to pass largely depends on the development of new algorithms as well as open-source software and strategies for efficient large-scale data handling.

 

Source:

http://doi.org/10.1007/s00216-016-9781-8

 

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