From single molecules to life: microscopy at the nanoscale

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.Keywords Super-resolution microscopy . Photophysics and photochemistry of fluorophores . Live cell imaging . Quantitative cell biology Spatial and temporal scales in the life sciences and microscopyThe 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.Observing and understanding all of these components of life requires us to be, at best, passive witnesses of undisturbed processes, but also to demand hard observational data that can allow us to quantitatively measure and trace all of the players involved-ranging from small molecules up to the interactions of whole cells in cellular communities-with the highest specificity and precision.To achieve this, instrumentation is needed that permits a wide three-dimensional view but also allows details to be

Advances in Imaging Plant Cell Dynamics

“…There is only one study available (Kleine-Vehn et al, 2011). This might relate to the extreme output of the laser lines used, rendering STED quite phototoxic and unsuitable for mid-to longterm observations of living plant samples (for review, see Turkowyd et al, 2016). The original commercial STED designs used continuous-wave (CW) lasers (CW-STED), increasing the photon load and phototoxicity.…”

Quantitative microscopy based on single-molecule fluorescence

“…They are especially useful for studying biological scaffold complexes or small organelles, whose sizes fall in the diffraction limit [18, 19, 27, 28, 29**]. This group of methods is based on the notion of photoswitchable single-molecule fluorescence and point-spread function localization: A single fluorophore will appear in an image as a blurred diffraction-limited spot, but the position of the fluorophore can be determined to within nanometers by performing centroid analysis, provided there aren’t any neighboring fluorophores within the diffraction-limited spot.…”

“…Currently, the tagging strategies available include the six-peptide sequence for labeling with a FlAsH dye (‘FlAsH tag’) inserted into a specific domain of proteins or a small SNAP/CLIP tag on C- or N- termini of proteins of interest combined with cell-permeable dyes for live-cell imaging [29]. However, the photophysical properties of FlAsH and SNAP/CLIP dyes were not rigorously tested for quantitative microscopy.…”

“…One obvious and important direction to improve these counting techniques is to expand their reach to measure dynamic events. However, dynamic molecular interactions in live cells can occur even within milliseconds [29**]. Counting single molecules at such fast rates will put severe demands on both the brightness of the probe and the sensitivity of the microscope.…”

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

“…In other SRM methods, the diffraction limit is overcome in two ways: The first one is spatial and/or temporal modulation of the transition between two molecular states of a fluorophore, and the second one is a narrowing of the point spread function of an ensemble image of many fluorophores located near each other. The main methods of the first group include stimulated emission depletion (STED), ground state depletion (GSD), structured illumination microscopy (SIM), and some of their combinations with I5M microscopy (4Pi variant) [6, 7]. The main methods of the second group are photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM), stochastic optical reconstruction microscopy (STORM), and binding-activated localization microscopy (BALM) [7, 8].…”