Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

Abstract

Single-molecule super-resolution fluorescence microscopy and single-particle tracking are two imaging modalities that illuminate the properties of cells and materials on spatial scales down to tens of nanometers, or with dynamical information about nanoscale particle motion in the millisecond range, respectively. These methods generally use wide-field microscopes and two-dimensional camera detectors to localize molecules to much higher precision than the diffraction limit. Given the limited total photons available from each single-molecule label, both modalities require careful mathematical analysis and image processing. Much more information can be obtained about the system under study by extending to three-dimensional (3D) single-molecule localization: without this capability, visualization of structures or motions extending in the axial direction can easily be missed or confused, compromising scientific understanding. A variety of methods for obtaining both 3D super-resolution images and 3D tracking information have been devised, each with their own strengths and weaknesses. These include imaging of multiple focal planes, point-spread-function engineering, and interferometric detection. These methods may be compared based on their ability to provide accurate and precise position information of single-molecule emitters with limited photons. To successfully apply and further develop these methods, it is essential to consider many practical concerns, including the effects of optical aberrations, field-dependence in the imaging system, fluorophore labeling density, and registration between different color channels. Selected examples of 3D super-resolution imaging and tracking are described for illustration from a variety of biological contexts and with a variety of methods, demonstrating the power of 3D localization for understanding complex systems.

Dynamic imaging of mitochondrial membrane proteins in specific sub-organelle membrane locations

“…On the other hand, in SPT/TALM, the diffusion coefficients are extracted from 2D projected movement, which results in an underestimation of mobility on tubular or spherical structures (Renner et al 2011). Albeit a variety of methods for obtaining both 3D super-resolution images and 3D tracking information have been devised (von Diezmann et al 2017), 3D tracking is still a challenging option and to our knowledge has not yet been applied to the analysis of the mobility of mitochondrial proteins. Hitherto, even 2D SPT/TALM has been feasible to elucidate sub-organelle localization from the pattern of the respective trajectory maps.…”

Through-focus or volumetric type of optical imaging methods: a review

“…Optical tools with numerous variations and techniques have become a major part of research and development in the bio-related fields. Usage of through-focus (TF) optical imaging is steadily gaining momentum, particularly in biological applications (9–16). TF imaging is sometimes informally and interchangeably referred to using terms such as volumetric, out-of-focus, blurred, defocused, extended-focused, extended-depth-of-field, axial scanning, and three-dimensional (3D) imaging.…”

Editorial Overview: Single-Molecule Approaches to Difficult Challenges in Folding and Dynamics

“…Single-molecule approaches have gained increasing acceptance in biomedical research and industry in the past two decades and have led to wide-ranging applications due to their unique abilities to study complex dynamics of macromolecules [1–4]. A variety of methods have become available, including single-molecule fluorescence microscopy [5, 6], single-molecule force spectroscopy [4], and nanopore technologies [7–9].…”

“…A variety of methods have become available, including single-molecule fluorescence microscopy [5, 6], single-molecule force spectroscopy [4], and nanopore technologies [7–9]. Single-molecule fluorescence detection has become the basis of many super-resolution imaging [1] and high-throughput DNA sequencing technologies [10]. In particular, single-molecule fluorescence resonance energy transfer (smFRET) [11] has been widely used to study conformational transitions of biomolecules.…”