Subdiffractive microscopy: techniques, applications, and challenges

Abstract

Cellular processes rely on the precise orchestration of signaling and effector molecules in space and time, yet it remains challenging to gain a comprehensive picture of the molecular organization underlying most basic biological functions. This organization often takes place at length scales below the resolving power of conventional microscopy. In recent years, several 'superresolution' fluorescence microscopic techniques have emerged that can surpass the diffraction limit of conventional microscopy by a factor of 2-20. These methods have been used to reveal previously unknown organization of macromolecular complexes and cytoskeletal structures. The resulting high-resolution view of molecular organization and dynamics is already changing our understanding of cellular processes at the systems level. However, current subdiffractive microscopic techniques are not without limitations; challenges remain to be overcome before these techniques achieve their full potential. Here, we introduce three primary types of subdiffractive microscopic techniques, consider their current limitations and challenges, and discuss recent biological applications.

Figure 1. The need for subdiffractive imaging and current commercially available subdiffractive approaches

A) Cartoon of two point sources of light separated by 150 nm and their overlapping PSFs (approximated by Gaussians) typical of confocal microscopy. Approximated PSF in the x-z plane is shown on the right at the same scale. B) Diffraction limited PSF overlaid to scale on an EM image of a dendritic spine, illustrating the difficulty of diffraction-limited microscopy in localizing synaptic proteins. C) The number of publications per year citing the use of each subdiffractive method (see Note for search terms). D) Appearance of two point sources separated by 150 nm via current commercially available subdiffractive microscopy methods. The PSF cartoons represent the effective precision associated with each method. Note: Data for Figure 1C were collected from scopus.com and limited to articles in journals using the search terms below. Data are plotted through 2012. Search terms (ar = article, j = journal) SIM: (TITLE-ABS-KEY(“structured illumination microscopy”) OR TITLE-ABS-KEY(“structured-illumination microscopy”)) AND (LIMIT-TO(DOCTYPE, “ar”)) AND (LIMIT-TO(SRCTYPE, “j”)) STED: ((TITLE-ABS-KEY(“stimulated-emission depletion” AND microscopy) OR TITLE-ABS-KEY(“stimulated emission depletion microscopy”) OR TITLE-ABS-KEY(“stimulated-emission-depletion fluorescence microscopy”) OR TITLE-ABS-KEY(“stimulated emission depletion nanoscopy”) OR TITLE-ABS-KEY(“RESOLFT”) OR TITLE-ABS-KEY(“stimulated emission depletion fluorescence nanoscopy”)) AND ( LIMIT-TO(DOCTYPE,”ar” ) ) AND ( LIMIT-TO(SRCTYPE,”j” ) ) ) LBM: ((TITLE-ABS-KEY(“stochastic optical reconstruction microscopy”) OR TITLE-ABS-KEY(“photoactivated localization microscopy”) OR TITLE-ABS-KEY(“photoactivation localization microscopy”) OR TITLE-ABS-KEY(“fluorescence photoactivation localization microscopy”) OR TITLE-ABS-KEY(“fluorescence photoactivated localization microscopy”) OR TITLE-ABS-KEY(“photoactivation-localization microscopy”)) AND ( LIMIT-TO(DOCTYPE,”ar” ) ) AND ( LIMIT-TO(DOCTYPE,”ar” ) ) AND ( LIMIT-TO(SRCTYPE,”j” ) ) )

Figure 2. The principles and representative images of the subdiffractive microscopic methods

Scale bars are 4 μm. Images in the top panel (actin in Hela cell) is used with permission and middle panel images (actin in embryonic chick fibroblast) are courtesy of Elise Stanley (Toronto Western Research Institute and University of Toronto). Bottom images (actin tagged with mEos3.2 in fox lung fibroblast) are our unpublished work.

Figure 3. Studies utilizing subdiffractive microscopic methods in neuroscience

A) The subsynaptic localization of both pre- and post-synaptic proteins examined using STORM. B) sptPALM was used to track actin and identify discrete regions of actin polymerization in dendritic spines. C) sptPALM measured AMPA receptor dynamics in cultured neurons. D) sptPALM data showed that the change in PKAc mobility upon increased cAMP concentration can be extracted from single molecule trajectories of PAFP-tagged PKAc. (BRL and HZ, unpublished) E) STORM revealed a previously unobserved periodic ring structure. containing actin, adducin and spectrin in the axons of cultured neurons. F) in vivo STED images showing the dynamic nature of EYPF labeled spines at high resolution, 10–15um below the surface of the cortex. All images used with permission from their respective publishers.

Figure 4. Subdiffractive imaging of protein complexes in eukaryote cell biology and microbiology

A) An iPALM system was used to map the spatial distribution of focal adhesion associated proteins in the z dimension. B) SIM was used to examine multiple centrosomal proteins. The images of each protein were aligned and averaged (left), and mapped to the center of the centriole (right) . C) STED microscopy combined with optimized sample preparation and antibody labeling showed that the centriolar protein cep164 makes a nine-cluster ring that can not be seen with confocal microscopy. D) SIM was used to map the arrangement of nuclear pore associated proteins. E) SIM was combined with particle averaging analysis to determine the organization of the y-shaped Nup107–160 complex (left) at the nuclear pore, which was further mapped onto cryo-EM structure of the pore complex (center, right). F) Live 3D SIM was used to track bacterial Z-ring proteins and show that they form dynamic clusters. All figures reproduced with permission from their respective publishers.