Super‐Resolution Microscopy: A Comparative Treatment

Abstract

One of the fundamental limitations of optical microscopy is that of diffraction, or in essence, how small a beam of light can be focused by using an optical lens system. This constraint, or barrier if you will, was theoretically described by Ernst Abbe in 1873 and is roughly equal to half the wavelength of light used to probe the system. Many structures, particularly those within cells, are much smaller than this limit and thus are difficult to visualize. Over the last two decades, a new field of super-resolution imaging has been created and been developed into a broad range of techniques that allow routine imaging beyond the far-field diffraction limit of light. In this unit we outline the basic principles of the various super-resolution imaging modalities, paying particular attention to the technical considerations for biological imaging. Furthermore, we discuss their various applications in the imaging of both fixed and live biological samples.

Comparative performance of airyscan and structured illumination superresolution microscopy in the study of the surface texture and 3D shape of pollen

“…Because the grid projection is structured on the Z-axis, the optical sectioning resolution is improved two-fold compared with the standard confocal. Therefore, resolutions of 120 nm laterally (XY) and 250 nm axially (Z) can be achieved depending on the wavelength used (Allen et al, 2014;Dan, Yao, & Lei, 2014;Gustafsson, 2000;Gustafsson et al, 2008;Jost & Heintzmann, 2013;Kasuboski, Sigal, Joens, Lillemeier, & Fitzpatrick, 2012;Long et al, 2014).…”

Optical microscopy with super-resolution by liquid-immersed high-index microspheres

“…According to Abbe [5] object features separated by less than 0.5λ/NA cannot be resolved, where λ is the free-space wavelength of illumination light and NA is the numerical aperture of the system defined by NA=n×sinθ, where n is the object-space refractive index and θ is the half-angle of the acceptance cone of the objective lens. For conventional white-light microscope systems the Abbe limit is estimated ~250 nm [6].…”

Superresolution live imaging of plant cells using structured illumination microscopy

“…protocol 1250 | VOL.10 NO.8 | 2015 | nature protocols SR methods. Exhaustive studies comparing SIM with PALM/ STORM, STED and other SR modes can be found in the literature 19,38,39 , and therefore will be only briefly summarized here.Currently, SIM seems to be the best compromise when considering speed, temporal resolution, field of view (FOV) and depth of view, phototoxicity, fluorophore specifications and ease of postacquisition image processing. By principle, SIM is a true WF approach, and therefore temporal imaging is only limited by grid rotations and phase shifts upon acquisition 19 .…”

“…protocol 1250 | VOL.10 NO.8 | 2015 | nature protocols SR methods. Exhaustive studies comparing SIM with PALM/ STORM, STED and other SR modes can be found in the literature 19,38,39 , and therefore will be only briefly summarized here.…”