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 two to twenty. 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.

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

“…The Elyra S1 SR-SIM light path (Carl Zeiss, Jena, Germany) is illustrated in Figure 1A. SR-SIM is a recently developed optical superresolution microscopy method with increasing applications in the life sciences (Habuchi, 2014;Han, Li, Fan, & Jiang, 2013;Huang, Bates, & Zhuang, 2010;Leung & Chou, 2011;Long, Robinson, & Zhong, 2014). It is a fluorescence technique with a light path similar to a widefield fluorescence microscope, but with a moving optical grid of parallel lines that is projected onto the sample in the lateral (XY) and axial (Z) directions.…”

“…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).…”

Chromosomes at Work: Organization of Chromosome Territories in the Interphase Nucleus

“…Conventional microscopy resolution is constrained by the diffraction limit of light. Super resolution microscopy [Markaki et al, 2012;Lakadamyali and Cosma, 2015] can increase resolution up to 20 fold over conventional microscopy [Long et al, 2014].…”

SmartScope2: Simultaneous Imaging and Reconstruction of Neuronal Morphology

“…The forefront of bioimaging technology is often driven by a desire for faster collection of larger image volumes, with increased spatial and temporal resolution. This has led to numerous technological developments, including great strides in optical engineering, new optical techniques such as multiphoton fluorescence microscopy 1 , light-sheet imaging 2 , and several modes of super-resolution microscopy 3 . This trend creates large and complex biological image data sets, leading to ongoing challenges in image visualization and analysis 4 .…”