Super-resolution Microscopy - Applications in Plant Cell Research.

Veit Schubert

doi:10.3389/fpls.2017.00531

Abstract

Most of the present knowledge about cell organization and function is based on molecular and genetic methods as well as cytological investigations. While electron microscopy allows identifying cell substructures until a resolution of ∼1 nm, the resolution of fluorescence microscopy is restricted to ∼200 nm due to the diffraction limit of light. However, the advantage of this technique is the possibility to identify and co-localize specifically labeled structures and molecules. The recently developed super-resolution microscopy techniques, such as Structured Illumination Microscopy, Photoactivated Localization Microscopy, Stochastic Optical Reconstruction Microscopy, and Stimulated Emission Depletion microscopy allow analyzing structures and molecules beyond the diffraction limit of light. Recently, there is an increasing application of these techniques in cell biology. This review evaluates and summarizes especially the data achieved until now in analyzing the organization and function of plant cells, chromosomes and interphase nuclei using super-resolution techniques.

Highlights

Light microscopy of DNA and proteins fluorescently labelled by fluorescence in situ hybridization (FISH) and immunostaining, respectively, as well as live cell imaging based on fluorescent recombinant proteins significantly increased our knowledge concerning cell organization and function, and is an important advantage compared to electron microscopy.due to the diffraction limit of light as defined by Abbe (1873) the spatial resolution of light microscopy including conventional fluorescence techniques is restricted, and reaches only ∼200 nm laterally and ∼600 nm in the axial dimension in biological specimens (Pawley, 1995)
Light microscopy of DNA and proteins fluorescently labelled by FISH and immunostaining, respectively, as well as live cell imaging based on fluorescent recombinant proteins significantly increased our knowledge concerning cell organization and function, and is an important advantage compared to electron microscopy
Most results obtained by super-resolution microscopy in plant cell research are concentrated in the fields of research groups with access to super-resolution microscopes

Summary

Introduction

Due to the diffraction limit of light as defined by Abbe (1873) the spatial resolution of light microscopy including conventional fluorescence techniques is restricted, and reaches only ∼200 nm laterally and ∼600 nm in the axial dimension in biological specimens (Pawley, 1995) This limited resolution did not allow identifying single molecules and structures with the resolution achieved by electron microscopy. To overcome this restriction and to bridge the resolution gap between light and electron microscopy the so-called super-resolution ( referred as optical nanoscopy) techniques SIM, PALM, STORM, and STED offering new insights into molecular structures, interactions and functions were developed. These “subdiffraction” methods can be divided into two different principles: (i) localization of individual fluorophores in the specimen with subdiffraction precision (PALM, STORM), and (ii) structuring the illumination light to collect high spatial frequencies in the image that contain high resolution information (SIM, STED) (Rego et al, 2012).

Methods

Results

Conclusion