Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes.

Abstract

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

Plant phenotyping relevance

植物染色体の構造を対象に、複数の顕微鏡法を比較評価し、解像度向上による表現型（染色体アーキテクチャ）取得を中心課題としている。

Code and data availability

The paper's super-resolution microscopy measurements (SIM/STED/PALM imaging of barley metaphase chromosomes) are supported by supplementary materials, including PALM movies (Movies S1–S4) and supplementary figures, publicly available at the MDPI supplement URL. The main datasets themselves are only 'available from the