Super‐resolution imaging for cell biologists

Fornasiero, Eugenio F.; Opazo, Felipe

doi:10.1002/bies.201400170

Cited by 126 publications

(82 citation statements)

References 134 publications

(128 reference statements)

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“…Although the combination of DNA-FISH and immunostaining resulted in apparently well-maintained chromatin morphology [4], it should be considered that the usually preferred conventional preparation techniques in super-resolution microscopy determine the practical limits of application [10]. Fornasiero et al [11] presented an overview of the advantages and disadvantages of the high-resolution microscopy methods regarding various biological tasks. A challenge that must not be underestimated in this context of circumventing possible limits is the development of adequate computer-based analysis methods for the evaluation of the high-resolution data.…”

Section: Introductionmentioning

confidence: 99%

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

Krufczik

Sievers

Hausmann

et al. 2017

IJMS

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Immunostaining and fluorescence in situ hybridization (FISH) are well established methods for specific labelling of chromatin in the cell nucleus. COMBO-FISH (combinatorial oligonucleotide fluorescence in situ hybridization) is a FISH method using computer designed oligonucleotide probes specifically co-localizing at given target sites. In combination with super resolution microscopy which achieves spatial resolution far beyond the Abbe Limit, it allows new insights into the nano-scaled structure and organization of the chromatin of the nucleus. To avoid nano-structural changes of the chromatin, the COMBO-FISH labelling protocol was optimized omitting heat treatment for denaturation of the target. As an example, this protocol was applied to ALU elements—dispersed short stretches of DNA which appear in different kinds in large numbers in primate genomes. These ALU elements seem to be involved in gene regulation, genomic diversity, disease induction, DNA repair, etc. By computer search, we developed a unique COMBO-FISH probe which specifically binds to ALU consensus elements and combined this DNA–DNA labelling procedure with heterochromatin immunostainings in formaldehyde-fixed cell specimens. By localization microscopy, the chromatin network-like arrangements of ALU oligonucleotide repeats and heterochromatin antibody labelling sites were simultaneously visualized and quantified. This novel approach which simultaneously combines COMBO-FISH and immunostaining was applied to chromatin analysis on the nanoscale after low-linear-energy-transfer (LET) radiation exposure at different doses. Dose-correlated curves were obtained from the amount of ALU representing signals, and the chromatin re-arrangements during DNA repair after irradiation were quantitatively studied on the nano-scale. Beyond applications in radiation research, the labelling strategy of immunostaining and COMBO-FISH with localization microscopy will also offer new potentials for analyses of subcellular elements in combination with other specific chromatin targets.

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Section: Introductionmentioning

confidence: 99%

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

Krufczik

Sievers

Hausmann

et al. 2017

IJMS

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“…Consequently, they are used to label structures and molecules of interest. After fixation of a specimen, which should alter the native structures as less as possible, specific fluorescent affinity probes of preferably small size (FAB fragments of antibodies, nanobodies, snap-tags) may be used for labeling (Fornasiero and Opazo, 2015). …”

Section: Specimen Preparation and The Super-resolution Methodsmentioning

confidence: 99%

“…However, due to phototoxicity live cell nanoscopy is much more challenging than imaging fixed specimens (Fornasiero and Opazo, 2015). Hence, fewer live cell imaging results have been published so far in plant cell research ( Table 1 ).…”

Section: Specimen Preparation and The Super-resolution Methodsmentioning

confidence: 99%

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Super-resolution Microscopy – Applications in Plant Cell Research

Schubert

2017

Front. Plant Sci.

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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.

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“…Fortunately, super-resolution°uores-cence imaging with rapid development is a promising valuable tool in the carbohydrate study of cells, because of its predominant resolution at the nanoscale level. 29,30 In this review, we mainly introduce the principle of super-resolution imaging, the°uorescent probes available for super-resolution imaging, some common labeling methods for carbohydrates, and the applications of super-resolution imaging in the study of glycoscience.…”

Section: Introductionmentioning

confidence: 99%

Super-resolution imaging in glycoscience: New developments and challenges

Chen

Tong

Wang

2016

J. Innov. Opt. Health Sci.

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Carbohydrates on cell surfaces play a crucial role in a wide variety of biological processes, including cell adhesion, recognition and signaling, viral and bacterial infection, in°ammation and metastasis. However, owing to the large diversity and complexity of carbohydrate structure and nongenetically synthesis, glycoscience is the least understood¯eld compared with genomics and proteomics. Although the structures and functions of carbohydrates have been investigated by various conventional analysis methods, the distribution and role of carbohydrates in cell membranes remain elusive. This review focuses on the developments and challenges of super-resolution imaging in glycoscience through introduction of imaging principle and the available°uorescent probes for super-resolution imaging, the labeling strategies of carbohydrates, and the recent applications of super-resolution imaging in glycoscience, which will promote the super-resolution imaging technology as a promising tool to provide new insights into the study of glycoscience.

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Super‐resolution imaging for cell biologists

Cited by 126 publications

References 134 publications

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

Super-resolution Microscopy – Applications in Plant Cell Research

Super-resolution imaging in glycoscience: New developments and challenges

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