Abstract
Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging.
Original language | English (US) |
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Pages (from-to) | 9716-9721 |
Number of pages | 6 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 113 |
Issue number | 35 |
DOIs | |
State | Published - Aug 30 2016 |
Funding
We thank Prof. Michael R. Wasielewski for the support in photochemistry studies and Dr. Ryan M. Young, Mr. Yilei Wu, and Ms. Jenna Logsdon for assistances in spectroscopic measurements. We acknowledge the generous financial support from the National Institutes of Health (Grants T32GM008152, T32HL076139, R01EY019951, R24EY022883, and R01CA165309), the National Science Foundation (Grants DBI-1353952, CBET-1055379, CBET-1066776, EFRI-1240416, and EEC-1530734), the Chicago Biomedical Consortium Lever Award L-006 for the Chicago Center for Physical Science Oncology Innovation and Translation, and a Research Catalyst Award by Northwestern McCormick School of Engineering.
Keywords
- Chromatin topology
- Chromosome
- Label-free imaging
- Nucleic acids
- Superresolution fluorescence microscopy
ASJC Scopus subject areas
- General