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Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast

  1. Vadim Backmana,2
  1. aBiomedical Engineering Department, Northwestern University, Evanston, IL 60208;
  2. bMechanical Engineering Department, Northwestern University, Evanston, IL 60208
  1. Edited by Gabriel Popescu, University of Illinois at Urbana–Champaign, Urbana, IL, and accepted by Editorial Board Member John A. Rogers July 8, 2016 (received for review February 9, 2016)

Significance

Fluorescence photoswitching of native, unmodified deoxyribonucleic acid (DNA) using visible light facilitates the label-free nanoscale imaging of chromatin structures based on the principle of single-molecule photon localization microscopy (PLM). With a demonstrated sub–20-nm resolution, DNA-PLM provides an ideal technique to visualize the spatial organization of single or groups of nucleosomes and quantitatively estimate the nucleosome occupancy level of DNA in unstained chromosomes and nuclei. This study paves a way for revealing nanoscopic features of chromatin without the need for exogenous labels and could substantially expand our understanding of the structure–function relationship of chromatin.

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.

Footnotes

  • ?1B.D. and L.M.A. contributed equally to this work.

  • ?2To whom correspondence may be addressed. Email: hfzhang{at}northwestern.edu or v-backman{at}northwestern.edu.
  • Author contributions: B.D., L.M.A., B.E.U., T.-Q.N., C.S., H.F.Z., and V.B. designed research; B.D., L.M.A., Y.S.-C., B.E.U., and J.E.C. performed research; B.D. and L.M.A. analyzed data; and B.D., L.M.A., H.F.Z., and V.B. wrote the paper.

  • Conflict of interest statement: H.F.Z. and C.S. have financial interests in Opticent Health; V.B. has financial interests in NanoCytomics. Neither of these companies funded this work.

  • This article is a PNAS Direct Submission. G.P. is a Guest Editor invited by the Editorial Board.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1602202113/-/DCSupplemental.

Freely available online through the PNAS open access option.

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