Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy
Luay M. Almassalha a,1, Greta M. Bauer a,1, John E. Chandler a,1, Scott Gladstein a,1, Lusik Cherkezyan a, Yolanda Stypula-Cyrus a, Samuel Weinberg b, Di Zhang a, Peder Thusgaard Ruhoff c, Hemant K. Roy a,d, Hariharan Subramanian a, Navdeep S. Chandel b, Igal Szleifer a,e,f, and Vadim Backman a,f,2
a Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208;
b Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611;
c Institute of Technology and Innovation, University of Southern Denmark, DK-5230 Odense M, Denmark;
d Section of Gastroenterology, Boston Medical Center, Boston University School of Medicine, Boston, MA 02118;
e Department of Chemistry, Northwestern University, Evanston, IL 60208;
f Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208
Edited by Rebecca R. Richards-Kortum, Rice University, Houston, TX, and approved August 23, 2016 (received for review May 25, 2016)
Chromatin is one of the most critical structures within the cell because it houses most genetic information. Its structure is well understood at the nucleosomal (<20-nm and="" chromosomal="">200-nm) levels; however, due to the lack of quantitative imaging modalities to study this organization, little is known about the higher-order structure between these length scales in live cells.20-nm> We present a label-free technique, live-cell partial-wave spectroscopic (PWS) microscopy, with sensitivity to structures between 20 and 200 nm that can quantify the nanoarchitecture in live cells. With this technique, we can detect DNA fragmentation and expand on the link between metabolic function and higher-order chromatin structure. Live-cell PWS allows high-throughput study of the relationship between nanoscale organization and molecular function.
The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure–function relationship in live cells.
chromatin microscopy DNA damage mitochondrial metabolism cell dynamics
1L.M.A., G.M.B., J.E.C., and S.G. contributed equally to this work.
2To whom correspondence should be addressed.
Author contributions: H.K.R., H.S., N.S.C., I.S., and V.B. designed research; L.M.A., G.M.B., J.E.C., S.G., Y.S.-C., and S.W. performed research; L.C., D.Z., and P.T.R. contributed new reagents/analytic tools; L.M.A., G.M.B., J.E.C., S.G., L.C., S.W., D.Z., and P.T.R. analyzed data; and L.M.A., G.M.B., J.E.C., and S.G. wrote the paper.
Conflict of interest statement: V.B., H.S., and H.K.R. are cofounders of Nanocytomics, LLC.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1608198113/-/DCSupplemental.
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