Localization microscopy of DNA in situ using Vybrant{sup ®} DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution

Journal Article · Sun May 01 00:00:00 EDT 2016 · Experimental Cell Research

DOI:https://doi.org/10.1016/J.YEXCR.2015.08.020· OSTI ID:22648563

Żurek-Biesiada, Dominika ^[1]; Szczurek, Aleksander T. ^[2]; Prakash, Kirti ^[2]; Mohana, Giriram K. ^[2]; Lee, Hyun-Keun ^[2]; Roignant, Jean-Yves ^[2]; Birk, Udo J. ^[2]; Cremer, Christoph ^[2]

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1. Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków (Poland)
2. Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz (Germany)

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant{sup ®} DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10{sup 6} signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100 nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. - Highlights: • Super-resolution imaging of nuclear DNA with Vybrant Violet and blue excitation. • 90nm resolution images of DNA structures in optically thick eukaryotic nuclei. • Enhanced resolution confirms the existence of DNA-free regions inside the nucleus. • Optimized imaging conditions enable multicolor super-resolution imaging.

• OSTI ID: 22648563
• Journal Information: Experimental Cell Research, Journal Name: Experimental Cell Research Journal Issue: 2 Vol. 343; ISSN 0014-4827; ISSN ECREAL
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
ACCURACY
APERTURES
BIOMEDICAL RADIOGRAPHY
BUFFERS
CELL NUCLEI
CHROMATIDS
CHROMATIN
CLONING
COMPACTS
COMPARTMENTS
CONTROL
DISTANCE
DNA
DNA REPLICATION
DYES
EXCITATION
FLUORESCENCE
GENES
ILLUMINANCE
IMAGES
MOLECULES
NANOSTRUCTURES
OPTICAL MICROSCOPY
PHOSPHATES
PROBES
PROCESSING
PRODUCTIVITY
RESOLUTION
RNA
SIGNALS
SPECIFICITY
STAINS
STANDARDS
VISIBLE RADIATION
WAVELENGTHS