We thank Dr. Sandra Ritz for letting us use the IMB Imaging Core Facility, Dr. Shih-Ya Chen for letting us use her custom-built SMLM microscope, Dr. Leonard Kubben (IMB) for providing human fibroblasts, Dr. Christof Niehrs (IMB) for providing the C3H10T1/2 cell line, and Dr. Jan Neumann for the MATLAB-Script that we have modified for this work. We also want to thank Dr. Marion Cremer, Dr. Thomas Cremer, and Dr. Christoph Cremer for fruitful discussions.

NOTE: Figure 3 gives an overview of the workflow described in this section. See the Table of Materials for details related to the reagents, materials, equipment, and software used in this protocol. The code used for this publication can be viewed and downloaded here:&#160;https://github.com/irradiator/Mapping-absolute-DNA-density-in-cell-nuclei-using-SMLM-microscopy.
1. Cell culture
<ol>
	<li>Cultivate C3H10T1/2, HFB, and HeLa c...

<ol>
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This article outlines how to measure absolute DNA densities in mammalian cell nuclei using SMLM. In addition, we have demonstrated how to determine the cell cycle stage of cultured cells and how to use this information to estimate the amount of DNA present in a light optical ultra-thin section. Also described in detail are the preparation of adherent cells for fBALM SMLM microscopy and how to process SMLM data to generate super-resolved microscopic images of genomic DNA in cell nuclei. Finally, the protocol shows how the...

Since the early days of cell biology, the cell nucleus-the seat of genetic information-has fascinated biologists. After applying self-developed cytological staining methods, Emil Heitz discovered, in 1928, differently, intensely stained areas within the cell nucleus1. He called the more intensely stained, dense areas &#34;heterochromatin&#34;, whereas he named the less strongly stained, less dense areas &#34;euchromatin&#34;. Over time, it became apparent that active genes are mostly located in euchromatin, while denser areas were found to be rich in repetitive elements and silent genes. The terms heterochromatin and euchromatin have survived to the present day although their definition has changed from structural to molecular properties (see below). Today we know that the cell nucleus is divided into two main phases. One liquid (the interchromatin compartment), which houses the nuclear bodies, the splicing compartment, and the nucleoplasm, and the other solid, hydrogel-like-the chromatin domain, which includes the chromosome territories and the nucleoli2. At the interface to the interchromatin space, chromatin is less dense, and this is where mostly the genetically active processes such as transcription, replication, and repair take place3,4,5, while adjacent to the lamina, around the nucleoli, and near the centromeres, chromatin shows high compaction levels and is in general transcriptionally rather inactive6. At the molecular level, chromatin is built from DNA wrapped around nucleosomes (an octamer of core histone proteins).
The histones possess intrinsically disordered tail domains that can be modified post-translationally by adding several chemical groups (methyl-, acetyl, phosphor-, biotin), amino-acids (arginine), or even proteins (SUMO, Ubiquitin)7. These modifications can be read and attract other proteins (e.g., chromatin remodeler, HP1, repair proteins, RNA) and thereby define the physiological state of the DNA sequence (transcriptionally permissive/restrictive) lying within, without directly changing it (therefore they are called &#34;epi&#34;genetic)7. The type and number of modifications around a specific sequence can differ profoundly between cell types, are reversible, and can change throughout development, senescence, and disease8,9,10. There are modifications of histone tails that are more prevalent in highly transcribed regions and modifications that predominate in regions of the genome that have little or no transcriptional activity.
For example, highly transcribed regions that are rich in the H3K4 modifications or whose histone tails are acetylated are usually described as euchromatin, whereas areas rich in repressive histone modifications (H3K9me3, H3K27me3, or H4K20me3) are referred to as heterochromatin, which is further divided into constitutive heterochromatin (transcriptionally inactive in all cells) (e.g., H4K20me3) and facultative heterochromatin (chromatin silenced depending on cell type, e.g., H3K27me3)6. Although biochemical and molecular biological methods are very well suited for measuring chemical changes at the nanoscale, it is much more challenging using them to make statements about spatial properties at the mesoscale using these methods.
For example, attempts have been made using contact data from Hi-C experiments, which detect and map close proximities of DNA between sequence regions, to reconstruct spatial models of the genome11. However, direct comparison with high-resolution light and electron microscopy images shows that this is only possible to a minor extent (Figure 1). Although methods such as Hi-C12, can detect chromosome territories in interphase cells correctly as separate entities, these models are rather &#34;near-sighted&#34;, because DNA sequence proximities alone are blind to reflect spatial relationships between more distant parts of the genome and thus are not very well suited for a proper 3D genome reconstruction. Therefore, density differences also cannot be reflected properly by contact frequency information. This leads to &#34;spaghettified&#34; representations of the genome in the nucleus (see Figure 1B), which appear to occur in wool-cluster-like, freely accessible loops.
To pave the way for an integrative, more realistic picture of the nucleus that combines biochemical information with biophysical and structural data, methods need to be developed that allow generating data on different aspects of nuclear organization. Until recently, it has also been difficult to determine absolute DNA densities in cell nuclei from structural data. The reasons for this were the limited resolution of conventional light microscopes, problems in electron microscopy to specifically stain DNA, and the small observation volumes in electron microscopic thin sections, which usually must be significantly thinner than a mammalian cell nucleus to be penetrable by electrons.
The resolution of conventional fluorescence microscopes is limited by the diffraction of light. The image of a point source of light is spread out due to the diffraction limit and can be described by the Point-Spread-Function (PSF). According to the PSF, the image of a fluorophore, which can be regarded in good approximation as a point source of light, occupies a volume of a certain size around its source of origin (fluorophore)13. When many fluorophores, located much closer to each other than the dimensions of their diffraction-limited images, are excited simultaneously, the imaged intensity distributions are superimposed, and the location of the single fluorophores cannot be resolved. Sequential, stochastic light emission from fluorophores (blinking) allows optical isolation of individual molecules and, thus, finding their exact locations by determining the intensity gravity centers of their signals.
This can be used to reconstruct structural information of samples by the accumulation of fluorophore localization data from many recorded images. This method is generally referred to as single-molecule localization microscopy (SMLM) (for further information see14). The more photons a fluorophore emits during its &#39;on-time,&#39; the more precisely can the intensity gravity centers be determined. Astigmatic lenses in the beam path transform signals of fluorophores above or below the optical focal plane into ellipses, which can be used to determine their position along the optical axis. The long axes of the ellipses originating from fluorescent signals below the focal plane are rotated at 90&#176; compared to the axes of the ellipses originating from fluorophores above the focal plane. Further, the axes-ratio of these ellipses allows determining the molecule&#39;s position along the optical axis relative to the focal plane within a range of &#177;300 nm15.
The quality of super-resolution images generated by the reconstruction of stochastic blinking events strongly depends on the labeling density and the number of blinking events. The latter depend on the photostability of the fluorophores and the number of blinking events before they eventually fail (number of on/off cycles). The method described here for obtaining super-resolution images of intranuclear DNA distribution is termed fBALM (DNA structure fluctuating-assisted binding-activated localization microscopy). It is based on fluorophores that transiently intercalate into nucleic acids and only fluoresce once they are bound to the DNA16,17. Owing to the charged residues of its phosphor-diester backbone, DNA is a highly negatively charged polymer. Stabilization of the complementary DNA strands in living cells requires neutralization by positively charged proteins (e.g., histones) and ions. By lowering the pH, the stability of the complementary pairing of the bases is reduced to allow intercalating dyes to diffuse in and out16,17.
Depending on the intercalating fluorescent dye, this state can be reached within a certain pH range. As fluorescent dyes such as YoYo-1 and SYTOX Orange only fluoresce when bound to DNA and because intercalating dyes (in contrast to dyes that bind the minor groove of the DNA such as Hoechst and 4&#39;,6-diamidino-2-phenylindole [DAPI]) usually bind in a sequence-independent manner, they are well-suited for mapping the distribution of DNA within cell-nuclei by localization microscopy.
Voronoi tessellation is a mathematical method allowing the subdivision of space into different partitions based on the location of the points. In 2D-Space, the size of the resulting tiles reflects the inverse of the density of points18. Because localization microscopy reconstructs images as a set of points that represent the location of fluorophores, Voronoi tessellation can help determinine&#160;the density of localization signals (Figure 2). Thus, using a DNA-specific dye as a fluorophore allows for measuring DNA densities.
A priori knowledge of the DNA content (number of base pairs) and the spatial dimension of the nucleus allows the transformation of the relative DNA densities into absolute DNA densities. The following protocol shows the mapping of absolute DNA densities in adherent cells using SMLM at a very high resolution and demonstrates that these densities are subject to large variations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>&#181;-Dish 35 mm, high Grid-500 Glass Bottom</td>
			<td>Ibidi</td>
			<td>81168</td>
			<td></td>
		</tr>
		<tr>
			<td>C3H 10T1/2</td>
			<td>IMB (Niehrs Lab)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>12320032</td>
			<td></td>
		</tr>
		<tr>
			<td>dPBS</td>
			<td>ThermoFisher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa</td>
			<td>Microscopy Core Facility (IMB)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HFB</td>
			<td>IMB (Kubben Lab)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential Aminoacids and vitamins for HFB</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>S8636</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Prep</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Merck</td>
			<td>2593710</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>ThermoFisher</td>
			<td>241922500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose Oxidase</td>
			<td>Merck</td>
			<td>49180</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Cocktail</td>
			<td>ThermoFisher</td>
			<td>AM2286</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Orange</td>
			<td>ThermoFisher</td>
			<td>S11368</td>
			<td></td>
		</tr>
		<tr>
			<td>TetraSpeck Fluorescent Microspheres Sampler Kit</td>
			<td>ThermoFisher</td>
			<td>T7284</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher</td>
			<td>327372500</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioFormats</td>
			<td>OpenMicroscopy.org</td>
			<td></td>
			<td>open source software https://www.openmicroscopy.org/bio-formats/</td>
		</tr>
		<tr>
			<td>CellProfiler v4.2.1</td>
			<td>CellProfiler.org</td>
			<td></td>
			<td>open source software https://cellprofiler.org</td>
		</tr>
		<tr>
			<td>FiJI</td>
			<td>nih.gov</td>
			<td></td>
			<td>open source software https://imagej.net/software/fiji/?Downloads</td>
		</tr>
		<tr>
			<td>LAND</td>
			<td>nih.gov</td>
			<td></td>
			<td>open source software https://github.com/Jan-NM/LAND</td>
		</tr>
		<tr>
			<td>MatLab 2021</td>
			<td>Math Works</td>
			<td></td>
			<td>commercial software - requires &#34;Image Processing Toolbox&#34;</td>
		</tr>
		<tr>
			<td>R v.4..0.3</td>
			<td>r-project.org</td>
			<td></td>
			<td>open source software https://www.r-project.org</td>
		</tr>
		<tr>
			<td>ThunderSTORM v1.3</td>
			<td></td>
			<td></td>
			<td>open source software https://zitmen.github.io/thunderstorm/</td>
		</tr>
		<tr>
			<td>Microscopes:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AF 7000</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica GSD</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SMLM Microscope</td>
			<td>Cremer Lab</td>
			<td></td>
			<td>custom-built by Dr. S-Y. Chen</td>
		</tr>
	</tbody>
</table>

mapping absolute dna density in cell nuclei using single-molecule localization microscopy

Within the cell nucleus, silent genes are generally located in chromatin areas of high density called heterochromatin, whereas active genes can be mostly found at the interface between chromatin and the interchromatin space called euchromatin. At present, the characterization of eu- and heterochromatin is mostly based on epigenetic modifications to histone proteins along the DNA sequence, while little is known about absolute DNA densities across the cell nucleus and their functional implications. Diagrams of the nucleus solely based on biochemical data and assumptions about the nature of chromatin as a polymer differ fundamentally from imaging data generated by high-resolution microscopy. This indicates that these methods are not well-suited for measuring density relationships in situ. We believe that spatial constraints might be involved in gene regulation and have therefore developed a method that allows the measurement of absolute DNA densities in mammalian cell nuclei by transforming super-resolution localization data into true-to-scale density maps by Voronoi tessellation.

The HeLa nucleus shown in Figure 5 was chosen from the table generated by CellProfiler4.2.1 in section 3 to have an integrated fluorescence intensity close to the first peak in the histogram shown in Figure 5 that represents nuclei in the G1 phase. Given its small size and pronounced inner structure, it is likely to be an early G1 nucleus that is still in the process of decondensing its chromatin after mitosis. Being in G1 means ...

The present protocol describes a method measuring absolute DNA densities within adherent cell nuclei using Voronoi tessellation of single-molecule localization microscopy data, known volume, genome size, and cell cycle stage.

Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy

Within the cell nucleus, silent genes are generally located in chromatin areas of high density called heterochromatin, whereas active genes can be mostly ...

Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy

Coming Soon!