Summary Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based on fluorescence microscopy with conventional resolution, however, suggested only modest compaction differences (∼2-10x) between active and inactive nuclear compartments (ANC and INC). Here, we present maps of nuclear landscapes with true-to-scale DNA-densities, ranging from &lt;5 Mbp/µm 3 to &gt;300 Mbp/µm 3 . Maps were generated from individual human and mouse cell nuclei with single-molecule localization microscopy at ∼20 nm lateral and ∼100 nm axial resolution and supplemented by electron spectroscopic imaging. Microinjection of fluorescent nanobeads with sizes corresponding to macromolecular assemblies for transcription and replication into nuclei of living cells, demonstrated their localization and movements within the ANC and exclusion from the INC.

True-to-scale DNA-density maps correlate with major accessibility differences between active and inactive chromatin

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