We have presented a new technology for the analysis of interphase chromosome structure and dynamics in living human cells. The study provides basic quantitative measures for physical properties of chromosomes in living cells. Our data reveal that chromosome volume and morphology are established rapidly after mitosis, changing only marginally after the first hour of G1. This contrasts with the behaviour of a locus on chromosome 11, which appeared to have a more gradual, progressive spatial reorganisation. Bulk chromosome morphology and volume showed tremendous resistance to inhibitors of various nuclear functions, such as transcription, histone deacetylation and chromatin remodelling, although local chromatin changes and effects on nuclear compartments could clearly be detected. We also measured heterogeneity in chromosome decondensation, which may reflect inherited features from the mother chromosome, in addition to structural impediments to full decondensation.
Labelling single mitotic chromosomes allowed observation of their decondensation. By measuring local fluorescence intensities of a GFP tagged histone H2B in HeLa cells, a previous study measured a decondensation factor of five 
. For the reverse process, the condensation of chromatin prior to mitosis, an estimated decrease in volume of 2–3 fold was measured, in NRK cells 
. The same study found an axial shortening after metaphase which peaked 8–12 min after anaphase onset, so the absolute decondensation level may depend upon when the time during mitosis at which the chromosome was labelled and measured. Our technique allows measurement of the decondensation factor for single chromosomes. On average we found decondensation of over 300%, in rough agreement with published data, yet we also detected a high variability in decondensation, ranging from almost zero to over 7 fold. This may reflect the different gene density and transcriptional status of chromosomes 
. In addition, the obstacles of nuclear compartments, the lamina and other chromosomes have the potential to cause stochastic chromosome-wide effects on measured bulk volume, which is perhaps why we observed sizeable differences in measured volumes between decondensing sister chromatids. This raises the question of whether such a structural impediment could impede gene expression.
Morphological definition of decondensing chromosomes was near complete within the first hour after mitosis (). Beyond this interval, bulk chromosome morphology and volume displayed only incremental changes. This implies the major physical reactions of the nucleus, such as nucleolus formation, space definition of chromosome territories, and establishment of nuclear space with respect to the cytoplasm, have been completed. It would be interesting to observe whether this fixity of position and morphology holds for motile cells, such as neutrophils, which must continuously redefine their cytoskeletons. This may place additional forced dynamicity on interphase chromosome morphology and relative position.
Labelling the chromosomes in mitosis also allows us to compare the daughter chromosomes, conditioned by the same inheritance and cell cycle stages. By eye it was obvious that daughter chromosomes could acquire very different morphologies, and this was supported by volume measurements. Morphology is therefore clearly neither an intrinsic property of chromosomes, nor a completely lineage-dependent one. A simple, perhaps less satisfying hypothesis, is that chromosomes simply fill the space they fall into, which will be defined by interchromosome interactions, chromatin interactions with the lamina and other nuclear compartments, and above all, by the constraints of the cytoplasm on the nucleus. Chromosome architecture will describe the balance between the needs for chromosome decondensation and accessibility with the constraints of the available space.
These balances may also explain the apparent resistance of chromosome architecture to treatments affecting major nuclear functions. Chromosome morphology was not significantly perturbed by blocking transcription, histone deacetylation, ATP synthesis or topisomerase II. These activities have previously been shown to be required for the positioning and dynamics of individual chromosome loci. And we have clearly seen perturbations of nuclear architecture by these treatments in the same cells which fail to lose chromosome structural integrity, in addition to anecdotal effects on morphology of a few chromosomes. These results may explain the ambivalent findings in the literature on the effects of transcription and acetylation on chromatin and chromosome morphology 
. Physical and functional constraints are likely to be multifactorial, and effects of depleting one or another of these factors will be masked by other stabilising forces. Perhaps more simplistically, physical constraints will mean there are places a single locus can go that a chromosome cannot.
Technical challenges that remain are generic to live cell imaging. How can cells be imaged at high spatial resolution, for long periods of time with minimal bleaching and phototoxicity? Another limitation of our approach, which is generic to many types of imaging, is the method used to define the edge of the chromosome region. Whether by automated script or by eye, there is always a user-defined aspect to thresholding. The existence of “looped-out” chromosome regions implies the threshold applied is generally too severe, with the edge defined to close to the chromosome centroid. How a user is supposed to improve upon current definition, without clear hybridisation or photolabel signal, is at present unclear. It is likely the unknown dispersal beyond the “visible” edge will impact on measurements, in this study and elsewhere.
A further issue with so-called caged fluorescent proteins is their tendency to activate (uncage) under normal illumination. In the present study, we have counteracted these issues by restricting the numbers of 3D stacks we captured after mitosis. This was especially important when carrying out 3 colour imaging, involving detection of fluors with overlapping spectra. Another persistent issue is the use of drugs to stall mitosis, to allow a greater number of chromosomes to be labelled in a given imaging routine. Greater automation of the photoactivation protocol would diminish the need for these treatments.
Live cell technologies provide greater temporal resolution of cellular events than extrapolations from fixed cells. They also provide mechanistic hypotheses not foreseeable from fixed material, or homogenous population extracts. It is easier to understand a process when one can observe the object of ones interest before, during and after a particular process or treatment. The ability to view single chromosome architecture in living cells is imperative to our future understanding of the functional nuclear landscape. This will, in turn, be fuelled by improvements in fluorescent protein photostability and efficiency, detection sensitivity, and automated capture and analysis routines, with the goal of imaging a chromosome and its loci through a whole cell cycle, from decondensation to condensation. This will allow us to begin to address the problem of how a chromosome folds.