Time-resolved Microscopy of Chromatin In Vitro and In Vivo¶

Abstract

In eukaryotic cell nuclei, double-stranded DNA is found in the form of chromatin, a large fiber made up of DNA complexed to histone proteins. In this article, recent studies using fluorescence techniques to look at the dynamics of chromatin, both in vivo and in vitro, are reviewed. Two-photon counterpropagating fluorescence recovery after patterned photobleaching is used to examine chromatin fluctuations on lengthscales ranging from less than 100 nm to microns. By combining in vivo studies with data on isolated nuclei and by measuring how these fluctuations depend on variables like ionic strength and photochemical cross-linking, it is demonstrated that the relatively large-scale motions of chromatin observed in vivo are consistent with smaller scale modifications of the histone-DNA interaction. This connection may provide a means to use conformational dynamics as an in vivo probe of the biochemical events involved in gene expression.

Dynamic light scattering as an investigating tool to study the global internal dynamics of a living cell nucleus

“…The smooth modulations of the scattered intensity occur over times typically of the order of 30-40 s. We believe they likely arise from the diffusion of the chromatin within the chromosomal territories; using the diffusion coefficients given in refs. [3,10] and [11], we find a characteristic time (1/Dq 2 ) that is of the order of 30-60 s.…”

“…The scattering volume has a size of about 20-30 µm 3 , hence we can estimate that the number of chromosomal territories inside is of about 6-8. Inside the chromosomal territories there are both motions of chromatin fibres [3,[10][11][12][13][14], with diffusion coefficients of the order of 10 of chromatin fibres [14,15]. In the interchromosomal domain specific dynamics are also observed; in particular, displacements and changes of structure of the various nuclear bodies that are inside this space [16][17][18][19], as well as diffusion of macromolecular "complexes" (proteinic complexes, proteins, nucleotides .…”

Molecular Imaging of Cancer Cells Using Plasmon‐Resonant‐Enhanced Third‐Harmonic‐Generation in Silver Nanoparticles

“…The strong epi-THG contrast should thus be contributed from a single nanoparticle. With a virtual transition characteristic, the silver-nanoparticle-induced THG signals do not suffer photobleaching [36,37] and blinking [38][39][40][41] problems, and are ideal for future long-term observations, after reduction of nano-toxicity with proper surface treatment. With accurate control of particle size, plasmon wavelength could be manipulated with a much narrower spectral width.…”