Direct Imaging of DNA in Living Cells Reveals the Dynamics of Chromosome Formation

Individual chromosomes are not directly visible within the interphase nuclei of most somatic cells; they can only be seen during mitosis. We have developed a method that allows DNA strands to be observed directly in living cells, and we use it to analyze how mitotic chromosomes form. A fluorescent analogue (e.g., Cy5-dUTP) of the natural precursor, thymidine triphosphate, is introduced into cells, which are then grown on the heated stage of a confocal microscope. The analogue is incorporated by the endogenous enzymes into DNA. As the mechanisms for recognizing and removing the unusual residues do not prevent subsequent progress around the cell cycle, the now fluorescent DNA strands can be followed as they assemble into chromosomes, and segregate to daughters and granddaughters. Movies of such strands in living cells suggest that chromosome axes follow simple recognizable paths through their territories during G2 phase, and that late replicating regions maintain their relative positions as prophase chromosomes form. Quantitative analysis confirms that individual regions move little during this stage of chromosome condensation. As a result, the gross structure of an interphase chromosome territory is directly related to that of the prophase chromosome

Spatially-controlled illumination microscopy: For prolonged live-cell and live-tissue imaging with extended dynamic range

Live-cell and live-tissue imaging using fluorescence optical microscopes presents an inherent trade-off between image quality and photodamage. Spatially-controlled illumination microscopy (SCIM) aims to strike the right balance between obtaining good image quality and minimizing the risk of photodamage. In traditional imaging, illumination is performed with a spatially-uniform light dose resulting in spatially-variable detected signals. SCIM adopts an alternative imaging approach where illumination is performed with a spatially-variable light dose resulting in spatially-uniform detected signals. The actual image information of the biological specimen in SCIM is predominantly encoded in the illumination profile. SCIM uses real-time spatial control of illumination in the imaging of fluorescent biological specimens. This alternative imaging paradigm reduces the overall illumination light dose during imaging, which facilitates prolonged imaging of live biological specimens by minimizing photodamage without compromising image quality. Additionally, the dynamic range of a SCIM image is no longer limited by the dynamic range of the detector (or camera), since it employs a uniform detection strategy. The large dynamic range of SCIM is predominantly determined by the illumination profile, and is advantageous for imaging both live and fixed biological specimens. In the present review, the concept and working mechanisms of SCIM are discussed, together with its application in various types of optical microscopes

Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules

Most chromatin in interphase nuclei is part of condensed chromatin domains. Previous work has indicated that transcription takes place predominantly at the surface of chromatin domains, that is, in the perichromatin region. It is possible that genes inside chromatin domains are silenced due to inaccessibility to macromolecular components of the transcription machinery. We have tested the accessibility of chromatin domains in nuclei of living cells with proteins and dextrans of different molecular sizes. Our results show that chromatin domains are readily accessible to large macromolecules, including proteins with a molecular weight of several hundred kilodaltons. Therefore, the silencing of genes that are incorporated into such domains is not due to the physical inaccessibility of condensed chromatin domains to transcription factors

Media 4: Re-scan confocal microscopy: scanning twice for better resolution

Originally published in Biomedical Optics Express on 01 November 2013 (boe-4-11-2644

Media 1: Re-scan confocal microscopy: scanning twice for better resolution

Originally published in Biomedical Optics Express on 01 November 2013 (boe-4-11-2644