Max-Planck-Institut für Multidisziplinäre Naturwissenschaften
Chromatin-Markierung und Bildgebung
Eine deutsche Beschreibung unserer Forschung wird in Kürze verfügbar sein.
Localization of chromosomes and even specific DNA sequences in the nuclear space play crucial role in the gene expression regulation during cell cycle, oncogenesis and neuronal signalling. Nucleus morphology has been used for diagnostics of various types of cancers for decades. However, these experiments are still performed mostly on fixed cells and suffer from such artifacts as structure modification or loss during sample preparation. The modern super-resolution fluorescent microscopy techniques allow imaging of nucleus in living cells at resolution which is only slightly lower compared to electron microscopy (Figure 1), thus providing an obvious advantage. For example, by using quantitative super-resolution microscopy it has been recently demonstrated that chromatin packing density (number and size of nanodomains) strongly correlates with the pluripotency potential of induced pluripotent stem cells.
Figure 1. Complexity levels of chromatin organization. Diffraction limit hampers detailed investigation of chromatin structures starting at chromatin loop level. Super-resolution microscopy (SIM, STED, PALM/STORM and MINFLUX) overcomes this barrier and permits imaging of chromatin structures down to nucleosome level.
Figure 1. Complexity levels of chromatin organization. Diffraction limit hampers detailed investigation of chromatin structures starting at chromatin loop level. Super-resolution microscopy (SIM, STED, PALM/STORM and MINFLUX) overcomes this barrier and permits imaging of chromatin structures down to nucleosome level.
Figure 2. Example image of DNA binding probe in nucleus of living cell showing more detailed structure in STED image as compared to confocal. Scale bar = 2 µm.
Figure 2. Example image of DNA binding probe in nucleus of living cell showing more detailed structure in STED image as compared to confocal. Scale bar = 2 µm.
A wider application of super-resolution imaging requires development of fluorescent probes for specific labelling. These probes are constructed by coupling fluorescent dye to a small molecule ligand, targeting nuclear components of interest. A large variety of inhibitors, cofactor analogues and binders targeting proteins and DNA are available, thus successful development of chromatin probes is feasible. However direct tagging of chromatin proteins, specific DNA sequences or DNA modifications impose a challenge of identifying correct combination of targeting moiety, linker and fluorophore.
Figure3. Biocompatibility of rhodamines can be enhanced by neighbouring group effect which changes the equilibrium between spirolactone and zwitterion. This enhances cell permeability and staining efficiency allowing acquisition of excellent quality 3D STED nanoscopy images.
Figure3. Biocompatibility of rhodamines can be enhanced by neighbouring group effect which changes the equilibrium between spirolactone and zwitterion. This enhances cell permeability and staining efficiency allowing acquisition of excellent quality 3D STED nanoscopy images.
Gražvydas Lukinavičius und seinem Team ist es gelungen, Farbstoffe für die Fluoreszenzmikroskopie so zu optimieren, dass sie besser in lebende Zellen gelangen. Durch Kombination mit der 3D-STED-Mikroskopie hat die Forschergruppe winzige Strukturen des Zellskeletts von nur 23 Nanometern Durchmesser sichtbar gemacht.
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