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Comparison of different excitation wavelengths for imaging: [left] MPM images of the same region at the same depth in the liver at different imaging wavelengths (green = Rhodamine 6G, blue = FITC-Dextran; scale bar 50 μm). [right] Diagrams considering penetration depths of different imaging wavelengths into hepatic tissue stained with FITC-Dextran (blue, graphs A and B) and Rhodamine 6G (green, graphs C and D).
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![Comparison of different excitation wavelengths for imaging: [left] MPM...](publication/344729350/figure/fig2/AS:11431281273891357@1724772887636/Comparison-of-different-excitation-wavelengths-for-imaging-left-MPM-images-of-the-same_Q320.jpg)
The liver is known to possess extensive regenerative capabilities, the processes and pathways of which are not fully understood. A necessary step towards a better understanding involves the analysis of regeneration on the microscopic level in the in vivo environment. We developed an evaluation method combining longitudinal imaging analysis in vivo...
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... Since the density of necessary photons occurs only in the focal plane, a precise separation from the environment can be reached, with no thermal or mechanical energy being released to the surrounding cells [17,20]. Based on the complexity and cost, femtosecond laser systems have rarely been used to study regeneration in a cellular [18,21,22] or even in an organoid context. ...
Organoids represent the cellular composition of natural tissue. So called colonoids, organoids derived from colon tissue, are a good model for understanding regeneration. However, next to the cellular composition, the surrounding matrix, the cell–cell interactions, and environmental factors have to be considered. This requires new approaches for the manipulation of a colonoid. Of key interest is the precise application of localized damage and the following cellular reaction. We have established multiphoton imaging in combination with femtosecond laser-based cellular nanosurgery in colonoids to ablate single cells in the colonoids’ crypts, the proliferative zones, and the differentiated zones. We observed that half of the colonoids recovered within six hours after manipulation. An invagination of the damaged cell and closing of the structure was observed. In about a third of the cases of targeted crypt damage, it caused a stop in crypt proliferation. In the majority of colonoids ablated in the crypt, the damage led to an increase in Wnt signalling, indicated via a fluorescent lentiviral biosensor. qRT-PCR analysis showed increased expression of various proliferation and Wnt-associated genes in response to damage. Our new model of probing colonoid regeneration paves the way to better understand organoid dynamics on a single cell level.
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An abdominal imaging window allowing the study of dynamic processes in the spleen of live mice over the course of several weeks.
