Konfokales Mikroskop mit spektraler Detektion für Langzeitaufnahmen am lebenden Objekt
Final Report Abstract
Heermann/Wittbrodt: In this project the microscope is used to visualize the closure of the optic fissure in living zebrafish embryos. Since the optic fissure is located at the ventral pole of the developing eye, which is facing the yolk at this developmental stage we are dependent on a high penetration depth. The multiphoton laser in combination with the long distance objective the Sp5 setup is equipped with facilitates imaging with deep tissue penetration. In addition the motorized stage of the Sp5 allows imaging of multiple embryos in parallel, increasing the effective use. The quality of the images obtained with the Sp5 is impressive and we are confident that with the help of this setup we will publish our findings in near future. Lust/Wittbrodt: Using the microscope combined with a Spectra-Physics Mai-Tai Ti:S laser we are able to perform in vivo imaging of the juvenile fish retina with single cell resolution for the first time. Using the laser for ablation of cells of interest and follow-up imaging allows to observe the reactions of stem cells in the retina after wounding in vivo in various genetic backgrounds. This facilitates the functional analysis of retinal regeneration by advanced imaging in vivo. Centanin/Wittbrodt: We have developed tools to label individual stem cells and to follow all of their descendants. We analysed the potential of individual retinal stem cells by studying their lineage through in vivo imaging in the context of the growing vertebrate retina. This analysis demonstrated a constitutive pluripotency of retinal stem cells. Poggi: In my research group we aim to understand how cell type diversification occurs through rounds of cell divisions in the in vivo developing zebrafish retina. The SP5 confocal microscope is therefore an essential piece of equipment in all our investigative areas. Along this line, application of both multi point 3D time-lapse profiling of transgenically labelled individual retinal progenitors, and simultaneous detection of several fluorophores, allowed us to provide first evidences of lineage-dependent logic of amacrine subtype diversification in the in vivo. Data from this study greatly improved our understanding as to the genetic and cell lineage-impact on neuronal subtype diversification in vivo and have therefore received already much attention in the research field - it has been selected as a highlight in “This week in the Journal” as well as in the Heidelberg University Press Releases (http://www.uni-heidelberg.de/presse/news2012/pm20121203_nervenzellen_en.html).
Publications
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(2011) Fate Restriction and Multipotency in Retinal Stem Cells. Cell Stem Cell 9: 553–562
Centanin, L., Hoeckendorf, B. & Wittbrodt, J.
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(2012) 'Biasing Amacrine Subtypes in the Atoh7 Lineage through Expression of Barhl2'. Journal of Neuroscience 32(40): 13929-13944
Jusuf, P. R., Albadri, S., Paolini, A., Currie, P. D., Argenton, F., Higashijima, S.-I., Harris, W. A. and Poggi, L.
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(2013) 'Imaging retinal progenitor lineages in developing zebrafish embryos'. Cold Spring Harb Protoc 2013(3)
Jusuf, P., Harris, W. A. and Poggi, L.
