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A photo-manipulation unit for high precision, 3-dimensional photoactivation and laser ablation on a multi-view light sheet microscope setup

English title A photo-manipulation unit for high precision, 3-dimensional photoactivation and laser ablation on a multi-view light sheet microscope setup
Applicant Brunner Damian
Number 189781
Funding scheme R'EQUIP
Research institution Institut für Molekulare Biologie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Embryology, Developmental Biology
Start/End 01.12.2019 - 30.11.2021
Approved amount 112'399.00
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All Disciplines (2)

Discipline
Embryology, Developmental Biology
Cellular Biology, Cytology

Keywords (9)

Laser-mediated microsurgery; Organismal development; Cell differentiation; Mechanics; Kidney development; Cytoskeleton; Cell- and Tissue connectivity; Optogenetics; Light Sheet Microscopy

Lay Summary (German)

Lead
Moderne Fluoreszenzmikroskopie kombiniert mit genetischer Manipulation ist eine schlagkräftige Werkzeugkombination zur Untersuchung biologischer Prozesse. Hier wird mittels einer neuen Photomanipulationseinheit eine fundamentale Limitierung bisheriger genetischer Manipulationen beseitigt - die ungenügende räumliche und zeitliche Kontrolle.
Lay summary

Inhalt und Ziele des Forschungsprojekts

Mittels lokalisierter Lichtaktivierung ermöglichen neueste molekularbiologische Methoden eine hochselektive Proteinmanipulation wobei Proteinfunktionen in Zellen aktiviert, inaktiviert und umprogrammiert und auch deren Lokalisation gezielt verändert werden können. Die hier finanzierte Photomanipulationseinheit produziert eine punktuelle Lichtaktivierung welche Proteinmanipulation mit beispielloser räumlicher und zeitlicher Präzision ermöglicht. Dies kann je nach Bedarf auf sub-zellulärer oder multi-zellulärer Ebene angewendet werden aber auch in ganzen Geweben und Organismen. Die Einheit ist an ein hochmodernes Lichtblattmikroskop gekoppelt. Dieses bietet im Vergleich zu herkömmlichen Fluoreszenzmikroskopen Bilder mit optimaler räumlicher Auflösung in Kombination mit massiv reduzierter Phototoxizität. Dadurch können die Auswirkungen der Proteinmanipulation hochpräzise, über deutlich verlängerte Zeiträume hinweg analysiert werden.

Da der eingesetzte Laser Typ genügend Energie liefert um auch Mikroschnitte an lebenden Zellen und Geweben durchzuführen können zudem Kräfte die auf Zellen und Gewebe wirken oder von diesen produziert werden analysiert werden. Auch hier eröffnen die zeitliche und räumliche Präzision des Systems neue experimentelle Möglichkeiten.

Anfänglich wird die Photomanipulationseinheit benutzt um Gewebeverschlussprozesse in Fruchtfliegenembryonen, kollektive Zellmigration in Fischembryonen, sowie die Entwicklung von Frosch- und Maus- Nierenorganoiden zu untersuchen.

 

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts

Die durch das Gerät ermöglichten Forschungsansätze dienen der Grundlagenforschung könnten aber längerfristig auch Projekten im Bereich des Gewebeengineerings dienen.

Direct link to Lay Summary Last update: 03.12.2019

Responsible applicant and co-applicants

Associated projects

Number Title Start Funding scheme
189102 Conserved transcriptional networks of tubulogenesis - towards a mechanistic understanding of renal malformations. 01.08.2020 Project funding (Div. I-III)
183550 Real time Exploration of GTPase-Cytoskeletal feedback underlying Contractile Actomyosin Systems 01.09.2019 Sinergia
176235 Cellular mechanisms of organ self-assembly in vivo 01.01.2018 Project funding (Div. I-III)

Abstract

Classical and modern methods of genetic interference have provided an extremely powerful approach to dissect the fundamental molecular processes underlying the development of multicellular organisms. Genetic studies have become even more powerful with the addition of live fluorescence imaging that enabled a more detailed, time resolved description of the mutants and comparison with the wild type. More recently, light sheet microscopy has further improved the observable readout by enabling much longer observation times and nearly isotropic 3D resolution, allowing quantitative analysis of developmental processes that were previously impossible. However, substantial technical limitations to genetic approaches remain. These have prevented tackling numerous, fundamental questions of cell- and developmental biology, which is why we are still far from having a holistic picture of the mechanisms driving organismal development. Their main drawback is a lack of precision in space and time. The problem is that many of the crucial cellular components (e.g. cytoskeleton, motors, regulatory proteins) are being used in a cell type-specific manner by all cells of a developing organism and often multiple ways within the same individual cells. Their perturbation thus has to be acute and requires a very high spatial and temporal resolution to avoid secondary effects obscuring the key function. The recent development of light-regulated proteins offers the possibility to overcome these long-standing technical limitations. The game changing innovation is that complex cellular protein networks can be selectively manipulated with light in a user-defined manner and with unprecedented spatial and temporal resolution. Such technology has already been successfully used to control neuron activities, to drive tissue invagination and is beginning to provide promising approaches in the treatment of medical conditions. The research groups of this grant application have already started generating various light regulated proteins in order to tackle key questions of current biological interest. While these tools can already be employed to selectively and acutely manipulate protein function in whole tissue with precise temporal control, they unfortunately cannot yet be used with sufficient spatial resolution to photoactivate the target proteins only in a subset of cells, or in individual cells within a given 3-dimensional tissue. Such high selectivity however, is fundamental for the presented projects and for many future projects. The main problem is that the commonly available activation light sources are not selective along the z-axis, penetrating deep into the tissue, and in addition produce massive light scattering in the X/Y imaging planes. Consequently, the light-sensitive probes become stimulated also in cells and tissue regions outside the targeted regions. While this can be solved by use of two-photon (2P) laser illumination, conventional 2P microscope systems are not-designed for high-resolution 3D live imaging of developing organisms and organoids. To overcome this problem and achieve the necessary precision in 3D, we aim to install a 2-photon laser unit on a preexisting cutting-edge light sheet microscope to combine high precision optical manipulation with simultaneous live imaging, with nearly isotropic resolution. The setup will in addition allow researchers to systematically address mechanochemical properties of tissue-tissue interactions as the novel 2-photon laser type is sufficiently powerful to enable selective cell ablation and tissue cutting. We will be amongst the very first world wide to apply such an experimental setup to pioneer new mechanistic insights into themes of considerable interest.
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