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Confocal Microscope with spectral detection, FLIM and laser ablation

English title Confocal Microscope with spectral detection, FLIM and laser ablation
Applicant Schroeder Timm
Number 177079
Funding scheme R'EQUIP
Research institution Computational Systems Biology Department of Biosystems, D-BSSE ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Cellular Biology, Cytology
Start/End 01.12.2017 - 30.11.2018
Approved amount 398'210.00
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All Disciplines (6)

Cellular Biology, Cytology
Embryology, Developmental Biology
Molecular Biology
Experimental Cancer Research

Keywords (8)

FCS; FLIM; spectral detection; optogenetics; FCCS; laser ablation; Confocal Microscope; point scanning microscope

Lay Summary (German)

Laser-Mikroskop für 3-D Schichtaufnahmen mit Spektralanalyse, Laserschere und Fluoreszenz-Zeitmessung im Nanosekundenbereich.
Lay summary

Das beantragte Gerät ist ein spezialisiertes Laser-Fluoreszenz Mikroskop welches in der Lage ist, mit hoher Auflösung ‘konfokale’ Schichtaufnahmen von lebenden und fixierten Zellen oder Gewebe anzufertigen, um so ihre räumliche Organisation zu erfassen. Spektralanalyse und Messungen kleinster zeitlicher Veränderungen von Fluoreszenz-Markierungen im Nanosekundenbereich mittels gepulster Laser erlauben Einblicke in Mechanismen auf der Ebene einzelner Moleküle. Projekt 1 wird eine neuentwickelte Färbemethode, mit der man 8 verschiedene Fluoreszenzfarbstoffe im gleichen Gewebe zur Markierung von Molekülen nutzt, anwenden. In Knochen- und Knochenmarkproben werden Stammzellen hinsichtlich ihrer Einbettung und Interaktionen mit dem umgebenden Gewebe und ihre Entwicklung untersucht. Projekt 2 nutzt das Mikroskop und die pulsierenden Laser, um molekulare Mechanismen in und an den Grenzflächen von künstlich hergestellten Tröpfchen mit definierten Inhaltsstoffen zu untersuchen. Projekt 3 nutzt die Laser des Mikroskops um Zellen zu stimulieren, die genetisch so verändert worden sind, dass sie auf verschiede Lichtfarben durch Veränderung ihres Stoffwechsels reagieren können. So eröffnet sich die Möglichkeit, über Licht Mechanismen in der Zelle zu beeinflussen und so mathematische Modelle dieser Mechanismen zu erstellen und zu prüfen. Projekt 4 nutzt eine Laserschere des Geräts in Gewebekulturen, um ausgewählte Zellen während der Entwicklung des Gewebes auszuschalten. Dadurch gewinnt man Informationen zur Funktion dieser Zellen, ihre Kommunikation mit anderen Zellen, zu Mustern der Entwicklung und zur räumlichen Organisation des Gewebes.  Das Mikroskop wird zur gemeinschaftlichen Nutzung für alle Forschergruppen in einer zentralen Einrichtung unseres Departements platziert.

Direct link to Lay Summary Last update: 22.11.2017

Responsible applicant and co-applicants

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We apply for a new confocal microscope with spectral detection, FLIM and laser ablation. Importantly, it contains pulsed white laser with much improved spectral flexibility and a very fast accusto-optical light patterning capability. This will enable future extension of spectral combinatorics for the simultaneous analysis of numerous molecular markers and thus cellular or molecular candidates of interest. The pulsed nature of the laser will allow improved signal to noise differentiation and thus the use also of inferior reporters, and FLIM analysis of multiple molecular candidates. This microscope will not only be crucial for continued operations at the imaging core facility of the Department of Biosystems Science and Engineering (D-BSSE) of the ETH Zürich in Basel by replacing an old and extremely heavily used confocal microscope. It also enables many exciting novel projects as described in this application. The first project uses a recently developed novel sample preparation, staining, scanning and data analysis pipeline allowing tissue imaging of very large volumes at subcellular resolution and with up to 8 simultaneous ‘colors’ to be detected. Quantitative analysis even of extremely difficult to prepare and analyze tissues, in particular of whole length thick bone sections, is possible with this approach. Together with custom-made software we programmed for the efficient analysis of the very large volumes of resulting imaging data, this approach allows unprecedented resolution in analyzing bone and bone marrow. We are planning to apply this internationally unique approach for central questions in bone and hematopoietic stem and progenitor cell biology. The improved spectral capabilities of the new microscope will be crucial to allow the required simultaneous detection of many cell types and molecular markers. In a second project, the spectral and pulsing capabilities of the planned white laser will be applied for FLIM quantification of molecular dynamics in custom developed droplet microenvironments. The spectral properties of the white laser will be crucial for much improved flexibility in analyzing multiple molecular candidates with the required efficiency.The third planned project aims to manipulate migration of individual cells by closed loop optogenetic control of cellular adhesion signaling and cytoskeleton organization. This approach promises much improved insights into and testing of existing computational models of the control of cell migration. It depends on the high spatial resolution of a confocal microscope, combined with the fast optical patterning through acousto-optic technology of the planned microscope for the required subcellular resolution, speed and versatility. Finally, this setup will be completed with an ablation laser to enable the manipulation of individual cells within cellular structures in the fourth project. This is a crucial prerequisite to understand the three-dimensional organization of cells, emergence of patterns, and their control by molecular gradients. The manipulation allows to test predictions of existing computational models. This important tool is currently missing in our departmental infrastructure. Since these ablation approaches are done on living cells within an ongoing experiment, they cannot be conducted by using a laser ablation system in other institutions. In combination, the planned microscope will enable a range of exciting and important projects, which would be impossible with currently available hardware. They range from large volume high dimensional imaging of fixed material to live cell and molecule imaging including manual or closed loop automated molecular manipulation through light. The planned projects address questions naturally arising from previous and current projects within ongoing funded projects or strategically important novel projects to be started in the coming months.