Project

Discipline

tissue imaging; organoid imaging; superresolution; high-resolution; in vivo imaging; spectral detection; multiphoton; laser scanning microscope; high-speed; Confocal Microscope; single-cell imaging

Lead
Wir beantragen ein konfokales Multiphoton-Mikroskop zur bildlichen Darstellung von Zellen, Zellsystemen, Organoiden und Organen. Dabei soll die Kombination eines neuen Detektorsystems mit Multiphotonenmikroskopie neue Erkenntnisse zum Aufbau und zur Funktion lebender Systeme in biotechnologischen und medizinischen Fragestellungen mit besonders hoher räumlicher und zeitlicher Auflösung liefern.
Lay summary
Das beantragte Mikroskop liefert aufgrund seiner neuartigen Bildaufnahmetechnologie eine neue Qualität von Daten, welche die hier aufgeführten Projekte zur Beantwortung ihrer Fragestellungen benötigen. Die Fragestellungen der vier repräsentativen Projekte der Antragsteller: Wie initiieren und regulieren Säugerzellen die Adhäsion an extrazellulären Matrixkomponenten? Welche Rolle und Funktion haben einzelne Zellen bei der normalen und pathologischen Entwicklung von Hirnorganoiden, die als Modell für das Zentralnervensystem verwendet werden? Welche örtliche Verteilung und funktionelle Rolle haben Stammzellen in großen Gewebevolumina (z.B. ganze Lymphknoten oder im Oberschenkelknochen im Mausmodell)? Wie kann man die Detektionsrate und Produktion von Biosignalen in synthetischen Zellsystemen, die zur Therapie und homöostatischer Kontrolle von komplexen Pathologien entwickelt werden, verbessern? Die erfolgreiche Implementierung des beantragten Mikroskops in oben genannten Projekten ermöglicht eine bisher nicht verfügbare zeitliche und örtliche Auflösung bei der bildlichen Darstellung der zu verfolgenden zellulären Prozesse. Dabei setzt das Mikroskop hochsensitive Detektoren ein, die eine besonders starke Vergrößerung und eine hohe zeitliche Auflösung der Aufnahmen im Millisekundenbreich realisieren. Zusätzlich ermöglicht die Multiphotonentechnik Bilder sehr tief im Gewebe aufzunehmen. Das Mikroskop ersetzt ein älteres sehr stark genutztes Mikroskop, welches nicht mehr dem Stand der Technik entspricht und altersbedingte Mängel aufweist. Das neue Mikroskop wird in einer zentralen wissenschaftlichen Einrichtung (Single Cell Facility) allen Arbeitsgruppen des D-BSSE zur Verfügung stehen.

Direct link to Lay Summary

Last update: 16.04.2020

Number	Title	Start	Funding scheme

We apply for a multiphoton confocal microscope for the fast and high-resolution imaging of single cells, cellular assemblies, and tissues to organoids and organs. The microscope contains lasers to considerably increase the thickness of the biological system imaged. The new detector technology enables high time resolution ˜ 1/42 frames/s and lateral (super-)resolution ˜ 120 nm (90 nm with deconvolution) with visible light excitation and improves multiphoton imaging beyond the diffraction limit. The availability of a 34-channel spectral detection device enables to freely choose the detected emission bands of the fluorophores and unmix otherwise difficult to separate fluorophores. The instrument will allow to perform the morphological and functional characterization of key living systems in biotechnological and medical applications with much higher spatial and time resolution, as required by the here presented exciting and novel projects. Moreover, the nature of the new confocal microscope, which replaces and extends a worn-out old and extremely heavily used confocal microscope, is crucial for the improved quantitative imaging operations for 13 research groups using the imaging core facility of the Department of Biosystems Science and Engineering (D-BSSE) of the ETH Zürich in Basel. The first project applies nanotechnological assays to characterize how mammalian cells initiate and regulate adhesion to extracellular matrix (ECM) components. The assays measure that i) in fibroblasts integrins adhering to ECM substrates signal other integrins to co-initiate and -strengthen adhesion, ii) fibroblasts use integrins to sense mechanical properties of the ECM and quickly (<1 s) signal other integrins to adjust adhesion, and iii) in fibroblasts GPCRs can signal integrins to initiate and strengthen adhesion. The insight of how different integrin types are regulated is fundamental to understand cell adhesion processes taking key roles in cell migration, development and maintenance of tissue and organs, and diseases including infection, immunology and cancer. To systematically characterize cells initiating and strengthening adhesion via the assembly of adhesion complexes, the considerably improved spatial resolution, time resolution and sensitivity of the new multiplex confocal microscope is crucial. The second project uses an integrative set of single-cell transcriptomic and imaging methodologies to reconstruct cerebral organoid development, and dissect single-cell behaviors that lead to human brain malformations. Using next generation multiphoton high resolution confocal microscopy, it will dynamically track the behavior of individual fluorescently labeled progenitors and neurons within live, intact organoid tissue generated from stem cells of patients with cortical malformations to directly measure the migratory behavior of healthy and mutant neurons and to capture and understand the origin of heterotopic neuronal clusters. The project further requires fast and sensitive super-resolution imaging across large fields of view. It is also crucial that the applied for instrument enables a depth penetration to several hundred micrometers at sufficiently high time resolution to follow cell migration in organoids. The third project, develops novel approaches to i) comprehensively quantify the spatial distribution of different (stem) cell types, their niches and their molecular regulators in very large volumes (e.g. whole lymph nodes or mouse femurs), ii) express signaling biosensors in transgenic cell and animal lines, including the design of required hard- and software allowing long-term live cell imaging and quantification of signaling pathway activities in different stem and progenitor cell types, and iii) transfer existing materials and know-how in quantitative imaging of signaling pathway dynamics in primary stem and progenitor cells to the in-vivo observation and quantification of relevant cell types in living tissues in mouse. The new microscope is essential for this project to enable imaging of large areas at much required improved speed, (super-)resolution, sensitivity and throughput. The fourth project improves the dynamics of human cells for detecting and producing biological signals at ultra-fast timescales. It requires the precise spatiotemporal control of target proteins at cellular compartments, such as organelles at defined times points. Since current confocal imaging technologies are limited in time and spatial resolution, rapid changes occurring within cells cannot be recorded at sufficiently high detail in 3D. The successful implementation of the project will greatly benefit from next-generation imaging technologies such as gentle multiplex imaging (time and fluorophores), which allow real-time monitoring of rapid cellular processes at specified cellular compartments.