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Multichannel confocal microscope with fluorescence lifetime imaging for life science samples

English title Multichannel confocal microscope with fluorescence lifetime imaging for life science samples
Applicant Stein Jens Volker
Number 189785
Funding scheme R'EQUIP
Research institution Microbiologie Département de Médecine Université de Fribourg
Institution of higher education University of Fribourg - FR
Main discipline Cellular Biology, Cytology
Start/End 01.03.2020 - 28.02.2021
Approved amount 401'028.00
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All Disciplines (4)

Discipline
Cellular Biology, Cytology
Embryology, Developmental Biology
Immunology, Immunopathology
Infectious Diseases

Keywords (4)

Life Science; FLIM; White light laser; FRET

Lay Summary (German)

Lead
Die heutigen Lebenswissenschaften inklusive biomedizinischer Forschung benötigen moderne bildgebende Verfahren, um komplexe Prozesse auf molekularer Ebene zu untersuchen. Diese Verfahren, wie beispielsweise laserunterstütze Konfokalmikroskopie (KM), ermöglichen es dynamische Abläufe mit hoher räumlicher und zeitlicher Auflösung aufzunehmen. Das von der UNIFR angeschaffte KM-Gerät ermöglicht die Entwicklung neuer Forschungsrichtungen für viele Arbeitsgruppen.
Lay summary

Inhalte und Ziele des Forschungsprojekts

Das angeschaffte KM-Gerät zeichnet sich durch ein Zusatzmodul aus, welches die Messung von enzymatischen Aktivitäten innerhalb lebender Zellen mit Hilfe von speziell entwickelten Proben ermöglicht. Diese Technologie kommt verschiedenen Arbeitsgruppen im Department Biologie und der Abteilung Medizin der UNIFR zu Gute. Die Gruppe Hoogewijs misst damit die Aktivität von Sauerstoff-bindenden Proteinen, während die Gruppe Stein dynamische Prozesse bei der Zellmigration analysiert. Die Gruppen Walch und Mantel werden den KM-Arbeitsplatz für Projekte mit dem Malariaerreger Plasmodium verwenden, während die Gruppe Jazwinska Wundheilung in einem Fischmodell untersucht. Die Gruppe Sprecher benutzt diese neue Technologie zur Analyse der synaptischen Plastizität in Fruchtfliegen, während die Gruppe Geisler den Transport von Pflanzenhormonen in Wurzeln untersucht.

 

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts

Alle aufgelisteten Projekte gehören dem Gebiet der Grundlagenforschung an und zeichnen sich durch ihre grosse Diversität aus, die die Forschung an der UNIFR wiederspiegelt. Die Anschaffung erhöht die Möglichkeiten der UNIFR, weiterhin kompetitive Forschung auf höchstem Niveau zu entwickeln. Dies wiederum wirkt sich positiv auf die Attraktivität für bestehende und zukünftige Forschungsaktivitäten aus und ermöglicht den Erwerb weiterer finanzieller Mittel.

Direct link to Lay Summary Last update: 14.02.2020

Responsible applicant and co-applicants

Associated projects

Number Title Start Funding scheme
169928 Characterization of cytotoxic lymphocyte responses against blood-stage human malaria 01.05.2017 Project funding (Div. I-III)
179213 Regenerative biology of zebrafish 01.10.2018 Project funding (Div. I-III)
182729 Investigating the role of cellular communication to promote bacterial infections during malaria 01.04.2019 Project funding (Div. I-III)
169993 Genetic and Molecular Mechanisms Controlling Photoreceptor Development 01.10.2016 Project funding (Div. I-III)
172994 Immunology in context: Analyzing adaptive immunity through advanced microscopy 01.04.2017 Project funding (Div. I-III)
165877 Impact of ABCG36 phosphorylation during root-microbe colonization 01.01.2017 Project funding (Div. I-III)
173000 Functional analysis of androglobin, a novel oxygen-binding protein associated with male infertility. 01.09.2017 Project funding (Div. I-III)
180316 Neural Processing of Distinct Prediction Errors: Theory, Mechanisms & Interventions 01.09.2018 Sinergia

Abstract

The fluorescence lifetime is an inherent property of a particular fluorophore and depends on its chemical environment such as for example the vicinity of a second photon absorbing fluorophore. Fluorescence lifetime imaging microscopy (FLIM) is a powerful method to measure the efficiency of Förster resonance energy transfer (FRET) and to analyze intramolecular conformational changes or protein-protein interactions. While intensity-based FRET measurements are susceptible to variations in fluorophore concentration and excitation intensity FLIM-FRET measurements are internally calibrated and therefore less affected by such factors. Although FRET was discovered over half a century ago, the technological barriers were only overcome recently to do FLIM-FRET studies with a broader spectrum of life-science samples.We request funding to purchase a high-end confocal microscope with a FLIM module to address a variety of current research questions. In our proposed projects, FLIM-FRET methods will be used to analyze Rac and Rho activity in memory T cell subsets (Stein), to measure the calpatin activity of androglobin in mammalian cells (Hoogewijs), to quantify the interaction of granzymes with plasmodial proteins (Walch), and to map the auxin transport in plant roots (Geisler). These experiments require a confocal laser scanning microscope (CLSM) that is capable of fast FLIM-FRET measurements. In further projects, a high-resolution confocal microscope is required to make multi-channel measurement over large volumes. Scaling from single cell level to a larger cell populations and tissues and small organs allows to study the molecular processes in a larger context. We will analyze the mechanisms of intracellular communication by malaria extracellular vesicles (Mantel), uncover molecular processes in wound healing in zebrafish tissues (Jazwinska) and endeavor a structural and functional characterization of synaptic plasticity in Drosophila memory formation (Sprecher).To exploit new powerful technologies and achieve our research goals in accord with the highest methodological standards, a microscope capable of performing quantitative fluorescent measurements is a necessity. Such an instrument is currently not available on our campus, which is a limiting factor to develop new research projects. Furthermore, offering state-of-the-art microscopy has become an important asset for recruitment of new staff in the coming years, which will be of central importance to the UNIFR. Soliciting similar infrastructure from other microscopy facilities in Switzerland is inefficient due to logistic difficulties and limited capacity for external researchers.To our knowledge only one instrument reunites 1) the technical requirements for our research goals, 2) the versatility to accommodate all our proposed projects, and 3) the usability to effectively perform these complex measurements. The SP8 FALCON (FAst Lifetime CONtrast - FLIM) from Leica Microsystems manages to take the leap from the technological feasibility of FLIM-FRET to creating a fully integrated system adapted to the needs of research in life sciences. The proposed system is equipped with a white light laser, an acusto-optical beam splitter and spectral detectors and accommodates excitation and detection configuration for any fluorophore in the visible spectrum. Therefore, newly developed fluorophores will not lead to costly and time-consuming upgrades of the light path.The instrument will be fully integrated in the Bioimage Light Microscopy and Image Analysis Facility (thereafter referred to as core facility). Our core facility will ensure professional operation of the equipment and provide state-of-the-art microscopy for researchers of the University of Fribourg and other research institutions.
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