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Full spectrum cytometry: the next generation cytometry for high-dimensional single cell analysis

English title Full spectrum cytometry: the next generation cytometry for high-dimensional single cell analysis
Applicant Rüegg Curzio Roberto
Number 205916
Funding scheme R'EQUIP
Research institution Dept Oncology, Microbiology, Immunology Faculty of Science and Medicine Université de Fribourg
Institution of higher education University of Fribourg - FR
Main discipline Immunology, Immunopathology
Start/End 01.01.2022 - 31.12.2022
Approved amount 245'083.00
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All Disciplines (5)

Discipline
Immunology, Immunopathology
Surgery
Experimental Cancer Research
Cardiovascular Research
Cellular Biology, Cytology

Keywords (7)

Tissue regeneration; Caridiovascular biology; Nanomaterials; Cancer research; Immunology; Biomerker discovery ; Ageing

Lay Summary (German)

Lead
Der Erwerb einer Vollspektrum-Durchflusszytometrie zur Förderung der Forschung in den Bereichen Krebs, Biomarkerforschung, Immunologie, Alterung, Geweberegeneration, zirkadiane Biologie und Materialwissenschaften an der Universität Freiburg.
Lay summary

Die fluoreszenzbasierte Durchflusszytometrie ist eine der am häufigsten verwendeten Techniken, um zelluläre Eigenschaften wie Zellidentität, Differenzierung, Proliferation und Tod zu untersuchen. Es kann auch zum Sortieren lebender Zellen für weitere Studien verwendet werden. Zu diesem Zweck werden Zellen mit fluoreszierenden Sonden oder Antikörpern markiert, oft in mehreren Kombinationen. Die konventionelle fluoreszenzbasierte Durchflusszytometrie ist aufgrund der spektralen Überlappung der Fluorophore und ihrer filterbasierten Trennung an technische Grenzen gestoßen. Die Vollspektrum-Durchflusszytometrie löst diese Einschränkung durch spektrales Entmischen der mehreren Fluorophore anstelle einer filterbasierten Trennung. Dies ermöglicht Kombinationen von multiplen Fluorophoren (bis zu 60), die in der konventionellen Durchflusszytometrie unerreichbar sind. Die spektrale Entmischung ermöglicht auch die Abschwächung der Hintergrundautofluoreszenz. An der Fakultät für Naturwissenschaften und Medizin unserer Universität haben wir in 2018 eine Durchflusszytometrie-Facility eingerichtet, um dem wachsenden Bedarf an Zellanalyse und -sortierung gerecht zu werden. Der Erfolg der Einrichtung stimulierte Forschungsprojekte, die komplexe Zellanalysen erforderten, die die Kapazitäten der derzeitigen Instrumente übersteigen. Um mit dem Forschungsbedarf Schritt zu halten, haben wir uns für die Anschaffung eines Vollspektrum-Durchflusszytometers entschieden. Nach sorgfältiger Überlegung haben wir uns für das Aurora-Durchflusszytometer von Cytek® entschieden. Sechs Projekte, die von unabhängigen Forschern geleitet werden und ein breites Spektrum an Forschungsthemen abdecken (Krebsforschung ,  Entdeckung von Biomarkern, T-Zell-Immunologie, Alterung und Geweberegeneration), werden sofort von diesem neuen Instrument profitieren. Weitere Anwender im Bereich der circadianen Biologie, Materialwissenschaften, Herzregeneration und Immunologie werden von dieser neuen Technologie zunehmend profitieren. Über die Ermöglichung dieser spezifischen Projekte hinaus wird der Erwerb dazu beitragen, unsere Facility an der Spitze des Wettbewerbs zu halten und die dynamische Entwicklung und Attraktivität unserer Universität zu steigern.

Direct link to Lay Summary Last update: 25.11.2021

Lay Summary (French)

Lead
Un cytométrie en flux à spectre complet pour promouvoir la recherche dans les domaines du cancer, de la recherche de biomarqueurs, de l'immunologie, du vieillissement, de la régénération tissulaire, de la biologie circadienne et de la science des matériaux à l'Université de Fribourg.
Lay summary

La cytométrie en flux basée sur la fluorescence est l'une des techniques les plus largement utilisées pour étudier des propriétés cellulaires telles que l'identité cellulaire, la différenciation, la prolifération, la mort. Il peut également être utilisé pour trier les cellules viables en vue d'études ultérieures. À cette fin, les cellules sont marquées avec des sondes fluorescentes ou des anticorps, souvent en combinaisons multiples. La cytométrie en flux conventionnelle basée sur la fluorescence a atteint des limites techniques en raison du chevauchement spectral des multiples fluorophores et de leur séparation par filtre. La cytométrie en flux à spectre complet résout cette limitation par un mélange spectral des fluorophores multiples au lieu d'une séparation basée sur un filtre. Cela permet des combinaisons de fluorophores (jusqu'à 60) qui sont inaccessibles en cytométrie de flux conventionnelle. Le démixage spectral permet également d'atténuer l'autofluorescence de fond. À la Faculté des sciences et de médecine de notre université, nous avons créé un service de cytométrie en flux en 2018, en réponse aux besoins croissants d'analyse et de tri cellulaire. Le succès de l'installation a stimulé des projets de recherche nécessitant des analyses cellulaires complexes dépassant les capacités des instruments actuels. Afin de répondre aux besoins de la recherche, nous avons décidé d'acquérir un cytomètre en flux à analyse spectrale. Après mûre réflexion, nous avons opté pour le cytomètre en flux Aurora de Cytek®. Six projets dirigés par des chercheurs indépendants et couvrant un large éventail de sujets de recherche, allant de la recherche sur le cancer à la découverte de biomarqueurs, en passant par l'immunologie des lymphocytes T, le vieillissement et la régénération tissulaire, bénéficieront immédiatement de ce nouvel instrument. D'autres utilisateurs dans le domaine de la biologie circadienne, des sciences des matériaux, de la régénération cardiaque et de l'immunologie bénéficieront également de cette nouvelle technologie.  Au-delà de la réalisation de ces projets spécifiques, l'acquisition de cet instrument contribuera à maintenir notre Facility à la pointe de la compétitivité, à stimuler la dynamique d'évolution et l'attractivité de notre Université.

Direct link to Lay Summary Last update: 25.11.2021

Lay Summary (Italian)

Lead
L'acquisition d'une cytométrie en flux à spectre complet stimulera la recherche dans les domaines du cancer, de la découverte de biomarqueurs, de l'immunologie, du vieillissement, de la régénération tissulaire, de la biologie circadienne, des sciences des matériaux, à l'Université de Fribourg.
Lay summary

La citometria a flusso basata sulla fluorescenza è una delle tecniche più utilizzate per studiare le proprietà cellulari come l'identità cellulare, la differenziazione, la proliferazione, la morte. Può anche essere usata per isolare cellule viventi per ulteriori studi. A tal fine, le cellule vengono marcate con sonde fluorescenti o anticorpi, spesso in combinazioni multiple. La citometria a flusso convenzionale basata sulla fluorescenza ha raggiunto limiti tecnici a causa della sovrapposizione spettrale dei fluorofori e della loro separazione con filtri. La citometria a flusso a spettro completo supera questa limitazione mediante la separazione spettrale di più fluorofori invece della separazione basata sul filtro. Ciò consente combinazioni di fluorofori (fino a 60) che sono inaccessibili nella citometria a flusso convenzionale. La separazione spettrale consente inoltre di attenuare l'autofluorescenza di fondo. Presso la Facoltà di Scienze e Medicina della nostra università, abbiamo creato una Facility di citometria a flusso nel 2018, in risposta alle crescenti esigenze di analisi e separazione cellulari. Il successo dell'installazione ha stimolato progetti di ricerca che richiedono analisi cellulari complesse oltre le capacità degli strumenti attuali. Per soddisfare le esigenze della ricerca, abbiamo deciso di acquistare un citometro a flusso a spettro completo. Dopo un'attenta valutazione, abbiamo optato per il citometro a flusso Aurora di Cytek®.  Sei progetti guidati da ricercatori indipendenti e che coprono un'ampia gamma di temi di ricerca, che vanno dalla ricerca sul cancro, alla scoperta di biomarcatori, all'immunologia delle cellule T, all'invecchiamento e alla rigenerazione dei tessuti, beneficeranno immediatamente di questo nuovo strumento. Altri utenti nel campo della biologia circadiana, della scienza dei materiali, della rigenerazione dei tessuti e della biologia delle infezioni beneficeranno di questa nuova tecnologia. Al di là della realizzazione di questi progetti specifici, l'acquisizione degli stessi contribuirà a mantenere il nostro istituto all'avanguardia della competitività, a stimolare la dinamica dell'evoluzione e l'attrattività del nostro Ateneo.

Direct link to Lay Summary Last update: 25.11.2021

Lay Summary (English)

Lead
The acquisition of a full spectrum flow cytometry will stimulate research in cancer biology, biomarker discovery, immunology, aging, tissue regeneration, circadian biology, material sciences, at University of Fribourg.
Lay summary

Fluorescence-based flow cytometry is one of the most widely used technique to study cellular properties such as cellidentifiy,  differentiation, proliferation, death. It can also be used for sorting viable cells for further studies. To this end cells are labeled with fluorescent probes or antibodies, often in multiple combinations. Conventional fluorescence-based flow cytometry has reached technical limitations due the spectral overlap of the fluorophores and their filter-based separation. Full spectrum flow cytometry resolves this limitation by spectral unmixing of the multiple fluorophores instead of filter based separation. This allows for combinations of fluorophores (up to 60) that are unattainable in conventional flow cytometry. Spectral unmixing also enables to attenuate background autofluorescence. At the Faculty of Science and Medicine of our University we established a flow cytometry facility in 2018, in response to growing needs for cell analysis and sorting. The success of the facility stimulated research projects requiring complex cell analyses beyond the capacities of the current instruments. In order to  keep up with the research needs, we decided to acquire a full spectrum flow cytometer. Aftre careful considerations we opted for the Aurora flow cytometer from Cytek. Six projects lead by independent investigators, and covering a broad spectrum of research topics, ranging from cancer research, biomarker discovery, T cell immunology, aging and tissue regeneration, will immediately benefit from this new instrument. Additional users in the field of circadianic biology, material sciences, cardiac regeneration and immunolgy will benefir from this new technology.  Beyond enabling these specific projects, the acquisition of this will contribute to maintain our facility at a competitive forefront, boost the dynamic evolution and attractivity of our University.

Direct link to Lay Summary Last update: 25.11.2021

Responsible applicant and co-applicants

Associated projects

Number Title Start Funding scheme
197711 Exploring lymphatic endothelium - innate immune system crosstalk in lymph nodes and its impact on adaptive immunity 01.02.2021 Project funding
208136 Myeloid-derived cells in breast cancer dormancy and metastasis 01.05.2022 Project funding
190848 Decoding cellular Communication during malaria infection 01.02.2020 Spark
179261 Arginse-II, chronic intermittent hypoxia, and aging 01.03.2019 Project funding
179248 Unravelling mechanisms of metastatic dormancy and colonization in breast cancer 01.05.2018 Project funding
196301 Intra-portal TGF-ß delivery by liposomes to stimulate liver regeneration 01.10.2020 Spark
182729 Investigating the role of cellular communication to promote bacterial infections during malaria 01.04.2019 Project funding
200406 Exploring tissue-specific CD8+ T cell-mediated host protection at single cell resolution 01.04.2021 Project funding
182881 NCCR Bio-Inspired Materials: Using Concepts from Nature to Create ‚Smart' Materials (phase II) 01.06.2018 National Centres of Competence in Research (NCCRs)

Abstract

Omics-based analyses of mRNA, protein and low-molecular weight metabolites, have revolutionized research in biology and medicine. Single-cell RNA sequencing is one of the salient examples of a technological leap that has revolutionized the analysis of complex cell populations in physiology and pathology. In parallel the need of high-dimensional, high-resolution phenotyping of living cells to study cell population dynamics, differentiation and function has grown dramatically. Fluorescence-based flow cytometry has been one of the most widely used methods to characterize the phenotype of various cell types, in particular of the immune system. Conventional flow cytometry, however, has reached technical limits due to the spectral overlap of the available fluorophores and the limitations for compensation to distinguish multiple fluorophores with comparable spectra. Spectral unmixing in full spectrum flow cytometry solves this problem to a large extent and frees users from the laborious task of compensation. This technology allows for combination of currently available fluorophores that are unattainable in conventional flow cytometry (up to over 60 combinations). Spectral unmixing also enables to attenuate high autofluorescence background signals, for example in neutrophils, macrophages or metabolically active cells, thereby facilitating detection of rare events. At our Faculty of Science and Medicine at University of Fribourg, we established a flow cytometry facility in 2018, in response to growing needs for cell analysis and sorting. The success of the facility further stimulated research projects requiring high-dimensional single cell analysis that go far beyond the capacities of the current instruments. This evolution calls for the next generation of flow cytometry instrument, namely the full spectrum flow cytometer. Six senior and junior applicants, Curzio Rüegg as main applicant and Jens V. Stein, Zhihong Yang, Simon Blanchoud, Pierre-Yves Mantel and Carmen Gonelle-Gispert as co-applicants, propose six research projects for which full spectrum flow cytometry is essential. These projects cover a broad spectrum of research topics, ranging from translational cancer research, antiviral T cell immunology, aging of the immune system, organism and tissue regeneration:i) High dimensional phenotypical profiling of circulating leukocytes in breast cancer; ii) Organ-specific high-dimensional analysis of antiviral tissue-resident memory CD8+ T cell populations;iii) In-depth profiling of the aging immune system in arginase II-deficient aging mouse model;iv) Regeneration-specific hemocytic features in colonial tunicates;v) Role of cellular communication on neutrophil dysfunction and bacterial infections during malaria;vi) Characterization of liver sinusoidal endothelial cells and immune cell subpopulations during mouse liver regeneration.Four additional potential users of full spectrum flow cytometry in the field of circadianic biology, material sciences, cardiac regeneration and immunolgy are included. Further, we describe in detail the uniqueness of full spectrum technology, its need for biomedical research at our Faculty and the reason for the choice of the Cytek® Aurora flow cytometer.Beyond enabling these specific projects, we see the acquisition of the full spectrum Cytek® Aurora as an essential investment for keeping up with technological evolution in flow cytometry to maintain our facility at a competitive forefront. It will further boost the dynamic evolution of our Faculty and render it attractive for the recruitments of future Faculty, SNSF and ERC grantees, postdocs and students to perform competitive biomedical research.
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