{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/343809"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/343809","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Super-resolved volumetric optical imaging of virus-infected cells","abstract":"Viruses are infectious agents that replicate within the cells of the organisms they infect. Most viruses are smaller than 200 nanometres, which makes them hard to visualise using diffraction-limited light microscopes. Yet, ‘seeing viruses’ is essential to unravel their infection mechanism within the organisms they highjack, which is key to understanding how such small entities can spark disruptive pandemics. This thesis shows how expansion microscopy is an excellent tool to achieve super-resolution when imaging virus-infected samples using optical microscopy. Expansion microscopy is a recently conceived sample preparation technique that physically expands fixed specimens after embedding them in water-absorbing polymers. By expanding virus-infected samples and imaging them on a light sheet microscope, it was possible to acquire super-resolved 3D images of virus-infected cells rapidly and in up to four colours simultaneously. The combination of expansion and light sheet microscopy was employed for studying the infection cycle of the emerging virus SARS-CoV-2 and for comparing the infection mechanism of two live attenuated influenza vaccine (LAIV) viruses characterised by differing vaccine effectiveness. In both cases, the increased resolution in three dimensions delivered details about the infection mechanism of these viruses that were not previously known. Imaging SARS-CoV-2 infected Vero cells revealed that the nucleocapsid protein of SARS-CoV-2 accumulates at the membrane of the viral replication organelles, strongly suggesting that this is where the encapsidation of the viral genome by the nucleocapsid protein occurs. Imaging the assembly of the viral genome of an optimal and suboptimal LAIV virus in A549 cells highlighted genome assembly faults for the suboptimal virus at the cytoplasmic level, which could be the main reason behind the reduced fitness of this LAIV strain.","abstract_html":"Viruses are infectious agents that replicate within the cells of the organisms they infect. Most viruses are smaller than 200 nanometres, which makes them hard to visualise using diffraction-limited light microscopes. Yet, ‘seeing viruses’ is essential to unravel their infection mechanism within the organisms they highjack, which is key to understanding how such small entities can spark disruptive pandemics. This thesis shows how expansion microscopy is an excellent tool to achieve super-resolution when imaging virus-infected samples using optical microscopy. Expansion microscopy is a recently conceived sample preparation technique that physically expands fixed specimens after embedding them in water-absorbing polymers. By expanding virus-infected samples and imaging them on a light sheet microscope, it was possible to acquire super-resolved 3D images of virus-infected cells rapidly and in up to four colours simultaneously. The combination of expansion and light sheet microscopy was employed for studying the infection cycle of the emerging virus SARS-CoV-2 and for comparing the infection mechanism of two live attenuated influenza vaccine (LAIV) viruses characterised by differing vaccine effectiveness. In both cases, the increased resolution in three dimensions delivered details about the infection mechanism of these viruses that were not previously known. 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Imaging the assembly of the viral genome of an optimal and suboptimal LAIV virus in A549 cells highlighted genome assembly faults for the suboptimal virus at the cytoplasmic level, which could be the main reason behind the reduced fitness of this LAIV strain.","abstract_has_math":false,"creators":["Mascheroni, Luca"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Kaminski, Clemens"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-08-16","date_published":"2022-08-16","updated_at":"2026-07-22T22:24:17Z","subjects":["Optical microscopy","Super-resolution microscopy","Virus infection mechanism","SARS-CoV-2","Live attenuated influenza vaccine"],"languages":["eng"],"rights":[],"rights_urls":["https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.91231","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Kaminski, Clemens"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["AstraZeneca"]},{"key":"dc:creator","label":"Author","values":["Mascheroni, Luca"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2022-08-16"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/343809"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Optical microscopy","Super-resolution microscopy","Virus infection mechanism","SARS-CoV-2","Live attenuated influenza vaccine"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.91231"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1ef9e9c6-ed3a-43b6-a992-427d22f0ecca/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Viruses are infectious agents that replicate within the cells of the organisms they infect. 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