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In the major fungal pathogens\n                    <jats:italic>Aspergillus fumigatus<\/jats:italic>\n                    and\n                    <jats:italic>Candida albicans<\/jats:italic>\n                    , we used microfluidic\u2010coupled, fluorescence\u2010mediated live\u2010cell imaging to capture the real\u2010time responses of fungal hyphae to clinical concentrations of AmBisome or Caspofungin. In both fungi, AmBisome exposure caused rapid growth arrest (&lt;15 min) and subcellular reorganisation and, in\n                    <jats:italic>C. albicans<\/jats:italic>\n                    , localised expansions of lipid\u2010like structures from the hyphal perimeter. Responses to Caspofungin exposure were slower, with initial lytic effects occurring after 1.5 or 4 h in\n                    <jats:italic>A. fumigatus<\/jats:italic>\n                    and\n                    <jats:italic>C. albicans<\/jats:italic>\n                    hyphae, respectively. While\n                    <jats:italic>C. albicans<\/jats:italic>\n                    hyphae undergo unsalvageable hyphal lysis in response to Caspofungin,\n                    <jats:italic>A. fumigatus<\/jats:italic>\n                    exhibits several compensatory growth behaviours, including a novel resuscitative growth response, that circumvents lytic events to maintain apical and sub\u2010apical hyphal growth. This study reveals how the differing biologies of the two pathogens affected outcomes and contributes to the highly disparate rates of antifungal efficacy amongst commonly used drugs, where spore\/yeast\u2010derived inhibitory doses may be underestimated to arrest\/kill the invasive hyphal morphotypes in vitro.\n                  <\/jats:p>\n                  <jats:p>Human pathogenic cause &gt;2\u00a0M deaths per year and we have a limited number of antifungals in the clinic to combat these infections. Those drugs are increasingly meeting resistance in killer fungi and our understanding of drug responses are limited. Our main assessment of antifungal resistance comes from end\u2010point 48 h drug culture of the fungus and is performed by eye for presence of absence of growth in a well. Further, these tests are performed on the pre\u2010invasive form of the fungus, the yeast or spore (for speed and simplicity), not the invasive filamentous form present during systemic infection of organs in humans.<\/jats:p>\n                  <jats:p>This work shows how the invasive filamentous form of two major human fungal pathogens respond to two frontline clinical antifungal drugs with real time microscopy. We describe the cell death and\/or adaptive growth responses via live\u2010cell microscopy to understand the morphological and cellular responses over time. We were able to do this by coupling fluorescently engineered pathogens and live\u2010cell 4D microscopy to microfluidic delivery of culture media and\/or antifungal drug. We used the triggerable microfluidics to first establish invasive filamentous growth without drug, and keep the cells in the same focal plane (with shallow roof that kept the filaments growing up out of the focal plane). We then triggered the switch to media with drug(s) to perfuse drug while observing comparative cellular responses live, up to 10\u00a0h in two pathogenic fungi.<\/jats:p>\n                  <jats:p>\n                    In both pathogens (\n                    <jats:italic>Aspergillus fumigatus<\/jats:italic>\n                    and\n                    <jats:italic>Candida albicans<\/jats:italic>\n                    ) we saw similar immediate responses to one drug (Ambisome), which inhibited filamentous growth almost immediately. In stark contrast, Caspofungin induced different but continued forms of growth to known inhibitory doses against invasive fungal filaments.\n                    <jats:italic>A. fumigatus<\/jats:italic>\n                    (a filamentous mould fungus) appeared hard\u2010wired to continue filamentous growth by various compensatory regenerative growth forms, including a novel \u2018resuscitative\u2019 form we describe which occurs after the drug causes filament bursting. However,\n                    <jats:italic>C. albicans<\/jats:italic>\n                    filaments (a polymorphic yeast) respond by transitioning from filamentous to yeast growth (all filaments eventually burst, leaving only budding yeast). This study details the short\u2010 and long\u2010term responses of invasive pathogenic fungal filaments to drugs and highlights that the traditional spore\/yeast\u2010derived inhibitory doses for these fungi may be insufficient for the invasive form of the fungus, where more attention to the filamentous form is needed.\n                  <\/jats:p>","DOI":"10.1111\/jmi.70053","type":"journal-article","created":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T10:37:41Z","timestamp":1766054261000},"page":"241-254","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Microfluidic live\u2010cell imaging of\n                    <i>Aspergillus fumigatus<\/i>\n                    and\n                    <i>Candida albicans<\/i>\n                    hyphal growth treated with AmBisome and Caspofungin"],"prefix":"10.1111","volume":"301","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4800-7717","authenticated-orcid":false,"given":"D. D.","family":"Thomson","sequence":"first","affiliation":[{"name":"Medical Research Council Centre for Medical Mycology at the University of Exeter Department of Biosciences, Faculty of Health and Life Sciences  Exeter UK"},{"name":"Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, Faculty of Biology, Medicine and Health University of Manchester  Manchester UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R.","family":"Inman","sequence":"additional","affiliation":[{"name":"Medical Research Council Centre for Medical Mycology at the University of Exeter Department of Biosciences, Faculty of Health and Life Sciences  Exeter UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S.","family":"Nye","sequence":"additional","affiliation":[{"name":"Medical Research Council Centre for Medical Mycology at the University of Exeter Department of Biosciences, Faculty of Health and Life Sciences  Exeter UK"},{"name":"Living Systems Institue Univeristy of Exeter  Exeter UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"E. M.","family":"Bignell","sequence":"additional","affiliation":[{"name":"Medical Research Council Centre for Medical Mycology at the University of Exeter Department of Biosciences, Faculty of Health and Life Sciences  Exeter UK"},{"name":"Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, Faculty of Biology, Medicine and Health University of Manchester  Manchester UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,12,18]]},"reference":[{"key":"e_1_2_8_2_1","unstructured":"EUCAST. (2023).EUCAST definitive document E.DEF 7.4: Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts. EUCAST antifungal microdilution method for yeasts.EUCAST."},{"key":"e_1_2_8_3_1","unstructured":"EUCAST. (2022).EUCAST definitive document E.DEF 9.4: Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia forming moulds. EUCAST antifungal microdilution method for moulds.EUCAST."},{"issue":"1","key":"e_1_2_8_4_1","article-title":"Hyphal growth in human fungal pathogens and its role in virulence","volume":"2012","author":"Brand A.","journal-title":"International Journal of Microbiology"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1439-0507.2002.00748.x"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.46.9.3001-3012.2002"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0035478"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00710-17"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00862-15"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.fgb.2015.08.001"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E15-06-0355"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.0020035"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1000227"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.44.3.760-763.2006"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.1496"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02383-17"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.03015-19"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mimet.2012.08.002"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-53517-3"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2008.06545.x"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1128\/EC.2.3.398-410.2003"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1002\/yea.1099"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1109\/MCSE.2007.55"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2019"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1109\/83.650848"},{"key":"e_1_2_8_26_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-019-0686-2"},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1029\/2005WR004804"}],"container-title":["Journal of Microscopy"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/jmi.70053","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1111\/jmi.70053","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/jmi.70053","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,9]],"date-time":"2026-02-09T10:46:01Z","timestamp":1770633961000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1111\/jmi.70053"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,18]]},"references-count":26,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2026,2]]}},"alternative-id":["10.1111\/jmi.70053"],"URL":"https:\/\/doi.org\/10.1111\/jmi.70053","archive":["Portico"],"relation":{},"ISSN":["0022-2720","1365-2818"],"issn-type":[{"value":"0022-2720","type":"print"},{"value":"1365-2818","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,18]]},"assertion":[{"value":"2024-11-12","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-11-24","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-12-18","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}