{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T13:47:16Z","timestamp":1773841636844,"version":"3.50.1"},"reference-count":21,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2021,10,13]],"date-time":"2021-10-13T00:00:00Z","timestamp":1634083200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Regione Toscana Bando FAS Salute 2014 Italy","award":["CUP B52I14005760002"],"award-info":[{"award-number":["CUP B52I14005760002"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Bacterial biofilm constitutes a strong barrier against the penetration of drugs and against the action of the host immune system causing persistent infections hardly treatable by antibiotic therapy. Helicobacter pylori (Hp), the main causative agent for gastritis, peptic ulcer and gastric adenocarcinoma, can form a biofilm composed by an exopolysaccharide matrix layer covering the gastric surface where the bacterial cells become resistant and tolerant to the commonly used antibiotics clarithromycin, amoxicillin and metronidazole. Antimicrobial PhotoDynamic Therapy (aPDT) was proposed as an alternative treatment strategy for eradicating bacterial infections, particularly effective for Hp since this microorganism produces and stores up photosensitizing porphyrins. The knowledge of the photophysical characteristics of Hp porphyrins in their physiological biofilm microenvironment is crucial to implement and optimize the photodynamic treatment. Fluorescence lifetime imaging microscopy (FLIM) of intrinsic bacterial porphyrins was performed and data were analyzed by the \u2018fit-free\u2019 phasor approach in order to map the distribution of the different fluorescent species within Hp biofilm. Porphyrins inside bacteria were easily distinguished from those dispersed in the matrix suggesting FLIM-phasor technique as a sensitive and rapid tool to monitor the photosensitizer distribution inside bacterial biofilms and to better orientate the phototherapeutic strategy.<\/jats:p>","DOI":"10.3390\/pharmaceutics13101674","type":"journal-article","created":{"date-parts":[[2021,10,13]],"date-time":"2021-10-13T21:48:39Z","timestamp":1634161719000},"page":"1674","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Fluorescence Lifetime Imaging Microscopy of Porphyrins in Helicobacter pylori Biofilms"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4431-7475","authenticated-orcid":false,"given":"Antonella","family":"Battisti","sequence":"first","affiliation":[{"name":"Istituto Nanoscienze\u2014CNR and NEST\u2014Scuola Normale Superiore, Piazza S. Silvestro 12, I-56127 Pisa, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4822-3601","authenticated-orcid":false,"given":"Paola","family":"Morici","sequence":"additional","affiliation":[{"name":"Istituto Nanoscienze\u2014CNR and NEST\u2014Scuola Normale Superiore, Piazza S. Silvestro 12, I-56127 Pisa, Italy"},{"name":"Ospedale Policlinico San Martino, Largo Rosanna Benzi 10, I-16132 Genova, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0858-3293","authenticated-orcid":false,"given":"Antonella","family":"Sgarbossa","sequence":"additional","affiliation":[{"name":"Istituto Nanoscienze\u2014CNR and NEST\u2014Scuola Normale Superiore, Piazza S. Silvestro 12, I-56127 Pisa, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,10,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1242\/dmm.027649","article-title":"Helicobacter pylori-induced gastric pathology: Insights from in vivo and ex vivo models","volume":"10","author":"Burkitt","year":"2017","journal-title":"Dis. Model. Mech."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Krzy\u017cek, P., Grande, R., Migda\u0142, P., Paluch, E., and Go\u015bciniak, G. (2020). Biofilm Formation as a Complex Result of Virulence and Adaptive Responses of Helicobacter pylori. Pathogens, 9.","DOI":"10.3390\/pathogens9121062"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2822","DOI":"10.1128\/AAC.49.7.2822-2827.2005","article-title":"Helicobacter pylori accumulates photoactive porphyrins and is killed by visible light","volume":"49","author":"Hamblin","year":"2005","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.bpc.2017.05.010","article-title":"Spectroscopic characterization and fluorescence imaging of Helicobacter pylori endogenous porphyrins","volume":"229","author":"Battisti","year":"2017","journal-title":"Biophys. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.bpc.2017.06.006","article-title":"Compositional analysis of endogenous porphyrins from Helicobacter pylori","volume":"229","author":"Battisti","year":"2017","journal-title":"Biophys. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"283","DOI":"10.3389\/fmicb.2020.00283","article-title":"The in vitro photoinactivation of Helicobacter pylori by a novel LED-based device","volume":"11","author":"Morici","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"260","DOI":"10.1002\/lsm.20161","article-title":"Helicobacter pylori in patients can be killed by visible light","volume":"36","author":"Ganz","year":"2005","journal-title":"Lasers Surg. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1002\/lsm.20770","article-title":"Treatment of Helicobacter pylori infection with intra-gastric violet light phototherapy: A pilot clinical trial","volume":"41","author":"Lembo","year":"2009","journal-title":"Lasers Surg. Med."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Rennie, M.Y., Dunham, D., Lindvere-Teene, L., Raizman, R., Hill, R., and Linden, R. (2019). Understanding Real-Time Fluorescence Signals from Bacteria and Wound Tissues Observed with the MolecuLight i:XTM. Diagnostics, 9.","DOI":"10.3390\/diagnostics9010022"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"20","DOI":"10.12968\/jowc.2018.27.1.20","article-title":"Efficacy of an imaging device at identifying the presence of bacteria in wounds at a plastic surgery outpatients clinic","volume":"27","author":"Blackshaw","year":"2018","journal-title":"J. Wound Care"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1159\/000459362","article-title":"Comparison of porphyrin and heme biosynthesis in various heterotrophic bacteria","volume":"16","author":"Doss","year":"1973","journal-title":"Enzyme"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/iwj.13564","article-title":"Detection of bacterial fluorescence from in vivo wound biofilms using a point-of-care fluorescence imaging device","volume":"18","author":"Lopez","year":"2021","journal-title":"Int. Wound J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"319","DOI":"10.2217\/fmb-2019-0279","article-title":"In vitro detection of porphyrin-producing wound bacteria with real-time fluorescence imaging","volume":"15","author":"Jones","year":"2020","journal-title":"Future Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1039\/b412924p","article-title":"Time-resolved fluorescence microscopy","volume":"4","author":"Suhling","year":"2005","journal-title":"Photochem. Photobiol. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1080\/05704928408081716","article-title":"The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry","volume":"20","author":"Jameson","year":"1984","journal-title":"Appl. Spectrosc. Rev."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1007\/s00284-012-0285-2","article-title":"Real-time Analysis of metabolic activity within Lactobacillus acidophilus by phasor fluorescence lifetime imaging microscopy of NADH","volume":"66","author":"Torno","year":"2013","journal-title":"Curr. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3743","DOI":"10.1038\/s41598-017-04032-w","article-title":"Metabolic fingerprinting of bacteria by fluorescence lifetime imaging microscopy","volume":"7","author":"Bhattacharjee","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1111\/j.1523-5378.2010.00815.x","article-title":"Proteomannans in biofilm of Helicobacter pylori ATCC 43504","volume":"16","author":"Yang","year":"2011","journal-title":"Helicobacter"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e1616","DOI":"10.21769\/BioProtoc.1616","article-title":"Bacterial porphyrin extraction and quantification by LC\/MS\/MS analysis","volume":"5","author":"Mancini","year":"2015","journal-title":"Bio-protocol"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"623","DOI":"10.1038\/nrmicro2415","article-title":"The biofilm matrix","volume":"8","author":"Flemming","year":"2010","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/1011-1344(95)07155-U","article-title":"Photophysical properties of porphyrins in biological membranes","volume":"29","author":"Ricchelli","year":"1995","journal-title":"J. Photochem. Photobiol. B Biol."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/13\/10\/1674\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:12:37Z","timestamp":1760166757000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/13\/10\/1674"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,13]]},"references-count":21,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2021,10]]}},"alternative-id":["pharmaceutics13101674"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics13101674","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,10,13]]}}}