{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,30]],"date-time":"2026-03-30T12:08:36Z","timestamp":1774872516398,"version":"3.50.1"},"reference-count":205,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2022,12,11]],"date-time":"2022-12-11T00:00:00Z","timestamp":1670716800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Portuguese Foundation for Science and Technology (FCT)","doi-asserted-by":"publisher","award":["FCT\/UIDB&P\/4950\/2020"],"award-info":[{"award-number":["FCT\/UIDB&P\/4950\/2020"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Portuguese Foundation for Science and Technology (FCT)","doi-asserted-by":"publisher","award":["PTDC\/EMD-EMD\/32162\/2017"],"award-info":[{"award-number":["PTDC\/EMD-EMD\/32162\/2017"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"name":"FEDER","award":["FCT\/UIDB&P\/4950\/2020"],"award-info":[{"award-number":["FCT\/UIDB&P\/4950\/2020"]}]},{"name":"FEDER","award":["PTDC\/EMD-EMD\/32162\/2017"],"award-info":[{"award-number":["PTDC\/EMD-EMD\/32162\/2017"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>Two-photon imaging (TPI) microscopy, namely, two-photon excited fluorescence (TPEF), fluorescence lifetime imaging (FLIM), and second-harmonic generation (SHG) modalities, has emerged in the past years as a powerful tool for the examination of biological tissues. These modalities rely on different contrast mechanisms and are often used simultaneously to provide complementary information on morphology, metabolism, and structural properties of the imaged tissue. The cornea, being a transparent tissue, rich in collagen and with several cellular layers, is well-suited to be imaged by TPI microscopy. In this review, we discuss the physical principles behind TPI as well as its instrumentation. We also provide an overview of the current advances in TPI instrumentation and image analysis. We describe how TPI can be leveraged to retrieve unique information on the cornea and to complement the information provided by current clinical devices. The present state of corneal TPI is outlined. Finally, we discuss the obstacles that must be overcome and offer perspectives and outlooks to make clinical TPI of the human cornea a reality.<\/jats:p>","DOI":"10.3390\/s22249699","type":"journal-article","created":{"date-parts":[[2022,12,12]],"date-time":"2022-12-12T05:10:19Z","timestamp":1670821819000},"page":"9699","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Two-Photon Imaging for Non-Invasive Corneal Examination"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5672-8266","authenticated-orcid":false,"given":"Ana","family":"Batista","sequence":"first","affiliation":[{"name":"Coimbra Institute for Biomedical Imaging and Translational Research (CIBIT), University of Coimbra, 3000-548 Coimbra, Portugal"},{"name":"Institute for Nuclear Sciences Applied to Health (ICNAS), University of Coimbra, 3000-548 Coimbra, Portugal"},{"name":"Department of Physics, Faculty of Science and Technology, University of Coimbra, 3004-516 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9465-4413","authenticated-orcid":false,"given":"Pedro","family":"Guimar\u00e3es","sequence":"additional","affiliation":[{"name":"Coimbra Institute for Biomedical Imaging and Translational Research (CIBIT), University of Coimbra, 3000-548 Coimbra, Portugal"},{"name":"Institute for Nuclear Sciences Applied to Health (ICNAS), University of Coimbra, 3000-548 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0562-8994","authenticated-orcid":false,"given":"Jos\u00e9 Paulo","family":"Domingues","sequence":"additional","affiliation":[{"name":"Coimbra Institute for Biomedical Imaging and Translational Research (CIBIT), University of Coimbra, 3000-548 Coimbra, Portugal"},{"name":"Institute for Nuclear Sciences Applied to Health (ICNAS), University of Coimbra, 3000-548 Coimbra, Portugal"},{"name":"Department of Physics, Faculty of Science and Technology, University of Coimbra, 3004-516 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5881-9161","authenticated-orcid":false,"given":"Maria Jo\u00e3o","family":"Quadrado","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Centro Hospitalar e Universit\u00e1rio de Coimbra, 3004-561 Coimbra, Portugal"},{"name":"Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9455-1206","authenticated-orcid":false,"given":"Ant\u00f3nio Miguel","family":"Morgado","sequence":"additional","affiliation":[{"name":"Coimbra Institute for Biomedical Imaging and Translational Research (CIBIT), University of Coimbra, 3000-548 Coimbra, Portugal"},{"name":"Institute for Nuclear Sciences Applied to Health (ICNAS), University of Coimbra, 3000-548 Coimbra, Portugal"},{"name":"Department of Physics, Faculty of Science and Technology, University of Coimbra, 3004-516 Coimbra, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"951","DOI":"10.1097\/ICO.0b013e318285c8f4","article-title":"High-Resolution Spectral Domain Anterior Segment Optical Coherence Tomography in Type 1 Boston Keratoprosthesis","volume":"32","author":"Shapiro","year":"2013","journal-title":"Cornea"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"68","DOI":"10.5005\/jp-journals-10008-1109","article-title":"Anterior Segment Optical Coherence Tomography and its Clinical Applications in Glaucoma","volume":"6","author":"Li","year":"2012","journal-title":"Curr. 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