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However, the laser exposure may lead to irreversible damage to cells during long-term optical imaging. Here, we developed a sensitive method to quantify and limit the photodamage during long-term in situ live-cell imaging by using the Raman band of cytochrome c (cyt c). This approach enables the detection of subtle chemical composition changes in the early stages of photodamage with over 5 times higher sensitivity than that of conventional morphological evaluation and mitochondrial fluorescence staining. By calibrating the cyt c Raman response against delivered light dosage across cells, we derive a dose\u2013damage relationship with a well-defined linear regime, enabling real-time quantification of photodamage during acquisition. Under the experimental conditions tested in this work, the model suggests that the cell can maintain good viability when cyt c fluctuations remain within a conservative tolerance (\u223c15\u201330%). Using this dosimetry as a decision rule, we implement practical mitigation strategies, i.e., interval time-lapse imaging to facilitate cellular self-recovery and prevent irreversible damage. With this method, we demonstrated that the lipid change during cell starvation can be faithfully recorded for up to 8 h. This work provides an efficient method to maintain cell activity during long-term in situ Raman imaging, which was difficult to achieve previously.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c02575","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T04:23:39Z","timestamp":1788323019000},"page":"26543-26554","source":"Crossref","is-referenced-by-count":0,"title":["Real-Time Quantification\nof Cellular Photodamage via\nIn Situ Raman Imaging by the Cytochrome\n                    <i>c<\/i>\n                    Marker\nBand"],"prefix":"10.1021","volume":"98","author":[{"given":"Jianfeng","family":"Chen","sequence":"first","affiliation":[{"name":"Xiamen University , , ,","place":["Xiamen, China, 361102"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengxi","family":"Xu","sequence":"additional","affiliation":[{"name":"Xiamen University , , ,","place":["Xiamen, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shiqi","family":"Li","sequence":"additional","affiliation":[{"name":"Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University , , ,","place":["Shenzhen, China, 518107"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yun","family":"Gao","sequence":"additional","affiliation":[{"name":"Anqing Normal University , , ,","place":["Anqing, China, 246133"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chun","family":"Lin","sequence":"additional","affiliation":[{"name":"Xiamen University , , ,","place":["Xiamen, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2223-1881","authenticated-orcid":true,"given":"Menglu","family":"Li","sequence":"additional","affiliation":[{"name":"Shenzhen Medical Academy of Research and Translation , , ,","place":["Shenzhen, Guangdong, China, 518017"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiyang","family":"Chen","sequence":"additional","affiliation":[{"name":"Xiamen University , , ,","place":["Xiamen, China, 361102"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daoxiang","family":"Lu","sequence":"additional","affiliation":[{"name":"Xiamen University , , ,","place":["Xiamen, China, 361102"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanping","family":"Li","sequence":"additional","affiliation":[{"name":"Shenzhen University , , ,","place":["Shenzhen, China, 518060"]},{"name":"Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Science , ,","place":["Suzhou, China, 215163"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3646-7396","authenticated-orcid":true,"given":"Zong","family":"Dai","sequence":"additional","affiliation":[{"name":"Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University , , ,","place":["Shenzhen, China, 518107"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7276-0832","authenticated-orcid":true,"given":"Lei","family":"Wang","sequence":"additional","affiliation":[{"name":"Xiamen University , , ,","place":["Xiamen, China, 361102"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9821-5864","authenticated-orcid":true,"given":"Bin","family":"Ren","sequence":"additional","affiliation":[{"name":"Xiamen University , , ,","place":["Xiamen, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9825-2681","authenticated-orcid":true,"given":"Hao","family":"He","sequence":"additional","affiliation":[{"name":"Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University , , ,","place":["Shenzhen, China, 518107"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,28]]},"reference":[{"key":"2026091511083036000_cit1","doi-asserted-by":"publisher","first-page":"3851","DOI":"10.1021\/acs.jpcb.3c01133","article-title":"Label-Free Tracking\nof Nanoprodrug Cellular Uptake and Metabolism Using Raman and Autofluorescence\nImaging","volume":"127","author":"Machida","year":"2023","journal-title":"J. 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U.S.A."},{"key":"2026091511083036000_cit4","doi-asserted-by":"publisher","first-page":"15032","DOI":"10.1021\/acsnano.0c05010","article-title":"Quantitative Drug Dynamics\nVisualized by Alkyne-Tagged Plasmonic-Enhanced Raman Microscopy","volume":"14","author":"Koike","year":"2020","journal-title":"ACS Nano"},{"key":"2026091511083036000_cit5","doi-asserted-by":"publisher","first-page":"14616","DOI":"10.1021\/acs.analchem.3c02129","article-title":"Multifocal Raman Spectrophotometer\nfor Examining Drug-Induced and Chemical-Induced Cellular Changes in\n3D Cell Spheroids","volume":"95","author":"Liao","year":"2023","journal-title":"Anal. Chem."},{"key":"2026091511083036000_cit6","doi-asserted-by":"publisher","first-page":"65","DOI":"10.1038\/nrc1529","article-title":"Chemotherapy\nand the war on cancer","volume":"5","author":"Chabner","year":"2005","journal-title":"Nat. Rev. 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Chem."},{"key":"2026091511083036000_cit9","doi-asserted-by":"publisher","first-page":"15765","DOI":"10.1021\/acs.analchem.4c03871","article-title":"Label-Free\nRaman Spectroscopy for Assessing\nPurity and Maturation of hiPSC-Derived Cardiac Tissue","volume":"96","author":"Li","year":"2024","journal-title":"Anal. Chem."},{"key":"2026091511083036000_cit10","doi-asserted-by":"publisher","first-page":"5259","DOI":"10.1021\/acs.analchem.4c06968","article-title":"Monitoring\nof Propiolamide-Mediated\nMolecular Crosstalk between Ferroptosis and Apoptosis by Raman Spectroscopy","volume":"97","author":"Yu","year":"2025","journal-title":"Anal. Chem."},{"key":"2026091511083036000_cit11","doi-asserted-by":"publisher","DOI":"10.1016\/j.addr.2024.115483","article-title":"Monitoring kinetic processes\nof drugs and metabolites: Surface-enhanced Raman spectroscopy","volume":"217","author":"Luo","year":"2025","journal-title":"Adv. Drug Deliver Rev."},{"key":"2026091511083036000_cit12","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0041990","article-title":"Mapping\nof Redox State\nof Mitochondrial Cytochromes in Live Cardiomyocytes Using Raman Microspectroscopy","volume":"7","author":"Brazhe","year":"2012","journal-title":"PLoS One"},{"key":"2026091511083036000_cit13","doi-asserted-by":"publisher","first-page":"664","DOI":"10.1038\/nprot.2016.036","article-title":"Using\nRaman spectroscopy\nto characterize biological materials","volume":"11","author":"Butler","year":"2016","journal-title":"Nat. Protoc."},{"key":"2026091511083036000_cit14","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-64737-3","article-title":"Raman spectroscopic\nsignatures of carotenoids and polyenes enable label-free visualization\nof microbial distributions within pink biofilms","volume":"10","author":"Horiue","year":"2020","journal-title":"Sci. Rep."},{"key":"2026091511083036000_cit15","doi-asserted-by":"publisher","first-page":"7070","DOI":"10.1021\/acs.analchem.8b05962","article-title":"Speeding Up the Line-Scan Raman Imaging of\nLiving Cells by Deep Convolutional Neural Network","volume":"91","author":"He","year":"2019","journal-title":"Anal. 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Raman Spectrosc."},{"key":"2026091511083036000_cit22","doi-asserted-by":"publisher","first-page":"0265","DOI":"10.1002\/bies.202300122","article-title":"New approaches for low\nphototoxicity imaging of living cells and tissues","volume":"46","author":"Kasprzycka","year":"2024","journal-title":"BioEssays"},{"key":"2026091511083036000_cit23","doi-asserted-by":"publisher","first-page":"1361","DOI":"10.1088\/1361-6463\/ab6b95","article-title":"Between life and death:\nstrategies to reduce phototoxicity in super-resolution microscopy","volume":"53","author":"Tosheva","year":"2020","journal-title":"J. Phys. D: Appl. Phys."},{"key":"2026091511083036000_cit24","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-017-00829-x","article-title":"Surpassing light-induced\ncell damage in vitro with novel cell culture media","volume":"7","author":"Stockley","year":"2017","journal-title":"Sci. Rep."},{"key":"2026091511083036000_cit25","doi-asserted-by":"publisher","first-page":"1778","DOI":"10.1529\/biophysj.106.096636","article-title":"Scattering of exciting\nlight by live cells in fluorescence Confocal imaging: Phototoxic effects\nand relevance for FRAP studies","volume":"93","author":"Dobrucki","year":"2007","journal-title":"Biophys. J."},{"key":"2026091511083036000_cit26","first-page":"1929","article-title":"The influence of sublethal\nblue light exposure on human RPE cells","volume":"15","author":"Roehlecke","year":"2009","journal-title":"Mol.\nVis."},{"key":"2026091511083036000_cit27","doi-asserted-by":"publisher","first-page":"552","DOI":"10.1002\/cyto.a.22291","article-title":"Standard fluorescent\nimaging of live cells is highly genotoxic","volume":"83A","author":"Ge","year":"2013","journal-title":"Cytometry,\nPart A"},{"key":"2026091511083036000_cit28","doi-asserted-by":"publisher","first-page":"74","DOI":"10.1002\/lsm.20271","article-title":"The role of laser fluence in cell viability, proliferation,\nand membrane integrity of wounded human skin fibroblasts following\nhelium-neon laser irradiation","volume":"38","author":"Hawkins","year":"2006","journal-title":"Lasers Surg.\nMed."},{"key":"2026091511083036000_cit29","unstructured":"Richmond, D.; Jost, A. P.-T.; Lambert, T.; et al.\n          DeadNet:\nIdentifying Phototoxicity from Label-Free Microscopy Images of Cells\nUsing Deep ConvNets, 2017. arXiv:1701.06109.\narXiv.org e-Print archive. https:\/\/arxiv.org\/abs\/1701.06109."},{"key":"2026091511083036000_cit30","doi-asserted-by":"publisher","first-page":"738","DOI":"10.3389\/fcell.2021.738786","article-title":"Assessing Phototoxicity\nin a Mammalian Cell Line: How Low Levels of Blue Light Affect Motility\nin PC3 Cells","volume":"9","author":"Alghamdi","year":"2021","journal-title":"Front. Cell Dev. Biol."},{"key":"2026091511083036000_cit31","doi-asserted-by":"publisher","DOI":"10.1002\/bies.201700003","article-title":"Phototoxicity in live\nfluorescence microscopy, and how to avoid it","volume":"39","author":"Icha","year":"2017","journal-title":"Bioessays"},{"key":"2026091511083036000_cit32","doi-asserted-by":"publisher","first-page":"1266","DOI":"10.1364\/BOE.3.001266","article-title":"Light-induced Ca2+ transients\nobserved in widefield epi-fluorescence microscopy of excitable cells","volume":"3","author":"McDonald","year":"2012","journal-title":"Biomed. Opt. Express"},{"key":"2026091511083036000_cit33","doi-asserted-by":"publisher","DOI":"10.1038\/srep30892","article-title":"Excitation Light Dose\nEngineering to Reduce Photo-bleaching and Photo-toxicity","volume":"6","author":"Boudreau","year":"2016","journal-title":"Sci. Rep."},{"key":"2026091511083036000_cit34","doi-asserted-by":"publisher","DOI":"10.1038\/srep05223","article-title":"Damage of photoreceptor-derived\ncells in culture induced by light emitting diode-derived blue light","volume":"4","author":"Kuse","year":"2014","journal-title":"Sci. Rep."},{"key":"2026091511083036000_cit35","doi-asserted-by":"publisher","first-page":"825","DOI":"10.1016\/j.cub.2006.03.059","article-title":"Long-range directional\nmovement of an interphase chromosome site","volume":"16","author":"Chuang","year":"2006","journal-title":"Curr.\nBiol."},{"key":"2026091511083036000_cit36","doi-asserted-by":"publisher","first-page":"242","DOI":"10.1242\/jcs.242834","article-title":"Optimizing live-cell\nfluorescence imaging conditions to minimize phototoxicity","volume":"133","author":"Kiepas","year":"2020","journal-title":"J. Cell Sci."},{"key":"2026091511083036000_cit37","doi-asserted-by":"publisher","first-page":"2531","DOI":"10.1039\/C8AN02213E","article-title":"Using redox-sensitive\nmitochondrial cytochrome Raman bands for label-free detection of mitochondrial\ndysfunction","volume":"144","author":"Morimoto","year":"2019","journal-title":"Analyst"},{"key":"2026091511083036000_cit38","doi-asserted-by":"publisher","first-page":"19651","DOI":"10.1021\/jacs.2c05359","article-title":"Raman Spectroscopy for\nChemical Biology Research","volume":"144","author":"Dodo","year":"2022","journal-title":"J. Am. Chem. Soc."},{"key":"2026091511083036000_cit39","doi-asserted-by":"publisher","first-page":"16374","DOI":"10.1021\/acs.analchem.5c02035","article-title":"The Raman Map of the\nHuman Cell","volume":"97","author":"Nowakowska","year":"2025","journal-title":"Anal. Chem."},{"key":"2026091511083036000_cit40","doi-asserted-by":"publisher","first-page":"256","DOI":"10.1002\/jbio.201200042","article-title":"Quantitative analysis\nof the redox states of cytochromes in a living L929 (NCTC) cell by\nresonance Raman microspectroscopy","volume":"6","author":"Kakita","year":"2013","journal-title":"J. Biophotonics."},{"key":"2026091511083036000_cit41","doi-asserted-by":"publisher","DOI":"10.1117\/1.2952192","article-title":"Raman microscopy for\ndynamic molecular imaging of living cells","volume":"13","author":"Hamada","year":"2008","journal-title":"J.\nBiomed. Opt."},{"key":"2026091511083036000_cit42","doi-asserted-by":"publisher","first-page":"1386","DOI":"10.1016\/j.saa.2022.121978","article-title":"Changes in the mitochondrial\nmembrane potential in endothelial cells can be detected by Raman microscopy","volume":"286","author":"Pieczara","year":"2023","journal-title":"Spectrochim. Acta, Part A"},{"key":"2026091511083036000_cit43","doi-asserted-by":"publisher","first-page":"7047","DOI":"10.1021\/acs.analchem.4c00236","article-title":"Imaging\nvs Nonimaging\nRaman Spectroscopy for High-Throughput Single-Cell Phenotyping","volume":"96","author":"Hobro","year":"2024","journal-title":"Anal. Chem."},{"key":"2026091511083036000_cit44","doi-asserted-by":"publisher","first-page":"3174","DOI":"10.3964\/j.issn.1000-0593(2022)10-3174-06","article-title":"A Spike Removal Algorithm\nBased on Median Filter\nand Statistic for Raman Spectra","volume":"42","author":"Ye","year":"2022","journal-title":"Spectrosc.\nSpectral Anal."},{"key":"2026091511083036000_cit45","doi-asserted-by":"publisher","first-page":"2552","DOI":"10.1002\/jrs.6024","article-title":"Automated weak signal extraction of hyperspectral\nRaman imaging data by adaptive low-rank matrix approximation","volume":"51","author":"He","year":"2020","journal-title":"J. Raman Spectrosc."},{"key":"2026091511083036000_cit46","doi-asserted-by":"publisher","first-page":"1138","DOI":"10.1039\/b922045c","article-title":"Baseline correction\nusing adaptive iteratively reweighted penalized least squares","volume":"135","author":"Zhang","year":"2010","journal-title":"Analyst"},{"key":"2026091511083036000_cit47","doi-asserted-by":"publisher","first-page":"193","DOI":"10.1016\/j.gene.2015.08.009","article-title":"Pitfalls of the MTT assay: Direct and off-target effects\nof inhibitors can result in over\/underestimation of cell viability","volume":"574","author":"Stepanenko","year":"2015","journal-title":"Gene"},{"key":"2026091511083036000_cit48","doi-asserted-by":"publisher","first-page":"3613","DOI":"10.1021\/acs.analchem.9b04410","article-title":"Light-Up Lipid Droplets\nDynamic Behaviors Using a Red-Emitting Fluorogenic Probe","volume":"92","author":"Zhang","year":"2020","journal-title":"Anal. Chem."},{"key":"2026091511083036000_cit49","doi-asserted-by":"publisher","first-page":"4491","DOI":"10.1039\/C9CC01404G","article-title":"An interface-targeting\nand H2O2-activatable probe liberating AIEgen: enabling on-site imaging\nand dynamic movement tracking of lipid droplets","volume":"55","author":"Li","year":"2019","journal-title":"Chem. Commun."},{"key":"2026091511083036000_cit50","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2321255121","article-title":"Unexpected omega-3 activities in intracellular\nlipolysis and macrophage foaming revealed by fluorescence lifetime\nimaging","volume":"121","author":"Tang","year":"2024","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026091511083036000_cit51","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms8176","article-title":"AMPK\nactivation promotes\nlipid droplet dispersion on detyrosinated microtubules to increase\nmitochondrial fatty acid oxidation","volume":"6","author":"Herms","year":"2015","journal-title":"Nat. Commun."},{"key":"2026091511083036000_cit52","doi-asserted-by":"publisher","first-page":"678","DOI":"10.1016\/j.devcel.2015.01.029","article-title":"Fatty Acid Trafficking\nin Starved Cells: Regulation by Lipid Droplet Lipolysis, Autophagy,\nand Mitochondrial Fusion Dynamics","volume":"32","author":"Rambold","year":"2015","journal-title":"Dev. Cell"}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c02575","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/36\/26543\/67605654\/acs.analchem.6c02575.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/36\/26543\/67605654\/acs.analchem.6c02575.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T15:52:21Z","timestamp":1789487541000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/36\/26543\/5349422\/Real-Time-Quantification-of-Cellular-Photodamage"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,28]]},"references-count":52,"journal-issue":{"issue":"36","published-online":{"date-parts":[[2026,8,28]]},"published-print":{"date-parts":[[2026,9,15]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c02575","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,28]]}}}