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Yet, their cellular responses exhibit highly heterogeneous and asynchronous pigment and morphological transitions under fluctuating light stress, making precise prediction and process optimization challenging. Existing analytical methods cannot resolve these transitions in living cells because they either require destructive sampling or lack spectral specificity. In this study, we introduce near-infrared (nIR) image cytometry (NIC), a label-free, nondestructive, and high-throughput platform that quantifies biophysicochemical heterogeneity at single-cell resolution. By positioning chlorophyll detection within the nIR window, NIC structurally removes spectral interference from visible pigments and achieves precise quantification of chlorophyll content comparable to liquid chromatography. Simultaneously, NIC extracts biophysical parameters such as cell size and shape together with biochemical states including carotenoid accumulation in a fully label-free manner, enabling direct observation of their mechanistic coupling in living cells. Using representative industrial microalgae including D. salina and H. pluvialis as test beds, NIC resolves species-specific photoprotective strategies through 3D heterogeneity mapping. Furthermore, we customized a machine learning model that enables the prediction of cultivation stages for completely unknown batches with up to 89.0% stage-variable accuracy, driven by the single-cell data and multivariate biophysicochemical features quantified by NIC. This generalizable framework links cellular heterogeneity to process behavior and provides a basis for real-time monitoring in next-generation microalgal biomanufacturing.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c02572","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T14:33:53Z","timestamp":1785854033000},"page":"23645-23661","source":"Crossref","is-referenced-by-count":0,"title":["Label-Free\nNear-Infrared Image Cytometry for Chlorophyll\nQuantification and Carotenoid Prediction with Multiparametric Biophysicochemical\nProfiling"],"prefix":"10.1021","volume":"98","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-8438-9497","authenticated-orcid":true,"given":"Seongcheol","family":"Park","sequence":"first","affiliation":[{"name":"Sungkyunkwan University , , ,","place":["Suwon, Republic of Korea, 16419"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gueeda","family":"Kim","sequence":"additional","affiliation":[{"name":"Hanyang University , , ,","place":["Seoul, Republic of Korea, 04763"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-4718-860X","authenticated-orcid":true,"given":"Youngho","family":"Song","sequence":"additional","affiliation":[{"name":"Sungkyunkwan University , , ,","place":["Suwon, Republic of Korea, 16419"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-6314-3519","authenticated-orcid":true,"given":"Changyu","family":"Tian","sequence":"additional","affiliation":[{"name":"Sungkyunkwan University , , ,","place":["Suwon, Republic of Korea, 16419"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-6404-7594","authenticated-orcid":true,"given":"Changi","family":"Baek","sequence":"additional","affiliation":[{"name":"Sungkyunkwan University , , ,","place":["Suwon, Republic of Korea, 16419"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-4403-3519","authenticated-orcid":true,"given":"Youngwook","family":"Cho","sequence":"additional","affiliation":[{"name":"Sungkyunkwan University , , ,","place":["Suwon, Republic of Korea, 16419"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6485-9936","authenticated-orcid":true,"given":"Jin Woong","family":"Kim","sequence":"additional","affiliation":[{"name":"Sungkyunkwan University , , ,","place":["Suwon, Republic of Korea, 16419"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"EonSeon","family":"Jin","sequence":"additional","affiliation":[{"name":"Hanyang University , , ,","place":["Seoul, Republic of Korea, 04763"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6294-1154","authenticated-orcid":true,"given":"Soo-Yeon","family":"Cho","sequence":"additional","affiliation":[{"name":"Sungkyunkwan University , , ,","place":["Suwon, Republic of Korea, 16419"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,3]]},"reference":[{"key":"2026081809084180500_cit1","doi-asserted-by":"publisher","first-page":"1396","DOI":"10.1016\/j.biotechadv.2016.10.005","article-title":"Carotenoids from microalgae:\nA review of recent developments","volume":"34","author":"Gong","year":"2016","journal-title":"Biotechnol.\nAdv."},{"key":"2026081809084180500_cit2","doi-asserted-by":"publisher","first-page":"128","DOI":"10.1016\/j.foodchem.2018.10.066","article-title":"Comparative assessment\non the extraction of carotenoids from microalgal sources: Astaxanthin\nfrom H. pluvialis and \u03b2-carotene from D. salina","volume":"277","author":"Rammuni","year":"2019","journal-title":"Food Chem."},{"key":"2026081809084180500_cit3","doi-asserted-by":"publisher","first-page":"272","DOI":"10.1186\/s13068-018-1275-9","article-title":"Microalgae\nfor the production of lipid and carotenoids: a review with focus on\nstress regulation and adaptation","volume":"11","author":"Sun","year":"2018","journal-title":"Biotechnol.\nBiofuels"},{"key":"2026081809084180500_cit4","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1186\/s13068-025-02676-9","article-title":"High-yield\nzeaxanthin production in Chlamydomonas reinhardtii via advanced metabolic\npathway engineering","volume":"18","author":"Jang","year":"2025","journal-title":"Biotechnol. Biofuels Bioprod."},{"key":"2026081809084180500_cit5","doi-asserted-by":"publisher","first-page":"29","DOI":"10.1016\/j.rser.2014.01.002","article-title":"Modeling maximum lipid productivity of microalgae:\nReview and next step","volume":"32","author":"Xu","year":"2014","journal-title":"Renewable Sustainable\nEnergy Rev."},{"key":"2026081809084180500_cit6","doi-asserted-by":"publisher","first-page":"1926","DOI":"10.1038\/ismej.2012.29","article-title":"Marine microalgae attack and feed on metazoans","volume":"6","author":"Berge","year":"2012","journal-title":"ISME J."},{"key":"2026081809084180500_cit7","doi-asserted-by":"publisher","first-page":"6257","DOI":"10.3390\/cimb44120427","article-title":"Microalgal Carotenoids: Therapeutic\nApplication and Latest Approaches to Enhance the Production","volume":"44","author":"Sirohi","year":"2022","journal-title":"Curr. Issues Mol. 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