{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T15:54:59Z","timestamp":1773071699295,"version":"3.50.1"},"reference-count":43,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2023,2,21]],"date-time":"2023-02-21T00:00:00Z","timestamp":1676937600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004329","name":"Slovenian Research Agency","doi-asserted-by":"publisher","award":["Z1-4384"],"award-info":[{"award-number":["Z1-4384"]}],"id":[{"id":"10.13039\/501100004329","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004329","name":"Slovenian Research Agency","doi-asserted-by":"publisher","award":["J2-8171"],"award-info":[{"award-number":["J2-8171"]}],"id":[{"id":"10.13039\/501100004329","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004329","name":"Slovenian Research Agency","doi-asserted-by":"publisher","award":["J3-3083"],"award-info":[{"award-number":["J3-3083"]}],"id":[{"id":"10.13039\/501100004329","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004329","name":"Slovenian Research Agency","doi-asserted-by":"publisher","award":["P1-0389"],"award-info":[{"award-number":["P1-0389"]}],"id":[{"id":"10.13039\/501100004329","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>Hyperspectral microscope imaging (HMI) is an emerging modality that integrates spatial information collected by standard laboratory microscopy and the spectral-based contrast obtained by hyperspectral imaging and may be instrumental in establishing novel quantitative diagnostic methodologies, particularly in histopathology. Further expansion of HMI capabilities hinges upon the modularity and versatility of systems and their proper standardization. In this report, we describe the design, calibration, characterization, and validation of the custom-made laboratory HMI system based on a Zeiss Axiotron fully motorized microscope and a custom-developed Czerny-Turner-type monochromator. For these important steps, we rely on a previously designed calibration protocol. Validation of the system demonstrates a performance comparable to classic spectrometry laboratory systems. We further demonstrate validation against a laboratory hyperspectral imaging system for macroscopic samples, enabling future comparison of spectral imaging results across length scales. An example of the utility of our custom-made HMI system on a standard hematoxylin and eosin-stained histology slide is also shown.<\/jats:p>","DOI":"10.3390\/s23052374","type":"journal-article","created":{"date-parts":[[2023,2,22]],"date-time":"2023-02-22T02:08:34Z","timestamp":1677031714000},"page":"2374","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Design and Validation of a Custom-Made Hyperspectral Microscope Imaging System for Biomedical Applications"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8629-4649","authenticated-orcid":false,"given":"Jo\u0161t","family":"Stergar","sequence":"first","affiliation":[{"name":"Jo\u017eef Stefan Institute, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia"},{"name":"Faculty of Mathematics and Physics, University of Ljubljana, Jadranska ulica 19, SI-1000 Ljubljana, Slovenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rok","family":"Hren","sequence":"additional","affiliation":[{"name":"Faculty of Mathematics and Physics, University of Ljubljana, Jadranska ulica 19, SI-1000 Ljubljana, Slovenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4417-0293","authenticated-orcid":false,"given":"Matija","family":"Milani\u010d","sequence":"additional","affiliation":[{"name":"Jo\u017eef Stefan Institute, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia"},{"name":"Faculty of Mathematics and Physics, University of Ljubljana, Jadranska ulica 19, SI-1000 Ljubljana, Slovenia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,2,21]]},"reference":[{"key":"ref_1","first-page":"1","article-title":"Programmable Hyperspectral Microscopy for High-Contrast Biomedical Imaging in a Snapshot","volume":"25","author":"Lu","year":"2020","journal-title":"J. Biomed. Opt."},{"key":"ref_2","unstructured":"Tomaszewski, J.E., and Ward, A.D. (2020). Medical Imaging 2020: Digital Pathology, SPIE."},{"key":"ref_3","first-page":"166","article-title":"Hyperspectral Imaging for Intraoperative Diagnosis of Colon Cancer Metastasis in a Liver","volume":"Volume 10956","author":"Kopriva","year":"2019","journal-title":"Medical Imaging 2019: Digital Pathology"},{"key":"ref_4","first-page":"201","article-title":"Digital Pathology with Hyperspectral Imaging for Colon and Ovarian Cancer","volume":"Volume 10956","author":"Nakaya","year":"2019","journal-title":"Medical Imaging 2019: Digital Pathology"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"17","DOI":"10.3389\/fonc.2018.00017","article-title":"Hyperspectral Imaging and K-Means Classification for Histologic Evaluation of Ductal Carcinoma In Situ","volume":"8","author":"Khouj","year":"2018","journal-title":"Front. Oncol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1364\/BOE.9.000818","article-title":"Detecting Brain Tumor in Pathological Slides Using Hyperspectral Imaging","volume":"9","author":"Ortega","year":"2018","journal-title":"Biomed. Opt. Express"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Ortega, S., Fabelo, H., Iakovidis, D.K., Koulaouzidis, A., and Callico, G.M. (2019). Use of Hyperspectral\/Multispectral Imaging in Gastroenterology. Shedding Some\u2013Different\u2013Light into the Dark. J. Clin. Med., 8.","DOI":"10.3390\/jcm8010036"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2818","DOI":"10.1039\/c0an00350f","article-title":"Accurate Histopathology from Low Signal-to-Noise Ratio Spectroscopic Imaging Data","volume":"135","author":"Reddy","year":"2010","journal-title":"Analyst"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1002\/1097-0320(20010401)43:4<239::AID-CYTO1056>3.0.CO;2-Z","article-title":"Hyperspectral Imaging: A Novel Approach for Microscopic Analysis","volume":"43","author":"Schultz","year":"2001","journal-title":"Cytometry"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"122473","DOI":"10.1109\/ACCESS.2019.2937729","article-title":"Hyperspectral Push-Broom Microscope Development and Characterization","volume":"7","author":"Ortega","year":"2019","journal-title":"IEEE Access"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"12293","DOI":"10.1364\/OE.17.012293","article-title":"Compact Image Slicing Spectrometer (ISS) for Hyperspectral Fluorescence Microscopy","volume":"17","author":"Gao","year":"2009","journal-title":"Opt. Express"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"14330","DOI":"10.1364\/OE.18.014330","article-title":"Snapshot Image Mapping Spectrometer (IMS) with High Sampling Density for Hyperspectral Microscopy","volume":"18","author":"Gao","year":"2010","journal-title":"Opt. Express"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"044004","DOI":"10.1117\/1.2003369","article-title":"Hyperspectral Imaging of Hemoglobin Saturation in Tumor Microvasculature and Tumor Hypoxia Development","volume":"10","author":"Sorg","year":"2005","journal-title":"J. Biomed. Opt."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1002\/jbio.201100066","article-title":"Hyperspectral Imaging Microscopy for Identification and Quantitative Analysis of Fluorescently-Labeled Cells in Highly Autofluorescent Tissue","volume":"5","author":"Leavesley","year":"2012","journal-title":"J. Biophoton."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"023707","DOI":"10.1063\/1.2885043","article-title":"An Excitation Wavelength\u2013Scanning Spectral Imaging System for Preclinical Imaging","volume":"79","author":"Leavesley","year":"2008","journal-title":"Rev. Sci. Instrum."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2404","DOI":"10.1364\/OPTCON.469040","article-title":"Dual-Modality Hyperspectral Microscopy for Transmission and Fluorescence Imaging","volume":"1","author":"Zhang","year":"2022","journal-title":"Opt. Contin."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"170636","DOI":"10.1016\/j.ijleo.2023.170636","article-title":"Design of MWIR Hyperspectral Imagers Based on Acousto-Optic Tunable Filters","volume":"276","author":"Li","year":"2023","journal-title":"Optik"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"E1679","DOI":"10.1073\/pnas.1119511109","article-title":"Compressive Fluorescence Microscopy for Biological and Hyperspectral Imaging","volume":"109","author":"Studer","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","unstructured":"Conchello, J.-A., Cogswell, C.J., and Wilson, T. (2006). Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII, SPIE."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"11032","DOI":"10.1364\/OE.16.011032","article-title":"High-Throughput, Multiplexed Pushbroom Hyperspectral Microscopy","volume":"16","author":"Gehm","year":"2008","journal-title":"Opt. Express"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3195","DOI":"10.1364\/BOE.386338","article-title":"Hyperspectral and Multispectral Imaging in Digital and Computational Pathology: A Systematic Review [Invited]","volume":"11","author":"Ortega","year":"2020","journal-title":"Biomed. Opt. Express"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2614","DOI":"10.1021\/acs.jpcc.1c08359","article-title":"Compressive Hyperspectral Microscopy of Scattering and Fluorescence of Nanoparticles","volume":"126","author":"Xu","year":"2022","journal-title":"J. Phys. Chem. C"},{"key":"ref_23","first-page":"046501","article-title":"Automatic Detection of Head and Neck Squamous Cell Carcinoma on Histologic Slides Using Hyperspectral Microscopic Imaging","volume":"27","author":"Ma","year":"2022","journal-title":"J. Biomed. Opt."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Tao, C., Du, J., Tang, Y., Wang, J., Dong, K., Yang, M., Hu, B., and Zhang, Z. (2022). A Deep-Learning Based System for Rapid Genus Identification of Pathogens under Hyperspectral Microscopic Images. Cells, 11.","DOI":"10.3390\/cells11142237"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1117\/1.JBO.23.2.021103","article-title":"Biophotonics: The Big Picture","volume":"23","author":"Marcu","year":"2017","journal-title":"J. Biomed. Opt."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Stergar, J., Hren, R., and Milani\u010d, M. (2022). Design and Validation of a Custom-Made Laboratory Hyperspectral Imaging System for Biomedical Applications Using a Broadband LED Light Source. Sensors, 22.","DOI":"10.3390\/s22166274"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"R37","DOI":"10.1088\/0031-9155\/58\/11\/R37","article-title":"Optical Properties of Biological Tissues: A Review","volume":"58","author":"Jacques","year":"2013","journal-title":"Phys. Med. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"879","DOI":"10.1364\/JOSA.54.000879","article-title":"Optimization of the Czerny\u2013Turner Spectrometer*","volume":"54","author":"Shafer","year":"1964","journal-title":"J. Opt. Soc. Am."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1088\/1475-4878\/33\/3\/301","article-title":"The C.I.E. Colorimetric Standards and Their Use","volume":"33","author":"Smith","year":"1931","journal-title":"Trans. Opt. Soc."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Welch, A.J., and van Gemert, M.J.C. (2011). Optical-Thermal Response of Laser-Irradiated Tissue, Springer. [2nd ed.].","DOI":"10.1007\/978-90-481-8831-4"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1155\/2012\/154735","article-title":"Color Standardization Method and System for Whole Slide Imaging Based on Spectral Sensing","volume":"35","author":"Tani","year":"2012","journal-title":"Anal. Cell Pathol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"712","DOI":"10.1002\/cyto.a.20242","article-title":"Imaging Spectrometer Fundamentals for Researchers in the Biosciences\u2014A Tutorial","volume":"69A","author":"Lerner","year":"2006","journal-title":"Cytometry"},{"key":"ref_33","unstructured":"Stergar, J. (2021). Zdru\u017eeno Hiperspektralno Slikanje Makroskopskih in Mikroskopskih Vzorcev Za Ocenjevanje Opti\u010dnih in Strukturnih Lastnosti Tkiv. [Ph.D. Thesis, University of Ljubljana]."},{"key":"ref_34","unstructured":"Stergar, J. (2016). Optotermina\u010dna Manipulacija Posameznih Submikronskih Delcev, University of Ljubljana."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1991","DOI":"10.1364\/BOE.387837","article-title":"Hyperspectral Evaluation of Peritoneal Fibrosis in Mouse Models","volume":"11","author":"Stergar","year":"2020","journal-title":"Biomed. Opt. Express"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"3461","DOI":"10.1364\/BOE.460288","article-title":"Hyperspectral Evaluation of Vasculature in Induced Peritonitis Mouse Models","volume":"13","author":"Stergar","year":"2022","journal-title":"Biomed. Opt. Express"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1002\/path.5921","article-title":"Developing Image Analysis Methods for Digital Pathology","volume":"257","author":"Bankhead","year":"2022","journal-title":"J. Pathol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"044007","DOI":"10.1088\/2040-8986\/aab0e8","article-title":"Multispectral Analysis Tools Can Increase Utility of RGB Color Images in Histology","volume":"20","author":"Fereidouni","year":"2018","journal-title":"J. Opt."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Tkaczyk, T.S. (2010). Field Guide to Microscopy, SPIE Press.","DOI":"10.1117\/3.798239"},{"key":"ref_40","unstructured":"Horowitz, P., and Hill, W. (1989). The Art of Electronics, Cambridge University Press. [2nd ed.]."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"e10","DOI":"10.14440\/jbm.2014.36","article-title":"Advanced Methods of Microscope Control Using \u039cManager Software","volume":"1","author":"Edelstein","year":"2014","journal-title":"J. Biol. Methods"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Rueden, C.T., Schindelin, J., Hiner, M.C., DeZonia, B.E., Walter, A.E., Arena, E.T., and Eliceiri, K.W. (2017). ImageJ2: ImageJ for the next Generation of Scientific Image Data. BMC Bioinform., 18.","DOI":"10.1186\/s12859-017-1934-z"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH Image to ImageJ: 25 Years of Image Analysis","volume":"9","author":"Schneider","year":"2012","journal-title":"Nat. Methods"}],"container-title":["Sensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8220\/23\/5\/2374\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:38:06Z","timestamp":1760121486000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8220\/23\/5\/2374"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,2,21]]},"references-count":43,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2023,3]]}},"alternative-id":["s23052374"],"URL":"https:\/\/doi.org\/10.3390\/s23052374","relation":{},"ISSN":["1424-8220"],"issn-type":[{"value":"1424-8220","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,2,21]]}}}