{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T18:03:56Z","timestamp":1791309836006,"version":"4.3.1"},"reference-count":57,"publisher":"American Chemical Society (ACS)","issue":"12","license":[{"start":{"date-parts":[[2016,9,30]],"date-time":"2016-09-30T00:00:00Z","timestamp":1475193600000},"content-version":"vor","delay-in-days":366,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_termsofuse.html"}],"funder":[{"DOI":"10.13039\/501100001164","name":"Australian Synchrotron","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001164","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001198","name":"Faculty of Science, Monash University","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001198","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2015,12,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Single molecule localization microscopy (SMLM) and synchrotron Fourier transform infrared (S-FTIR) spectroscopy are two techniques capable of elucidating unique and valuable biological detail. SMLM provides images of the structures and distributions of targeted biomolecules at spatial resolutions up to an order of magnitude better than the diffraction limit, whereas IR spectroscopy objectively measures the holistic biochemistry of an entire sample, thereby revealing any variations in overall composition. Both tools are currently applied extensively to detect cellular response to disease, chemical treatment, and environmental change. Here, these two techniques have been applied correlatively at the single cell level to probe the biochemistry of common fixation methods and have detected various fixation-induced losses of biomolecular composition and cellular ultrastructure. Furthermore, by extensive honing and optimizing of fixation protocols, many fixation artifacts previously considered pervasive and regularly identified using IR spectroscopy and fluorescence techniques have been avoided. Both paraformaldehyde and two-step glutaraldehyde fixation were identified as best preserving biochemistry for both SMLM and IR studies while other glutaraldehyde and methanol fixation protocols were demonstrated to cause significant biochemical changes and higher variability between samples. Moreover, the potential complementarity of the two techniques was strikingly demonstrated in the correlated detection of biochemical changes as well as in the detection of fixation-induced damage that was only revealed by one of the two techniques.<\/jats:p>","DOI":"10.1021\/acschembio.5b00754","type":"journal-article","created":{"date-parts":[[2015,9,30]],"date-time":"2015-09-30T18:27:01Z","timestamp":1443637621000},"page":"2874-2883","source":"Crossref","is-referenced-by-count":16,"title":["Correlative Synchrotron Fourier Transform Infrared\nSpectroscopy and Single Molecule Super Resolution Microscopy for the\nDetection of Composition and Ultrastructure Alterations in Single\nCells"],"prefix":"10.1021","volume":"10","author":[{"given":"Donna R.","family":"Whelan","sequence":"first","affiliation":[{"name":"Monash University , , , ,","place":["Victoria, Australia, 3800"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toby D. M.","family":"Bell","sequence":"additional","affiliation":[{"name":"Monash University , , , ,","place":["Victoria, Australia, 3800"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2015,9,30]]},"reference":[{"key":"2026092909084186900_cit1","doi-asserted-by":"publisher","first-page":"561","DOI":"10.1007\/s00418-014-1184-3","article-title":"Eight years of single-molecule localization\nmicroscopy","volume":"141","author":"Klein","year":"2014","journal-title":"Histochem. Cell Biol."},{"key":"2026092909084186900_cit2","doi-asserted-by":"publisher","first-page":"7924","DOI":"10.1038\/srep07924","article-title":"Image artifacts\nin single molecule localization microscopy:\nwhy optimization of sample preparation protocols matters","volume":"5","author":"Whelan","year":"2015","journal-title":"Sci. Rep."},{"key":"2026092909084186900_cit3","doi-asserted-by":"publisher","first-page":"1183","DOI":"10.1038\/nmeth.3206","article-title":"Seeing in super-resolution","volume":"11","year":"2014","journal-title":"Nat. Methods"},{"key":"2026092909084186900_cit4","doi-asserted-by":"publisher","first-page":"647","DOI":"10.1002\/cphc.201300853","article-title":"Correlative atomic force microscopy\nand localization-based super-resolution microscopy: revealing labelling\nand image reconstruction artefacts","volume":"15","author":"Monserrate","year":"2014","journal-title":"ChemPhysChem"},{"key":"2026092909084186900_cit5","doi-asserted-by":"publisher","first-page":"993","DOI":"10.1146\/annurev.biochem.77.061906.092014","article-title":"Super-resolution\nfluorescence microscopy","volume":"78","author":"Huang","year":"2009","journal-title":"Annu. Rev. Biochem."},{"key":"2026092909084186900_cit6","doi-asserted-by":"publisher","first-page":"152","DOI":"10.1038\/nmeth.1855","article-title":"Immunolabeling\nartifacts and the need for live-cell imaging","volume":"9","author":"Schnell","year":"2012","journal-title":"Nat. Methods"},{"key":"2026092909084186900_cit7","doi-asserted-by":"publisher","first-page":"6136","DOI":"10.1073\/pnas.1121558109","article-title":"Correlative\n3D superresolution fluorescence and electron microscopy reveal the\nrelationship of mitochondrial nucleoids to membranes","volume":"109","author":"Kopek","year":"2012","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2026092909084186900_cit8","doi-asserted-by":"publisher","first-page":"4351","DOI":"10.1242\/jcs.156620","article-title":"Correlative super-resolution fluorescence\nand electron microscopy\nof the nuclear pore complex with molecular resolution","volume":"127","author":"Loschberger","year":"2014","journal-title":"J. Cell Sci."},{"key":"2026092909084186900_cit9","doi-asserted-by":"publisher","first-page":"305","DOI":"10.1038\/nmeth.2816","article-title":"Correlative\nsuper-resolution fluorescence and metal-replica transmission electron\nmicroscopy","volume":"11","author":"Sochacki","year":"2014","journal-title":"Nat. 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Biol."},{"key":"2026092909084186900_cit13","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1016\/B978-0-12-333920-1.50022-7","article-title":"Artifacts","volume-title":"Fixation for Electron Microscopy","author":"Hayat","year":"1981"},{"key":"2026092909084186900_cit14","doi-asserted-by":"publisher","first-page":"285","DOI":"10.1084\/jem.95.3.285","article-title":"A study\nof fixation for electron microscopy","volume":"95","author":"Palade","year":"1952","journal-title":"J. Exp.\nMed."},{"key":"2026092909084186900_cit15","doi-asserted-by":"publisher","first-page":"1771","DOI":"10.1038\/nprot.2014.110","article-title":"Using Fourier transform IR spectroscopy to analyze\nbiological materials","volume":"9","author":"Baker","year":"2014","journal-title":"Nat. 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Chem., Int. Ed."},{"key":"2026092909084186900_cit26","doi-asserted-by":"publisher","first-page":"346","DOI":"10.1097\/00129039-200112000-00010","article-title":"Effects of acetone, methanol, or\nparaformaldehyde on cellular structure, visualized by reflection contrast\nmicroscopy and transmission and scanning electron microscopy","volume":"9","author":"Hoetelmans","year":"2001","journal-title":"Appl. Immunohistochem."},{"key":"2026092909084186900_cit27","doi-asserted-by":"publisher","first-page":"115","DOI":"10.1016\/0739-6260(86)90042-0","article-title":"Glutaraldehyde\n\u2013 role in electron microscopy","volume":"17","author":"Hayat","year":"1986","journal-title":"Micron\nMicrosc. Acta"},{"key":"2026092909084186900_cit28","doi-asserted-by":"publisher","first-page":"921","DOI":"10.1002\/bip.21036","article-title":"Infrared microscopy\nfor the study of biological cell monolayers. I. Spectral effects of\nacetone and formalin fixation","volume":"89","author":"Hastings","year":"2008","journal-title":"Biopolymers"},{"key":"2026092909084186900_cit29","doi-asserted-by":"publisher","first-page":"e0116491","DOI":"10.1371\/journal.pone.0116491","article-title":"Importance of tissue\npreparation methods in FTIR micro-spectroscopical\nanalysis of biological tissues: \u2018traps for new users\u2019","volume":"10","author":"Zohdi","year":"2015","journal-title":"PLoS One"},{"key":"2026092909084186900_cit30","doi-asserted-by":"publisher","first-page":"3665","DOI":"10.1016\/S0006-3495(02)75366-6","article-title":"Atomic force microscopy investigation of fibroblasts\ninfected with wild-type and mutant murine leukemia virus (MuLV)","volume":"83","author":"Kuznetsov","year":"2002","journal-title":"Biophys. J."},{"key":"2026092909084186900_cit31","article-title":"Fixation and Embedding","volume-title":"Microscopy, Immunohistochemistry,\nand Antigen Retrieval Methods for LIght and Electron Microscopy","author":"Hayat","year":"2002"},{"key":"2026092909084186900_cit32","doi-asserted-by":"publisher","first-page":"773","DOI":"10.1177\/002215540305100608","article-title":"Fixation\nmethods for the study of lipid droplets by\nimmunofluorescence microscopy","volume":"51","author":"DiDonato","year":"2003","journal-title":"J. Histochem.\nCytochem."},{"key":"2026092909084186900_cit33","doi-asserted-by":"publisher","first-page":"1781","DOI":"10.1007\/s00216-009-3411-7","article-title":"Studies of chemical\nfixation effects in human cell lines using Raman microspectroscopy","volume":"396","author":"Meade","year":"2010","journal-title":"Anal. Bioanal. Chem."},{"key":"2026092909084186900_cit34","doi-asserted-by":"publisher","first-page":"775","DOI":"10.1002\/jbio.201200112","article-title":"Quantification of DNA in simple eukaryotic cells using\nFourier transform infrared spectroscopy","volume":"6","author":"Whelan","year":"2013","journal-title":"Journal\nof Biophotonics"},{"key":"2026092909084186900_cit35","doi-asserted-by":"publisher","first-page":"477","DOI":"10.1016\/S0301-4622(03)00035-8","article-title":"A library of IR bands of nucleic\nacids in solution","volume":"104","author":"Banyay","year":"2003","journal-title":"Biophys. Chem."},{"key":"2026092909084186900_cit36","doi-asserted-by":"publisher","first-page":"183","DOI":"10.1016\/j.yexmp.2005.10.002","article-title":"Assessment of fixatives,\nfixation, and tissue processing on morphology and RNA integrity","volume":"80","author":"Cox","year":"2006","journal-title":"Exp. Mol. Pathol."},{"key":"2026092909084186900_cit37","doi-asserted-by":"publisher","first-page":"1961","DOI":"10.1016\/S0002-9440(10)64472-0","article-title":"Effect of fixatives\nand tissue processing\non the content and integrity of nucleic acids","volume":"161","author":"Srinivasan","year":"2002","journal-title":"Am. J. Pathol."},{"key":"2026092909084186900_cit38","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1242\/jcs.107.4.881","article-title":"Quantitative-determination of the\nproportion of microtubule\npolymer present during the mitosis-interphase transition","volume":"107","author":"Zhai","year":"1994","journal-title":"J. Cell Sci."},{"key":"2026092909084186900_cit39","volume-title":"Multivariate\nCalibration","author":"Martens","year":"1989"},{"key":"2026092909084186900_cit40","doi-asserted-by":"publisher","first-page":"329","DOI":"10.1002\/1097-0282(2000)57:6&lt;329::AID-BIP20&gt;3.0.CO;2-2","article-title":"IR spectroscopic characteristics of cell cycle and\ncell death probed by synchrotron radiation based fourier transform\nIR spectromicroscopy","volume":"57","author":"Holman","year":"2000","journal-title":"Biopolymers"},{"key":"2026092909084186900_cit41","doi-asserted-by":"publisher","first-page":"3957","DOI":"10.1039\/c3an00507k","article-title":"Exploring\nthe spectroscopic differences\nof Caki-2 cells progressing through the cell cycle while proliferating\nin vitro","volume":"138","author":"Jimenez-Hernandez","year":"2013","journal-title":"Analyst"},{"key":"2026092909084186900_cit42","doi-asserted-by":"publisher","first-page":"908","DOI":"10.1016\/j.bbamem.2006.04.018","article-title":"Cell-cycle-dependent\nvariations in FTIR micro-spectra of single proliferating\nHeLa cells: Principal component and artificial neural network analysis","volume":"1758","author":"Boydston-White","year":"2006","journal-title":"Biochim. Biophys. Acta, Biomembr."},{"key":"2026092909084186900_cit43","doi-asserted-by":"publisher","first-page":"8265","DOI":"10.1021\/ac3017407","article-title":"Evaluating different\nfixation protocols for spectral cytopathology,\npart 2: cultured cells","volume":"84","author":"Mazur","year":"2012","journal-title":"Anal. Chem."},{"key":"2026092909084186900_cit44","doi-asserted-by":"publisher","first-page":"1259","DOI":"10.1021\/ac202046d","article-title":"Evaluating different\nfixation protocols for spectral cytopathology,\npart 1","volume":"84","author":"Mazur","year":"2012","journal-title":"Anal. Chem."},{"key":"2026092909084186900_cit45","doi-asserted-by":"publisher","first-page":"3094","DOI":"10.1039\/c0an00543f","article-title":"Characterization of\nmicroRNA-125b expression in MCF7 breast cancer cells by ATR-FTIR spectroscopy","volume":"135","author":"Simsek Ozek","year":"2010","journal-title":"Analyst"},{"key":"2026092909084186900_cit46","doi-asserted-by":"publisher","first-page":"134","DOI":"10.1080\/05704920701829043","article-title":"Fourier transform infrared (FTIR)\nspectroscopy of biological tissues","volume":"43","author":"Movasaghi","year":"2008","journal-title":"Appl. Spectrosc.\nRev."},{"key":"2026092909084186900_cit47","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1369\/jhc.2010.957217","article-title":"Comparison of fixation methods for\npreservation of morphology, RNAs, and proteins from paraffin-embedded\nhuman cancer cell-implanted mouse models","volume":"59","author":"Matsuda","year":"2011","journal-title":"J.\nHistochem. Cytochem."},{"key":"2026092909084186900_cit48","doi-asserted-by":"publisher","first-page":"10","DOI":"10.1186\/1471-2121-2-10","article-title":"Biochemical,\nelectron microscopic and immunohistological observations of cationic\ndetergent-extracted cells: detection and improved preservation of\nmicroextensions and ultramicroextensions","volume":"2","author":"Nakamura","year":"2001","journal-title":"BMC\nCell Biol."},{"key":"2026092909084186900_cit49","doi-asserted-by":"crossref","first-page":"790","DOI":"10.2144\/04375RV01","article-title":"Glutaraldehyde:\nbehavior in aqueous solution, reaction with proteins, and application\nto enzyme crosslinking","volume":"37","author":"Migneault","year":"2004","journal-title":"Biotechniques"},{"key":"2026092909084186900_cit50","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1016\/S0006-3495(63)86803-4","article-title":"Physicaochemical\nstudies on reaction between formaldehyde\nand DNA","volume":"3","author":"Freifelder","year":"1963","journal-title":"Biophys. J."},{"key":"2026092909084186900_cit51","doi-asserted-by":"publisher","first-page":"399","DOI":"10.1155\/2012\/649094","article-title":"Influence of fixation\nproducts used in the histological processing in the FTIR spectra of\nlung cells","volume":"27","author":"Pereira","year":"2012","journal-title":"Spectroscopy"},{"key":"2026092909084186900_cit52","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1039\/C0SC00409J","article-title":"Ultrafast infrared\nchemical imaging of live cells","volume":"2","author":"Amrania","year":"2011","journal-title":"Chemical Science"},{"key":"2026092909084186900_cit53","doi-asserted-by":"publisher","first-page":"3856","DOI":"10.1039\/C4AN00638K","article-title":"Large scale infrared\nimaging of tissue micro arrays (TMAs) using a tunable Quantum Cascade\nLaser (QCL) based microscope","volume":"139","author":"Bassan","year":"2014","journal-title":"Analyst"},{"key":"2026092909084186900_cit54","doi-asserted-by":"publisher","first-page":"4768","DOI":"10.1021\/ac300313x","article-title":"Infrared microspectroscopy\nof live cells in microfluidic devices (MD-IRMS): toward a powerful\nlabel-free cell-based assay","volume":"84","author":"Vaccari","year":"2012","journal-title":"Anal. Chem."},{"key":"2026092909084186900_cit55","doi-asserted-by":"publisher","first-page":"34","DOI":"10.1016\/j.vibspec.2010.02.005","article-title":"FTIR spectroscopy\nof single live cells in aqueous media by synchrotron IR microscopy\nusing microfabricated sample holders","volume":"53","author":"Tobin","year":"2010","journal-title":"Vib. Spectrosc."},{"key":"2026092909084186900_cit56","doi-asserted-by":"publisher","first-page":"603A","DOI":"10.1016\/j.bpj.2013.11.3338","article-title":"Super-resolution\nmicroscopy of cells expressing rhabdovirus proteins","volume":"106","author":"Bell","year":"2014","journal-title":"Biophys. J."},{"key":"2026092909084186900_cit57","doi-asserted-by":"publisher","first-page":"179","DOI":"10.1071\/CH13499","article-title":"Focus on super-resolution\nimaging with direct stochastic optical reconstruction microscopy (dSTORM)","volume":"67","author":"Whelan","year":"2014","journal-title":"Aust. J. Chem."}],"container-title":["ACS Chemical Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acschembio.5b00754","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/10\/12\/2874\/67280142\/acschembio.5b00754.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/10\/12\/2874\/67280142\/acschembio.5b00754.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T15:03:22Z","timestamp":1790694202000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/acbcct\/article\/10\/12\/2874\/776929\/Correlative-Synchrotron-Fourier-Transform-Infrared"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2015,9,30]]},"references-count":57,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2015,9,30]]},"published-print":{"date-parts":[[2015,12,18]]}},"URL":"https:\/\/doi.org\/10.1021\/acschembio.5b00754","relation":{},"ISSN":["1554-8929","1554-8937"],"issn-type":[{"value":"1554-8929","type":"print"},{"value":"1554-8937","type":"electronic"}],"subject":[],"published":{"date-parts":[[2015,9,30]]}}}