{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,2]],"date-time":"2026-01-02T07:49:24Z","timestamp":1767340164852,"version":"3.41.2"},"reference-count":39,"publisher":"AIP Publishing","issue":"8","content-domain":{"domain":["pubs.aip.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2024,8,1]]},"abstract":"<jats:p>Super-resolution imaging techniques have fundamentally changed our understanding of cellular architecture and dynamics by surpassing the diffraction limit and enabling the visualization of subcellular details. The popular super-resolution method known as stochastic optical reconstruction microscopy (STORM) relies on the exact localization of single fluorescent molecules. The significance of employing Vectashield as a mounting medium for the super-resolution imaging scheme called direct STORM has recently been explored. Alexa Fluor 647 (AF647), one of the most popular dyes, shows significant blinking in Vectashield. However, to observe prominent blinking of the fluorophore for the reconstruction of super-resolved images, the power of the excitation laser needs to be tuned. This work demonstrates the tuning of excitation power density in the sample plane for superior imaging performance using AF647 in Vectashield. Samples comprising MDA-MB-231 breast cancer cell line are used for the experiments. The actin filaments of the cell are stained with phalloidin-conjugated AF647 dye. For the experiment, we employ a low-cost openFrame-based STORM system equipped with a programmable Arduino-regulated laser source emitting at 638\u00a0nm. An excitation power density of 0.60 kW\/cm2 at 638\u00a0nm in the sample plane is observed to maximize the signal-to-noise ratio, the number of switching events, and the number of photons detected per event during image acquisition, thereby leading to the best imaging performance in terms of resolution. The outcome of this work will promote further STORM-based super-resolved imaging applications in cell biology using Alexa Fluor 647 in Vectashield.<\/jats:p>","DOI":"10.1063\/5.0217409","type":"journal-article","created":{"date-parts":[[2024,8,1]],"date-time":"2024-08-01T12:39:45Z","timestamp":1722515985000},"update-policy":"https:\/\/doi.org\/10.1063\/aip-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Tuning the excitation laser power in a stochastic optical reconstruction microscope for Alexa Fluor 647 dye in Vectashield mounting media"],"prefix":"10.1063","volume":"95","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2613-2189","authenticated-orcid":false,"given":"Amalesh","family":"Kumar","sequence":"first","affiliation":[{"name":"Department of Physics, IIT Guwahati 1 , Guwahati 781039, Assam, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9004-6998","authenticated-orcid":false,"given":"Anupam","family":"Bharadwaj","sequence":"additional","affiliation":[{"name":"Department of Physics, IIT Guwahati 1 , Guwahati 781039, Assam, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4883-7005","authenticated-orcid":false,"given":"Pranjal","family":"Choudhury","sequence":"additional","affiliation":[{"name":"Department of Physics, IIT Guwahati 1 , Guwahati 781039, Assam, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7720-1613","authenticated-orcid":false,"given":"Sam P.","family":"Mathew","sequence":"additional","affiliation":[{"name":"Department of Bioscience and Bioengineering, IIT Guwahati 2 , Guwahati 781039, Assam, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5955-1371","authenticated-orcid":false,"given":"Bithiah Grace","family":"Jaganathan","sequence":"additional","affiliation":[{"name":"Department of Bioscience and Bioengineering, IIT Guwahati 2 , Guwahati 781039, Assam, India"},{"name":"Jyoti and Bhupat Mehta School of Health Sciences and Technology, IIT Guwahati 3 , Guwahati 781039, Assam, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4573-0248","authenticated-orcid":false,"given":"Bosanta R.","family":"Boruah","sequence":"additional","affiliation":[{"name":"Department of Physics, IIT Guwahati 1 , Guwahati 781039, Assam, India"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"317","published-online":{"date-parts":[[2024,8,1]]},"reference":[{"issue":"9","key":"2024080112391484400_c1","doi-asserted-by":"publisher","first-page":"e104","DOI":"10.1038\/lsa.2013.60","article-title":"Label-free super-resolution imaging of adenoviruses by submerged microsphere optical nanoscopy","volume":"2","year":"2013","journal-title":"Light Sci. Appl."},{"issue":"2","key":"2024080112391484400_c2","doi-asserted-by":"publisher","first-page":"366","DOI":"10.1111\/os.12637","article-title":"Optical microscopy and electron microscopy for the morphological evaluation of tendons: A mini review","volume":"12","year":"2020","journal-title":"Orthop. Surg."},{"issue":"7","key":"2024080112391484400_c3","doi-asserted-by":"publisher","first-page":"1047","DOI":"10.1016\/j.cell.2010.12.002","article-title":"Breaking the diffraction barrier: Super-resolution imaging of cells","volume":"143","year":"2010","journal-title":"Cell"},{"issue":"12","key":"2024080112391484400_c4","doi-asserted-by":"publisher","first-page":"834","DOI":"10.1016\/j.tplants.2015.08.013","article-title":"Super-resolution microscopy in plant cell imaging","volume":"20","year":"2015","journal-title":"Trends Plant Sci."},{"issue":"1","key":"2024080112391484400_c5","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1038\/s43586-021-00038-x","article-title":"Single-molecule localization microscopy","volume":"1","year":"2021","journal-title":"Nat. Rev. Methods Primers"},{"issue":"1","key":"2024080112391484400_c6","doi-asserted-by":"publisher","first-page":"12.46.1","DOI":"10.1002\/cpcy.23","article-title":"Stochastic optical reconstruction microscopy (STORM)","volume":"81","year":"2017","journal-title":"Curr. Protoc. Cytom."},{"issue":"1","key":"2024080112391484400_c7","doi-asserted-by":"publisher","first-page":"99","DOI":"10.1002\/cphc.201100735","article-title":"Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection","volume":"13","year":"2012","journal-title":"ChemPhysChem"},{"issue":"5","key":"2024080112391484400_c8","doi-asserted-by":"publisher","first-page":"438","DOI":"10.1002\/cjp2.217","article-title":"Application of direct stochastic optical reconstruction microscopy (dSTORM) to the histological analysis of human glomerular disease","volume":"7","year":"2021","journal-title":"J.\u00a0Pathol.: Clin. Res."},{"issue":"1","key":"2024080112391484400_c9","doi-asserted-by":"publisher","first-page":"163","DOI":"10.1038\/nprot.2016.002","article-title":"Correlated confocal and super-resolution imaging by VividSTORM","volume":"11","year":"2016","journal-title":"Nat. Protoc."},{"issue":"1","key":"2024080112391484400_c10","doi-asserted-by":"publisher","first-page":"6441","DOI":"10.1038\/s41598-020-63418-5","article-title":"Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy","volume":"10","year":"2020","journal-title":"Sci. Rep."},{"issue":"1","key":"2024080112391484400_c11","doi-asserted-by":"publisher","first-page":"8749","DOI":"10.1038\/s41598-018-27059-z","article-title":"Optimized protocol for combined PALM-dSTORM imaging","volume":"8","year":"2018","journal-title":"Sci. Rep."},{"key":"2024080112391484400_c12","doi-asserted-by":"publisher","first-page":"37","DOI":"10.1007\/978-1-4939-9818-0_4","article-title":"Cytological characterization of Arabidopsis arenosa polyploids by SIM","volume":"2061","year":"2020","journal-title":"Methods Mol. Biol."},{"issue":"6","key":"2024080112391484400_c13","doi-asserted-by":"publisher","first-page":"885","DOI":"10.1364\/boe.4.000885","article-title":"Simple buffers for 3D STORM microscopy","volume":"4","year":"2013","journal-title":"Biomed. Opt. Express"},{"key":"2024080112391484400_c14","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1007\/978-1-4939-7759-8","article-title":"Correlative 3D structured illumination microscopy and single-molecule localization microscopy for imaging cancer invasion","volume-title":"Protein Complex Assembly: Methods and Protocols","author":"Marsh","year":"2018"},{"issue":"21","key":"2024080112391484400_c15","doi-asserted-by":"publisher","first-page":"218103","DOI":"10.1103\/physrevlett.98.218103","article-title":"Breaking the diffraction barrier in fluorescence microscopy by optical shelving","volume":"98","year":"2007","journal-title":"Phys. Rev. Lett."},{"issue":"1","key":"2024080112391484400_c16","doi-asserted-by":"publisher","first-page":"6287","DOI":"10.1038\/ncomms7287","article-title":"Visualization of molecular fluorescence point spread functions via remote excitation switching fluorescence microscopy","volume":"6","year":"2015","journal-title":"Nat. Commun."},{"key":"2024080112391484400_c17"},{"key":"2024080112391484400_c18"},{"issue":"2","key":"2024080112391484400_c19","doi-asserted-by":"publisher","first-page":"244","DOI":"10.18520\/cs%2Fv126%2Fi2%2F244-254","article-title":"A cost-effective, modular, research-grade optical microscope","volume":"126","year":"2024","journal-title":"Curr.Sci."},{"key":"2024080112391484400_c20","doi-asserted-by":"publisher","first-page":"112790","DOI":"10.1016\/j.cma.2019.112790","article-title":"An energy approach to the solution of partial differential equations in computational mechanics via machine learning: Concepts, implementation and applications","volume":"362","year":"2020","journal-title":"Comput. Methods Appl. Mech. Eng."},{"key":"2024080112391484400_c21","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1016\/j.ccr.2018.08.006","article-title":"Organic fluorescent probes for stochastic optical reconstruction microscopy (STORM): Recent highlights and future possibilities","volume":"380","year":"2019","journal-title":"Coord. Chem. Rev."},{"issue":"29","key":"2024080112391484400_c22","doi-asserted-by":"publisher","first-page":"5465","DOI":"10.1002\/anie.200801518","article-title":"A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes","volume":"47","year":"2008","journal-title":"Angew. Chem., Int. Ed."},{"issue":"7","key":"2024080112391484400_c23","doi-asserted-by":"publisher","first-page":"e0158884","DOI":"10.1371\/journal.pone.0158884","article-title":"Optimizing imaging conditions for demanding multi-color super resolution localization microscopy","volume":"11","year":"2016","journal-title":"PLoS One"},{"issue":"14","key":"2024080112391484400_c24","doi-asserted-by":"publisher","first-page":"6699","DOI":"10.1039\/c0cp01919d","article-title":"Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy","volume":"13","year":"2011","journal-title":"Phys. Chem. Chem. Phys."},{"issue":"1","key":"2024080112391484400_c25","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1002\/0471142727.mb1420s92","article-title":"Computer control of microscopes using \u00b5Manager","volume":"92","year":"2010","journal-title":"Curr. Protoc. Mol. Biol."},{"issue":"7","key":"2024080112391484400_c26","doi-asserted-by":"publisher","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An open-source platform for biological-image analysis","volume":"9","year":"2012","journal-title":"Nat. Methods"},{"issue":"7","key":"2024080112391484400_c27","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH image to ImageJ: 25 years of image analysis","volume":"9","year":"2012","journal-title":"Nat. Methods"},{"issue":"9","key":"2024080112391484400_c28","doi-asserted-by":"publisher","first-page":"948","DOI":"10.1002\/jbio.201500324","article-title":"easySTORM: a robust, lower-cost approach to localisation and TIRF microscopy","volume":"9","year":"2016","journal-title":"J.\u00a0Biophot."},{"issue":"3","key":"2024080112391484400_c29","doi-asserted-by":"publisher","first-page":"035007","DOI":"10.1088\/2050-6120\/ad4ae6","article-title":"Enhanced fluorescence blinking of AF647 fluorophores in Mowiol via violet and UV light induced recovery for superior localization microscopy","volume":"12","year":"2024","journal-title":"Methods Appl. Fluoresc."},{"key":"2024080112391484400_c30"},{"issue":"21","key":"2024080112391484400_c31","doi-asserted-by":"publisher","first-page":"4699","DOI":"10.3390\/s19214699","article-title":"Radiometric assessment of a UAV-based push-broom hyperspectral camera","volume":"19","year":"2019","journal-title":"Sensors"},{"key":"2024080112391484400_c32","doi-asserted-by":"publisher","first-page":"114112","DOI":"10.1016\/j.enconman.2021.114112","article-title":"A review of sensitivity analysis practices in wind resource assessment","volume":"238","year":"2021","journal-title":"Energy Convers. Manage."},{"key":"2024080112391484400_c33","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1016\/j.advengsoft.2016.06.005","article-title":"A software framework for probabilistic sensitivity analysis for computationally expensive models","volume":"100","year":"2016","journal-title":"Adv. Eng. Software"},{"issue":"9","key":"2024080112391484400_c34","doi-asserted-by":"publisher","first-page":"97","DOI":"10.21105\/joss.00097","article-title":"SALib: An open-source Python library for Sensitivity Analysis","volume":"2","year":"2017","journal-title":"J.\u00a0Open Source Software"},{"issue":"12","key":"2024080112391484400_c35","doi-asserted-by":"publisher","first-page":"1027","DOI":"10.1038\/nmeth.1768","article-title":"Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging","volume":"8","year":"2011","journal-title":"Nat. Methods"},{"issue":"1","key":"2024080112391484400_c36","doi-asserted-by":"publisher","first-page":"29687","DOI":"10.1038\/srep29687","article-title":"Methodology for quantitative characterization of fluorophore photoswitching to predict superresolution microscopy image quality","volume":"6","year":"2016","journal-title":"Sci. Rep."},{"issue":"1","key":"2024080112391484400_c37","doi-asserted-by":"publisher","first-page":"3103","DOI":"10.1038\/s41467-019-11024-z","article-title":"Fourier ring correlation simplifies image restoration in fluorescence microscopy","volume":"10","year":"2019","journal-title":"Nat. Commun."},{"issue":"6","key":"2024080112391484400_c38","doi-asserted-by":"publisher","first-page":"557","DOI":"10.1038\/nmeth.2448","article-title":"Measuring image resolution in optical nanoscopy","volume":"10","year":"2013","journal-title":"Nat. Methods"},{"key":"2024080112391484400_c39","doi-asserted-by":"publisher","first-page":"393","DOI":"10.3389\/fcell.2020.00393","article-title":"Effect of nuclear stiffness on cell mechanics and migration of human breast cancer cells","volume":"8","year":"2020","journal-title":"Front. Cell Dev. Biol."}],"container-title":["Review of Scientific Instruments"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.aip.org\/aip\/rsi\/article-pdf\/doi\/10.1063\/5.0217409\/20087553\/083701_1_5.0217409.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.aip.org\/aip\/rsi\/article-pdf\/doi\/10.1063\/5.0217409\/20087553\/083701_1_5.0217409.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,8,1]],"date-time":"2024-08-01T12:39:50Z","timestamp":1722515990000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.aip.org\/rsi\/article\/95\/8\/083701\/3305742\/Tuning-the-excitation-laser-power-in-a-stochastic"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,8,1]]},"references-count":39,"journal-issue":{"issue":"8","published-print":{"date-parts":[[2024,8,1]]}},"URL":"https:\/\/doi.org\/10.1063\/5.0217409","relation":{},"ISSN":["0034-6748","1089-7623"],"issn-type":[{"type":"print","value":"0034-6748"},{"type":"electronic","value":"1089-7623"}],"subject":[],"published-other":{"date-parts":[[2024,8,1]]},"published":{"date-parts":[[2024,8,1]]},"article-number":"083701"}}