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The bleaching time can be controlled through the repetition rate of a pulsed excitation source. We show that reducing the excitation repetition rate from 40 to 2.5 MHz delays photobleaching and therefore extends the observation window of a single-molecule fluorophore by more than an order of magnitude. Furthermore, we demonstrate that photon-stream analyses that depend on emitted photon flux suffer a decrease in signal-to-noise ratio with decreasing repetition rate, while analyses that depend on total photon number have a signal-to-noise ratio that is independent of excitation repetition rate.<\/jats:p>","DOI":"10.1021\/acs.jpcb.6c03371","type":"journal-article","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T05:01:46Z","timestamp":1789534906000},"page":"10140-10145","source":"Crossref","is-referenced-by-count":0,"title":["Extending Observation\nWindows While Preserving Signal-to-Noise\nin Single-Molecule Spectroscopy"],"prefix":"10.1021","volume":"130","author":[{"given":"Quyen B.","family":"Le","sequence":"first","affiliation":[{"name":"University of New Mexico , , 210 University Blvd , , ,","place":["Albuquerque, New Mexico, United States, 87102"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adrienne M.","family":"Garcia","sequence":"additional","affiliation":[{"name":"University of New Mexico , , 210 University Blvd , , ,","place":["Albuquerque, New Mexico, United States, 87102"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cynthia A.","family":"Taylor","sequence":"additional","affiliation":[{"name":"University of New Mexico , , 210 University Blvd , , ,","place":["Albuquerque, New Mexico, United States, 87102"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5444-7436","authenticated-orcid":true,"given":"John T.","family":"King","sequence":"additional","affiliation":[{"name":"University of New Mexico , , 210 University Blvd , , ,","place":["Albuquerque, New Mexico, United States, 87102"]},{"name":"University of New Mexico , , 210 University Blvd , , ,","place":["Albuquerque, New Mexico, United States, 87102"]},{"name":"University of New Mexico , , 210 University Blvd , , ,","place":["Albuquerque, New Mexico, United States, 87102"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,16]]},"reference":[{"key":"2026100109061243900_cit1","doi-asserted-by":"publisher","first-page":"1670","DOI":"10.1126\/science.283.5408.1670","article-title":"Illuminating Single Molecules in Condensed Matter","volume":"283","author":"Moerner","year":"1999","journal-title":"Science"},{"key":"2026100109061243900_cit2","doi-asserted-by":"publisher","first-page":"2048","DOI":"10.1126\/science.288.5473.2048","article-title":"A Single-Molecule Study of RNA Catalysis\nand Folding","volume":"288","author":"Zhuang","year":"2000","journal-title":"Science"},{"key":"2026100109061243900_cit3","doi-asserted-by":"publisher","first-page":"743","DOI":"10.1038\/nature01060","article-title":"Probing\nthe Free-Energy Surface for\nProtein Folding with Single-Molecule Fluorescence Spectroscopy","volume":"419","author":"Schuler","year":"2002","journal-title":"Nature"},{"key":"2026100109061243900_cit4","doi-asserted-by":"publisher","first-page":"9302","DOI":"10.1073\/pnas.1133280100","article-title":"Single-Molecule Transition-State\nAnalysis of RNA Folding","volume":"100","author":"Bokinsky","year":"2003","journal-title":"Proc. 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