Photobleaching and stabilization of. fluorophores used for single-molecule analysis. with one- and two-photon excitation

Fluorescence spectroscopic measurements at the single-molecule level usually require large absorption cross sections and fluorescence quantum yields for the dyes under study. In addition to these parameters, the collectable number of fluorescence photons and, thus, the signal-to-noise ratio of the m...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Applied physics. B, Lasers and optics Lasers and optics, 2001-12, Vol.73 (8), p.829-837
Hauptverfasser: Dittrich, P.S., Schwille, P.
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
container_end_page 837
container_issue 8
container_start_page 829
container_title Applied physics. B, Lasers and optics
container_volume 73
creator Dittrich, P.S.
Schwille, P.
description Fluorescence spectroscopic measurements at the single-molecule level usually require large absorption cross sections and fluorescence quantum yields for the dyes under study. In addition to these parameters, the collectable number of fluorescence photons and, thus, the signal-to-noise ratio of the measurement, is influenced by processes like triplet-state population and photobleaching, shifting the saturation threshold of the dye to lower excitation intensities. Confocal fluorescence correlation spectroscopy (FCS) is a versatile method to precisely determine photon emission rates of single molecules but also gives access to rate constants of dynamic bleaching and intersystem crossing. In recent FCS studies in solution and living cells, two-photon excitation with its inherent spatial sectioning has proven to be a very valuable alternative to minimize background and cumulative signal loss. However, there is evidence that in many dye systems, the photobleaching rates with two-photon excitation are significantly enhanced with respect to one-photon excitation at comparable photon-emission yields. The reasons have so far remained mainly speculative. In the present study, potential photobleaching pathways are investigated by adding chemical stabilizers and by working at different oxygen concentrations. The results suggest that the population of triplet states does not appear to be responsible for the limited emission rate with two-photon excitation. Rather, photobleaching pathways via the formation of radicals seem to be plausible causes for the signal limitation. Favorable conditions are discussed to maximize the overall photon-collection yield in two-photon experiments.
doi_str_mv 10.1007/s003400100737
format Article
fullrecord <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_1671451971</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>1671451971</sourcerecordid><originalsourceid>FETCH-LOGICAL-c268t-2c07d61daa7fe844e6db7df81b790c17f5e524b78ae0e7b09688fac0648e839c3</originalsourceid><addsrcrecordid>eNpVkL1PwzAQxS0EEqUwsntkcbET13ZGVPElVYIB5shxzsTIjYPtqIW_nrRl4Za74b3f6T2ErhldMErlbaK05JTu71KeoBnjZUGo4NUpmtGKC1Iwyc7RRUqfdBqh1AztXruQQ-NBm871H1j3LU5ZN867H51d6HGwC2z9GGIYuhAh4TFBi22IOE0GD2QTPJjRw-TV_ju5tMBblzsceiAHXt4GMuzf9Bh2xuUD9xKdWe0TXP3tOXp_uH9bPZH1y-Pz6m5NTCFUJoWhshWs1VpaUJyDaBvZWsUaWVHDpF3CsuCNVBooyIZWUyqrzZRagSorU87RzZE7xPA1Qsr1xiUD3usewphqJiTjS1ZJNknJUWpiSCmCrYfoNjp-14zW-1brfw2Xv5ihcIs</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1671451971</pqid></control><display><type>article</type><title>Photobleaching and stabilization of. fluorophores used for single-molecule analysis. with one- and two-photon excitation</title><source>SpringerLink Journals</source><creator>Dittrich, P.S. ; Schwille, P.</creator><creatorcontrib>Dittrich, P.S. ; Schwille, P.</creatorcontrib><description>Fluorescence spectroscopic measurements at the single-molecule level usually require large absorption cross sections and fluorescence quantum yields for the dyes under study. In addition to these parameters, the collectable number of fluorescence photons and, thus, the signal-to-noise ratio of the measurement, is influenced by processes like triplet-state population and photobleaching, shifting the saturation threshold of the dye to lower excitation intensities. Confocal fluorescence correlation spectroscopy (FCS) is a versatile method to precisely determine photon emission rates of single molecules but also gives access to rate constants of dynamic bleaching and intersystem crossing. In recent FCS studies in solution and living cells, two-photon excitation with its inherent spatial sectioning has proven to be a very valuable alternative to minimize background and cumulative signal loss. However, there is evidence that in many dye systems, the photobleaching rates with two-photon excitation are significantly enhanced with respect to one-photon excitation at comparable photon-emission yields. The reasons have so far remained mainly speculative. In the present study, potential photobleaching pathways are investigated by adding chemical stabilizers and by working at different oxygen concentrations. The results suggest that the population of triplet states does not appear to be responsible for the limited emission rate with two-photon excitation. Rather, photobleaching pathways via the formation of radicals seem to be plausible causes for the signal limitation. Favorable conditions are discussed to maximize the overall photon-collection yield in two-photon experiments.</description><identifier>ISSN: 0946-2171</identifier><identifier>EISSN: 1432-0649</identifier><identifier>DOI: 10.1007/s003400100737</identifier><language>eng</language><subject>Absorption cross sections ; Dyes ; Dynamical systems ; Dynamics ; Excitation ; Fluorescence ; Pathways ; Spectroscopy</subject><ispartof>Applied physics. B, Lasers and optics, 2001-12, Vol.73 (8), p.829-837</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c268t-2c07d61daa7fe844e6db7df81b790c17f5e524b78ae0e7b09688fac0648e839c3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27903,27904</link.rule.ids></links><search><creatorcontrib>Dittrich, P.S.</creatorcontrib><creatorcontrib>Schwille, P.</creatorcontrib><title>Photobleaching and stabilization of. fluorophores used for single-molecule analysis. with one- and two-photon excitation</title><title>Applied physics. B, Lasers and optics</title><description>Fluorescence spectroscopic measurements at the single-molecule level usually require large absorption cross sections and fluorescence quantum yields for the dyes under study. In addition to these parameters, the collectable number of fluorescence photons and, thus, the signal-to-noise ratio of the measurement, is influenced by processes like triplet-state population and photobleaching, shifting the saturation threshold of the dye to lower excitation intensities. Confocal fluorescence correlation spectroscopy (FCS) is a versatile method to precisely determine photon emission rates of single molecules but also gives access to rate constants of dynamic bleaching and intersystem crossing. In recent FCS studies in solution and living cells, two-photon excitation with its inherent spatial sectioning has proven to be a very valuable alternative to minimize background and cumulative signal loss. However, there is evidence that in many dye systems, the photobleaching rates with two-photon excitation are significantly enhanced with respect to one-photon excitation at comparable photon-emission yields. The reasons have so far remained mainly speculative. In the present study, potential photobleaching pathways are investigated by adding chemical stabilizers and by working at different oxygen concentrations. The results suggest that the population of triplet states does not appear to be responsible for the limited emission rate with two-photon excitation. Rather, photobleaching pathways via the formation of radicals seem to be plausible causes for the signal limitation. Favorable conditions are discussed to maximize the overall photon-collection yield in two-photon experiments.</description><subject>Absorption cross sections</subject><subject>Dyes</subject><subject>Dynamical systems</subject><subject>Dynamics</subject><subject>Excitation</subject><subject>Fluorescence</subject><subject>Pathways</subject><subject>Spectroscopy</subject><issn>0946-2171</issn><issn>1432-0649</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><recordid>eNpVkL1PwzAQxS0EEqUwsntkcbET13ZGVPElVYIB5shxzsTIjYPtqIW_nrRl4Za74b3f6T2ErhldMErlbaK05JTu71KeoBnjZUGo4NUpmtGKC1Iwyc7RRUqfdBqh1AztXruQQ-NBm871H1j3LU5ZN867H51d6HGwC2z9GGIYuhAh4TFBi22IOE0GD2QTPJjRw-TV_ju5tMBblzsceiAHXt4GMuzf9Bh2xuUD9xKdWe0TXP3tOXp_uH9bPZH1y-Pz6m5NTCFUJoWhshWs1VpaUJyDaBvZWsUaWVHDpF3CsuCNVBooyIZWUyqrzZRagSorU87RzZE7xPA1Qsr1xiUD3usewphqJiTjS1ZJNknJUWpiSCmCrYfoNjp-14zW-1brfw2Xv5ihcIs</recordid><startdate>200112</startdate><enddate>200112</enddate><creator>Dittrich, P.S.</creator><creator>Schwille, P.</creator><scope>AAYXX</scope><scope>CITATION</scope><scope>7SP</scope><scope>7U5</scope><scope>8FD</scope><scope>H8D</scope><scope>L7M</scope></search><sort><creationdate>200112</creationdate><title>Photobleaching and stabilization of. fluorophores used for single-molecule analysis. with one- and two-photon excitation</title><author>Dittrich, P.S. ; Schwille, P.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c268t-2c07d61daa7fe844e6db7df81b790c17f5e524b78ae0e7b09688fac0648e839c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Absorption cross sections</topic><topic>Dyes</topic><topic>Dynamical systems</topic><topic>Dynamics</topic><topic>Excitation</topic><topic>Fluorescence</topic><topic>Pathways</topic><topic>Spectroscopy</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Dittrich, P.S.</creatorcontrib><creatorcontrib>Schwille, P.</creatorcontrib><collection>CrossRef</collection><collection>Electronics &amp; Communications Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Technology Research Database</collection><collection>Aerospace Database</collection><collection>Advanced Technologies Database with Aerospace</collection><jtitle>Applied physics. B, Lasers and optics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Dittrich, P.S.</au><au>Schwille, P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Photobleaching and stabilization of. fluorophores used for single-molecule analysis. with one- and two-photon excitation</atitle><jtitle>Applied physics. B, Lasers and optics</jtitle><date>2001-12</date><risdate>2001</risdate><volume>73</volume><issue>8</issue><spage>829</spage><epage>837</epage><pages>829-837</pages><issn>0946-2171</issn><eissn>1432-0649</eissn><abstract>Fluorescence spectroscopic measurements at the single-molecule level usually require large absorption cross sections and fluorescence quantum yields for the dyes under study. In addition to these parameters, the collectable number of fluorescence photons and, thus, the signal-to-noise ratio of the measurement, is influenced by processes like triplet-state population and photobleaching, shifting the saturation threshold of the dye to lower excitation intensities. Confocal fluorescence correlation spectroscopy (FCS) is a versatile method to precisely determine photon emission rates of single molecules but also gives access to rate constants of dynamic bleaching and intersystem crossing. In recent FCS studies in solution and living cells, two-photon excitation with its inherent spatial sectioning has proven to be a very valuable alternative to minimize background and cumulative signal loss. However, there is evidence that in many dye systems, the photobleaching rates with two-photon excitation are significantly enhanced with respect to one-photon excitation at comparable photon-emission yields. The reasons have so far remained mainly speculative. In the present study, potential photobleaching pathways are investigated by adding chemical stabilizers and by working at different oxygen concentrations. The results suggest that the population of triplet states does not appear to be responsible for the limited emission rate with two-photon excitation. Rather, photobleaching pathways via the formation of radicals seem to be plausible causes for the signal limitation. Favorable conditions are discussed to maximize the overall photon-collection yield in two-photon experiments.</abstract><doi>10.1007/s003400100737</doi><tpages>9</tpages></addata></record>
fulltext fulltext
identifier ISSN: 0946-2171
ispartof Applied physics. B, Lasers and optics, 2001-12, Vol.73 (8), p.829-837
issn 0946-2171
1432-0649
language eng
recordid cdi_proquest_miscellaneous_1671451971
source SpringerLink Journals
subjects Absorption cross sections
Dyes
Dynamical systems
Dynamics
Excitation
Fluorescence
Pathways
Spectroscopy
title Photobleaching and stabilization of. fluorophores used for single-molecule analysis. with one- and two-photon excitation
url https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-25T15%3A05%3A22IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Photobleaching%20and%20stabilization%20of.%20fluorophores%20used%20for%20single-molecule%20analysis.%20with%20one-%20and%20two-photon%20excitation&rft.jtitle=Applied%20physics.%20B,%20Lasers%20and%20optics&rft.au=Dittrich,%20P.S.&rft.date=2001-12&rft.volume=73&rft.issue=8&rft.spage=829&rft.epage=837&rft.pages=829-837&rft.issn=0946-2171&rft.eissn=1432-0649&rft_id=info:doi/10.1007/s003400100737&rft_dat=%3Cproquest_cross%3E1671451971%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1671451971&rft_id=info:pmid/&rfr_iscdi=true