Photobleaching reduction in modulated super-resolution microscopy

Abstract Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the exc...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Microscopy 2021-06, Vol.70 (3), p.278-288
Hauptverfasser: Ghithan, Jafar H, Noel, Jennifer M, Roussel, Thomas J, McCall, Maureen A, Alphenaar, Bruce W, Mendes, Sergio B
Format: Artikel
Sprache:eng
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
container_end_page 288
container_issue 3
container_start_page 278
container_title Microscopy
container_volume 70
creator Ghithan, Jafar H
Noel, Jennifer M
Roussel, Thomas J
McCall, Maureen A
Alphenaar, Bruce W
Mendes, Sergio B
description Abstract Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.
doi_str_mv 10.1093/jmicro/dfaa062
format Article
fullrecord <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_2451857048</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><oup_id>10.1093/jmicro/dfaa062</oup_id><sourcerecordid>2451857048</sourcerecordid><originalsourceid>FETCH-LOGICAL-c306t-68cf681695c9970cadfa9b6697709c1ae1bd9a4c410ae6459654e0ede7c7626d3</originalsourceid><addsrcrecordid>eNqFUD1PwzAQtRBIVKUrc0YY0p5T24nHquJLqgQDzJZjX2iqJA52PPTfkzZl5pY76d679-4Rck9hSUGuV4e2Nt6tbKU1iOyKzDLgkPIc6PXfLGRxSxYhHGCsglNgYkY2H3s3uLJBbfZ19514tNEMteuSuktaZ2OjB7RJiD361GNwTTxvz3LBuP54R24q3QRcXPqcfD0_fW5f0937y9t2s0vNGsSQisJUoqBCciNlDkaPVmUphMxzkIZqpKWVmhlGQaNgXArOENBibnKRCbuek4fpbu_dT8QwqLYOBptGd-hiUBnjtBgfZsUIXU7Qk8fgsVK9r1vtj4qCOsWlprjUJa6R8DgRXOz_w_4CtZVvHQ</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2451857048</pqid></control><display><type>article</type><title>Photobleaching reduction in modulated super-resolution microscopy</title><source>Oxford University Press Journals All Titles (1996-Current)</source><source>Alma/SFX Local Collection</source><creator>Ghithan, Jafar H ; Noel, Jennifer M ; Roussel, Thomas J ; McCall, Maureen A ; Alphenaar, Bruce W ; Mendes, Sergio B</creator><creatorcontrib>Ghithan, Jafar H ; Noel, Jennifer M ; Roussel, Thomas J ; McCall, Maureen A ; Alphenaar, Bruce W ; Mendes, Sergio B</creatorcontrib><description>Abstract Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.</description><identifier>ISSN: 2050-5698</identifier><identifier>EISSN: 2050-5701</identifier><identifier>DOI: 10.1093/jmicro/dfaa062</identifier><language>eng</language><publisher>UK: Oxford University Press</publisher><ispartof>Microscopy, 2021-06, Vol.70 (3), p.278-288</ispartof><rights>The Author(s) 2020. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com 2020</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c306t-68cf681695c9970cadfa9b6697709c1ae1bd9a4c410ae6459654e0ede7c7626d3</citedby><cites>FETCH-LOGICAL-c306t-68cf681695c9970cadfa9b6697709c1ae1bd9a4c410ae6459654e0ede7c7626d3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,777,781,1579,27905,27906</link.rule.ids></links><search><creatorcontrib>Ghithan, Jafar H</creatorcontrib><creatorcontrib>Noel, Jennifer M</creatorcontrib><creatorcontrib>Roussel, Thomas J</creatorcontrib><creatorcontrib>McCall, Maureen A</creatorcontrib><creatorcontrib>Alphenaar, Bruce W</creatorcontrib><creatorcontrib>Mendes, Sergio B</creatorcontrib><title>Photobleaching reduction in modulated super-resolution microscopy</title><title>Microscopy</title><description>Abstract Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.</description><issn>2050-5698</issn><issn>2050-5701</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><recordid>eNqFUD1PwzAQtRBIVKUrc0YY0p5T24nHquJLqgQDzJZjX2iqJA52PPTfkzZl5pY76d679-4Rck9hSUGuV4e2Nt6tbKU1iOyKzDLgkPIc6PXfLGRxSxYhHGCsglNgYkY2H3s3uLJBbfZ19514tNEMteuSuktaZ2OjB7RJiD361GNwTTxvz3LBuP54R24q3QRcXPqcfD0_fW5f0937y9t2s0vNGsSQisJUoqBCciNlDkaPVmUphMxzkIZqpKWVmhlGQaNgXArOENBibnKRCbuek4fpbu_dT8QwqLYOBptGd-hiUBnjtBgfZsUIXU7Qk8fgsVK9r1vtj4qCOsWlprjUJa6R8DgRXOz_w_4CtZVvHQ</recordid><startdate>20210606</startdate><enddate>20210606</enddate><creator>Ghithan, Jafar H</creator><creator>Noel, Jennifer M</creator><creator>Roussel, Thomas J</creator><creator>McCall, Maureen A</creator><creator>Alphenaar, Bruce W</creator><creator>Mendes, Sergio B</creator><general>Oxford University Press</general><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20210606</creationdate><title>Photobleaching reduction in modulated super-resolution microscopy</title><author>Ghithan, Jafar H ; Noel, Jennifer M ; Roussel, Thomas J ; McCall, Maureen A ; Alphenaar, Bruce W ; Mendes, Sergio B</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c306t-68cf681695c9970cadfa9b6697709c1ae1bd9a4c410ae6459654e0ede7c7626d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ghithan, Jafar H</creatorcontrib><creatorcontrib>Noel, Jennifer M</creatorcontrib><creatorcontrib>Roussel, Thomas J</creatorcontrib><creatorcontrib>McCall, Maureen A</creatorcontrib><creatorcontrib>Alphenaar, Bruce W</creatorcontrib><creatorcontrib>Mendes, Sergio B</creatorcontrib><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Microscopy</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ghithan, Jafar H</au><au>Noel, Jennifer M</au><au>Roussel, Thomas J</au><au>McCall, Maureen A</au><au>Alphenaar, Bruce W</au><au>Mendes, Sergio B</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Photobleaching reduction in modulated super-resolution microscopy</atitle><jtitle>Microscopy</jtitle><date>2021-06-06</date><risdate>2021</risdate><volume>70</volume><issue>3</issue><spage>278</spage><epage>288</epage><pages>278-288</pages><issn>2050-5698</issn><eissn>2050-5701</eissn><abstract>Abstract Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.</abstract><cop>UK</cop><pub>Oxford University Press</pub><doi>10.1093/jmicro/dfaa062</doi><tpages>11</tpages></addata></record>
fulltext fulltext
identifier ISSN: 2050-5698
ispartof Microscopy, 2021-06, Vol.70 (3), p.278-288
issn 2050-5698
2050-5701
language eng
recordid cdi_proquest_miscellaneous_2451857048
source Oxford University Press Journals All Titles (1996-Current); Alma/SFX Local Collection
title Photobleaching reduction in modulated super-resolution microscopy
url https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-19T02%3A31%3A52IST&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%20reduction%20in%20modulated%20super-resolution%20microscopy&rft.jtitle=Microscopy&rft.au=Ghithan,%20Jafar%20H&rft.date=2021-06-06&rft.volume=70&rft.issue=3&rft.spage=278&rft.epage=288&rft.pages=278-288&rft.issn=2050-5698&rft.eissn=2050-5701&rft_id=info:doi/10.1093/jmicro/dfaa062&rft_dat=%3Cproquest_cross%3E2451857048%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=2451857048&rft_id=info:pmid/&rft_oup_id=10.1093/jmicro/dfaa062&rfr_iscdi=true