Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy

Imaging molecular structures separated by distances of a few nanometers still represents a complex challenge. Moreover, it is normally restricted to observations on thin (few micrometers) samples. In this work, we rotate the polarization of the excitation beam of two-photon excited fluorescence (TPE...

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Detalles Bibliográficos
Autores: Artigas García, David|||0000-0001-5508-2873, Merino, David, Polzer, Christoph, Loza Álvarez, Pablo
Tipo de recurso: artículo
Fecha de publicación:2017
País:España
Institución:Universitat Politècnica de Catalunya (UPC)
Repositorio:UPCommons. Portal del coneixement obert de la UPC
Idioma:inglés
OAI Identifier:oai:upcommons.upc.edu:2117/107461
Acceso en línea:https://hdl.handle.net/2117/107461
https://dx.doi.org/10.1364/OPTICA.4.000911
Access Level:acceso abierto
Palabra clave:Photonics
Fotònica
Àrees temàtiques de la UPC::Enginyeria de la telecomunicació::Telecomunicació òptica::Fotònica
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spelling Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopyArtigas García, David|||0000-0001-5508-2873Merino, DavidPolzer, ChristophLoza Álvarez, PabloPhotonicsFotònicaÀrees temàtiques de la UPC::Enginyeria de la telecomunicació::Telecomunicació òptica::FotònicaImaging molecular structures separated by distances of a few nanometers still represents a complex challenge. Moreover, it is normally restricted to observations on thin (few micrometers) samples. In this work, we rotate the polarization of the excitation beam of two-photon excited fluorescence (TPEF) images to show that fluorescent structures at the molecular scale can be discriminated in a living organism. The polarization rotation generates a modulation of the signal intensity in each pixel of the TPEF images that carry information related to the fluorophore orientation. We analyze the signal modulation in every pixel of the polarization-resolved (PR) TPEF images through a Fourier analysis and generate images for the different Fourier components. Doing that, we show that two fluorophores oriented in different directions can be distinguished. Although by imaging the Fourier components the resolution of the optical system restricts the exact localization of two close molecules, discrimination is still possible even when the molecules are located at sub-diffraction distances. We propose a model that predicts this behavior, and demonstrate it experimentally in the neurons of a living Caenorhabditis elegans nematode, where we distinguish the walls of an axon with a diameter below the objective resolution. Since the technique is based in TPEF, the method can be extended to deep tissue imaging and has potential applications in single molecule detection, biological sensors, or super-resolution imaging techniques.Peer ReviewedOptical Society of American (OSA)20172017-08-2020172017-09-06journal articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/2117/107461https://dx.doi.org/10.1364/OPTICA.4.000911reponame:UPCommons. Portal del coneixement obert de la UPCinstname:Universitat Politècnica de Catalunya (UPC)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2info:eu-repo/semantics/openAccessoai:upcommons.upc.edu:2117/1074612026-05-27T15:37:01Z
dc.title.none.fl_str_mv Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy
title Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy
spellingShingle Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy
Artigas García, David|||0000-0001-5508-2873
Photonics
Fotònica
Àrees temàtiques de la UPC::Enginyeria de la telecomunicació::Telecomunicació òptica::Fotònica
title_short Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy
title_full Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy
title_fullStr Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy
title_full_unstemmed Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy
title_sort Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy
dc.creator.none.fl_str_mv Artigas García, David|||0000-0001-5508-2873
Merino, David
Polzer, Christoph
Loza Álvarez, Pablo
author Artigas García, David|||0000-0001-5508-2873
author_facet Artigas García, David|||0000-0001-5508-2873
Merino, David
Polzer, Christoph
Loza Álvarez, Pablo
author_role author
author2 Merino, David
Polzer, Christoph
Loza Álvarez, Pablo
author2_role author
author
author
dc.subject.none.fl_str_mv Photonics
Fotònica
Àrees temàtiques de la UPC::Enginyeria de la telecomunicació::Telecomunicació òptica::Fotònica
topic Photonics
Fotònica
Àrees temàtiques de la UPC::Enginyeria de la telecomunicació::Telecomunicació òptica::Fotònica
description Imaging molecular structures separated by distances of a few nanometers still represents a complex challenge. Moreover, it is normally restricted to observations on thin (few micrometers) samples. In this work, we rotate the polarization of the excitation beam of two-photon excited fluorescence (TPEF) images to show that fluorescent structures at the molecular scale can be discriminated in a living organism. The polarization rotation generates a modulation of the signal intensity in each pixel of the TPEF images that carry information related to the fluorophore orientation. We analyze the signal modulation in every pixel of the polarization-resolved (PR) TPEF images through a Fourier analysis and generate images for the different Fourier components. Doing that, we show that two fluorophores oriented in different directions can be distinguished. Although by imaging the Fourier components the resolution of the optical system restricts the exact localization of two close molecules, discrimination is still possible even when the molecules are located at sub-diffraction distances. We propose a model that predicts this behavior, and demonstrate it experimentally in the neurons of a living Caenorhabditis elegans nematode, where we distinguish the walls of an axon with a diameter below the objective resolution. Since the technique is based in TPEF, the method can be extended to deep tissue imaging and has potential applications in single molecule detection, biological sensors, or super-resolution imaging techniques.
publishDate 2017
dc.date.none.fl_str_mv 2017
2017-08-20
2017
2017-09-06
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/2117/107461
https://dx.doi.org/10.1364/OPTICA.4.000911
url https://hdl.handle.net/2117/107461
https://dx.doi.org/10.1364/OPTICA.4.000911
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Optical Society of American (OSA)
publisher.none.fl_str_mv Optical Society of American (OSA)
dc.source.none.fl_str_mv reponame:UPCommons. Portal del coneixement obert de la UPC
instname:Universitat Politècnica de Catalunya (UPC)
instname_str Universitat Politècnica de Catalunya (UPC)
reponame_str UPCommons. Portal del coneixement obert de la UPC
collection UPCommons. Portal del coneixement obert de la UPC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15.300719

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