Fast, large-field fluorescence and second-harmonic generation imaging with a single-spinning disk two-photon microscope
Confocal microscopes have been the workhorses of 3-D biological imaging, but they are slow, offer limited depth penetration and collect only ballistic photons. With their inefficient use of excitation photons they expose biological samples to an often intolerably high light burden. The speed limitat...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
EDP Sciences
2023-01-01
|
| Series: | EPJ Web of Conferences |
| Online Access: | https://www.epj-conferences.org/articles/epjconf/pdf/2023/13/epjconf_eosam2023_03002.pdf |
| _version_ | 1797634938238926848 |
|---|---|
| author | Deeg Andreas Trigo Federico Hazart Doriane Delhomme Brigitte Zar Tchyia Naiser Thomas Seebacher Christian Salomon Adi Ricard Clément Uhl Rainer Oheim Martin |
| author_facet | Deeg Andreas Trigo Federico Hazart Doriane Delhomme Brigitte Zar Tchyia Naiser Thomas Seebacher Christian Salomon Adi Ricard Clément Uhl Rainer Oheim Martin |
| author_sort | Deeg Andreas |
| collection | DOAJ |
| description | Confocal microscopes have been the workhorses of 3-D biological imaging, but they are slow, offer limited depth penetration and collect only ballistic photons. With their inefficient use of excitation photons they expose biological samples to an often intolerably high light burden. The speed limitation and photo-bleaching risk can be somewhat relaxed in a spinning-disk geometry, due to shorter pixel dwell times and rapid re-scans during image capture. Alternatively, light-sheet microscopes rapidly image large volumes of transparent or chemically cleared samples. Finally, with infrared excitation and efficient scattered-light collection, 2-photon microscopy allows deep-tissue imaging, but it remains slow. Here, we describe a new optical scheme that borrows the best from three different worlds: the speed and direct-view from a spinning-disk confocal, deep tissue-penetration and intrinsic optical sectioning from 2-photon excitation, and a large field of view and a low invasiveness of a selective-plane illumination microscope – all with a single objective lens. We validate the performance of our 2-photon spinning-disk microscope in various applications that have in common to simultaneously require a large depth penetration, high speed and larger fields of view. Beyond biological fluorescence, we demonstrate an application in material science, imaging coherent non-linear scattering from a 3-D nano-porous network. |
| first_indexed | 2024-03-11T12:14:19Z |
| format | Article |
| id | doaj.art-f740a4cba27f4db8aeb372b619f6e75e |
| institution | Directory Open Access Journal |
| issn | 2100-014X |
| language | English |
| last_indexed | 2024-03-11T12:14:19Z |
| publishDate | 2023-01-01 |
| publisher | EDP Sciences |
| record_format | Article |
| series | EPJ Web of Conferences |
| spelling | doaj.art-f740a4cba27f4db8aeb372b619f6e75e2023-11-07T10:20:28ZengEDP SciencesEPJ Web of Conferences2100-014X2023-01-012870300210.1051/epjconf/202328703002epjconf_eosam2023_03002Fast, large-field fluorescence and second-harmonic generation imaging with a single-spinning disk two-photon microscopeDeeg Andreas0Trigo Federico1Hazart Doriane2Delhomme Brigitte3Zar Tchyia4Naiser Thomas5Seebacher Christian6Salomon Adi7Ricard Clément8Uhl Rainer9Oheim Martin10TILL I.D. GmbH, Am Klopferspitz 19aUniversité Paris Cité, Saint-Pères Paris Institute for the Neurosciences, CNRSUniversité Paris Cité, Saint-Pères Paris Institute for the Neurosciences, CNRSUniversité Paris Cité, Saint-Pères Paris Institute for the Neurosciences, CNRSInstitute of Nanotechnology and Advanced Materials (BINA), Department of Chemistry, Bar-Ilan UniversityTILL I.D. GmbH, Am Klopferspitz 19aTILL I.D. GmbH, Am Klopferspitz 19aUniversité Paris Cité, Saint-Pères Paris Institute for the Neurosciences, CNRSUniversité Paris Cité, Saint-Pères Paris Institute for the Neurosciences, CNRSTILL I.D. GmbH, Am Klopferspitz 19aUniversité Paris Cité, Saint-Pères Paris Institute for the Neurosciences, CNRSConfocal microscopes have been the workhorses of 3-D biological imaging, but they are slow, offer limited depth penetration and collect only ballistic photons. With their inefficient use of excitation photons they expose biological samples to an often intolerably high light burden. The speed limitation and photo-bleaching risk can be somewhat relaxed in a spinning-disk geometry, due to shorter pixel dwell times and rapid re-scans during image capture. Alternatively, light-sheet microscopes rapidly image large volumes of transparent or chemically cleared samples. Finally, with infrared excitation and efficient scattered-light collection, 2-photon microscopy allows deep-tissue imaging, but it remains slow. Here, we describe a new optical scheme that borrows the best from three different worlds: the speed and direct-view from a spinning-disk confocal, deep tissue-penetration and intrinsic optical sectioning from 2-photon excitation, and a large field of view and a low invasiveness of a selective-plane illumination microscope – all with a single objective lens. We validate the performance of our 2-photon spinning-disk microscope in various applications that have in common to simultaneously require a large depth penetration, high speed and larger fields of view. Beyond biological fluorescence, we demonstrate an application in material science, imaging coherent non-linear scattering from a 3-D nano-porous network.https://www.epj-conferences.org/articles/epjconf/pdf/2023/13/epjconf_eosam2023_03002.pdf |
| spellingShingle | Deeg Andreas Trigo Federico Hazart Doriane Delhomme Brigitte Zar Tchyia Naiser Thomas Seebacher Christian Salomon Adi Ricard Clément Uhl Rainer Oheim Martin Fast, large-field fluorescence and second-harmonic generation imaging with a single-spinning disk two-photon microscope EPJ Web of Conferences |
| title | Fast, large-field fluorescence and second-harmonic generation imaging with a single-spinning disk two-photon microscope |
| title_full | Fast, large-field fluorescence and second-harmonic generation imaging with a single-spinning disk two-photon microscope |
| title_fullStr | Fast, large-field fluorescence and second-harmonic generation imaging with a single-spinning disk two-photon microscope |
| title_full_unstemmed | Fast, large-field fluorescence and second-harmonic generation imaging with a single-spinning disk two-photon microscope |
| title_short | Fast, large-field fluorescence and second-harmonic generation imaging with a single-spinning disk two-photon microscope |
| title_sort | fast large field fluorescence and second harmonic generation imaging with a single spinning disk two photon microscope |
| url | https://www.epj-conferences.org/articles/epjconf/pdf/2023/13/epjconf_eosam2023_03002.pdf |
| work_keys_str_mv | AT deegandreas fastlargefieldfluorescenceandsecondharmonicgenerationimagingwithasinglespinningdisktwophotonmicroscope AT trigofederico fastlargefieldfluorescenceandsecondharmonicgenerationimagingwithasinglespinningdisktwophotonmicroscope AT hazartdoriane fastlargefieldfluorescenceandsecondharmonicgenerationimagingwithasinglespinningdisktwophotonmicroscope AT delhommebrigitte fastlargefieldfluorescenceandsecondharmonicgenerationimagingwithasinglespinningdisktwophotonmicroscope AT zartchyia fastlargefieldfluorescenceandsecondharmonicgenerationimagingwithasinglespinningdisktwophotonmicroscope AT naiserthomas fastlargefieldfluorescenceandsecondharmonicgenerationimagingwithasinglespinningdisktwophotonmicroscope AT seebacherchristian fastlargefieldfluorescenceandsecondharmonicgenerationimagingwithasinglespinningdisktwophotonmicroscope AT salomonadi fastlargefieldfluorescenceandsecondharmonicgenerationimagingwithasinglespinningdisktwophotonmicroscope AT ricardclement fastlargefieldfluorescenceandsecondharmonicgenerationimagingwithasinglespinningdisktwophotonmicroscope AT uhlrainer fastlargefieldfluorescenceandsecondharmonicgenerationimagingwithasinglespinningdisktwophotonmicroscope AT oheimmartin fastlargefieldfluorescenceandsecondharmonicgenerationimagingwithasinglespinningdisktwophotonmicroscope |