Two photon imaging probe with highly efficient autofluorescence collection at high scattering and deep imaging conditions

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dc.citation.firstpage3163en_US
dc.citation.issueNumber5en_US
dc.citation.journalTitleBiomedical Optics Expressen_US
dc.citation.lastpage3182en_US
dc.citation.volumeNumber15en_US
dc.contributor.authorCamli, Berken_US
dc.contributor.authorAndrus, Liamen_US
dc.contributor.authorRoy, Adityaen_US
dc.contributor.authorMishra, Biswajiten_US
dc.contributor.authorXu, Chrisen_US
dc.contributor.authorGeorgakoudi, Ireneen_US
dc.contributor.authorTkaczyk, Tomaszen_US
dc.contributor.authorBen-Yakar, Adelaen_US
dc.date.accessioned2024-11-04T16:25:10Zen_US
dc.date.available2024-11-04T16:25:10Zen_US
dc.date.issued2024en_US
dc.description.abstractIn this paper, we present a 2-photon imaging probe system featuring a novel fluorescence collection method with improved and reliable efficiency. The system aims to miniaturize the potential of 2-photon imaging in the metabolic and morphological characterization of cervical tissue at sub-micron resolution over large imaging depths into a flexible and clinically viable platform towards the early detection of cancers. Clinical implementation of such a probe system is challenging due to inherently low levels of autofluorescence, particularly when imaging deep in highly scattering tissues. For an efficient collection of fluorescence signals, our probe employs 12 0.5 NA collection fibers arranged around a miniaturized excitation objective. By bending and terminating a multitude of collection fibers at a specific angle, we increase collection area and directivity significantly. Positioning of these fibers allows the collection of fluorescence photons scattered away from their ballistic trajectory multiple times, which offers a system collection efficiency of 4%, which is 55% of what our bench-top microscope with 0.75 NA objective achieves. We demonstrate that the collection efficiency is largely maintained even at high scattering conditions and high imaging depths. Radial symmetry of arrangement maintains uniformity of collection efficiency across the whole FOV. Additionally, our probe can image at different tissue depths via axial actuation by a dc servo motor, allowing depth dependent tissue characterization. We designed our probe to perform imaging at 775 nm, targeting 2-photon autofluorescence from NAD(P)H and FAD molecules, which are often used in metabolic tissue characterization. An air core photonic bandgap fiber delivers laser pulses of 100 fs duration to the sample. A miniaturized objective designed with commercially available lenses of 3 mm diameter focuses the laser beam on tissue, attaining lateral and axial imaging resolutions of 0.66 µm and 4.65 µm, respectively. Characterization results verify that our probe achieves collection efficiency comparable to our optimized bench-top 2-photon imaging microscope, minimally affected by imaging depth and radial positioning. We validate autofluorescence imaging capability with excised porcine vocal fold tissue samples. Images with 120 µm FOV and 0.33 µm pixel sizes collected at 2 fps confirm that the 300 µm imaging depth was achieved.en_US
dc.identifier.citationCamli, B., Andrus, L., Roy, A., Mishra, B., Xu, C., Georgakoudi, I., Tkaczyk, T., & Ben-Yakar, A. (2024). Two photon imaging probe with highly efficient autofluorescence collection at high scattering and deep imaging conditions. Biomedical Optics Express, 15(5), 3163–3182. https://doi.org/10.1364/BOE.520729en_US
dc.identifier.digitalboe-15-5-3163en_US
dc.identifier.doihttps://doi.org/10.1364/BOE.520729en_US
dc.identifier.urihttps://hdl.handle.net/1911/117990en_US
dc.language.isoengen_US
dc.publisherOptica Publishing Groupen_US
dc.rightsPublished under the terms of the Optica Open Access Publishing Agreementen_US
dc.rights.urihttps://opg.optica.org/library/license_v2.cfm#VOR-OAen_US
dc.titleTwo photon imaging probe with highly efficient autofluorescence collection at high scattering and deep imaging conditionsen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpublisher versionen_US
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