title: 3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation.
abstract: Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane. This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. The accuracy and performance of the proposed method is demonstrated in several imaging studies important for understanding peripheral nerve regeneration processes. Most importantly, the parallelism of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude.
authors.affiliation: Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02193, USA.
authors.forename: Heejin
authors.initials: H
authors.lastname: Choi
authors.forename: Dimitrios S
authors.initials: DS
authors.lastname: Tzeranis
authors.forename: Jae Won
authors.initials: JW
authors.lastname: Cha
authors.forename: Philippe
authors.initials: P
authors.lastname: Clémenceau
authors.forename: Sander J G
authors.initials: SJ
authors.lastname: de Jong
authors.forename: Lambertus K
authors.initials: LK
authors.lastname: van Geest
authors.forename: Joong Ho
authors.initials: JH
authors.lastname: Moon
authors.forename: Ioannis V
authors.initials: IV
authors.lastname: Yannas
authors.forename: Peter T C
authors.initials: PT
authors.lastname: So
mesh_terms: D003593:Cytoplasm / Q000648:ultrastructure*; D005453:Fluorescence; D006801:Humans; D021621:Imaging, Three-Dimensional; D008856:Microscopy, Fluorescence / Q000379:methods*; D017785:Photons