Open Access3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation
Author(s) -
Heejin Choi,
Dimitrios S. Tzeranis,
Jae Yon Won,
Philippe Clémenceau,
Sander J. G. de Jong,
Lambertus K. van Geest,
Joong Ho Moon,
Ioannis V. Yannas,
Peter T. C. So
Publication year - 2012
Publication title -
optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.394
H-Index - 271
ISSN - 1094-4087
DOI - 10.1364/oe.20.026219
Subject(s) - biophotonics , fluorescence lifetime imaging microscopy , phosphorescence , optics , materials science , microscopy , biological imaging , temporal resolution , photon counting , excitation , detector , fluorescence , optoelectronics , laser , physics , quantum mechanics
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.