Fibre-optic two-photon scanning fluorescence microscopy.

Abstract

Two geometries of a novel two-photon fluorescence microscope incorporating single-mode fibre optics for the delivery of ultrashort-pulsed illumination to a remote sample are characterized. First, a 785 nm single-mode optical fibre is implemented in a scanning microscope, which demonstrates that an improvement in axial resolution is achieved due to the non-linear response of the fibre to intense ultrashort-pulsed light. Second, a 785 nm single-mode optical fibre coupler is adapted, in which case spectral broadening and blue shifting of the ultrashort-pulsed laser beam caused by the non-linear response of the fibre to ultrashort-pulsed illumination are experimentally characterized. An investigation into the impact of temporal broadening of the ultrashort-pulsed beam on the systems is also considered. The coupling efficiency of both geometries for various illumination wavelengths is also presented. The introduction of the fibre coupler to the system has significant advantages, including an improved optical sectioning effect and a reduction in the number of bulk optical components resulting in a low-cost, compact instrument. Sets of three-dimensional images of fluorescent polymer microspheres and biological material confirm these features.