Epifluorescence light collection for multiphoton microscopic endoscopy

Multiphoton microscopic endoscopy (MPM-E) is a promising medical in vivo diagnostic imaging technique because it captures intrinsic fluorescence and second harmonic generation signals to reveal anatomical and histological information about disease states in tissue. However, maximizing light collection from multiphoton endoscopes remains a challenge: weak nonlinear emissions from endogenous structures, miniature optics, large imaging depths, and light scattering in tissue all hamper light collection. The quantity of light that may be collected using a dual-clad fiber system from scattering phantoms that mimic the properties of the in vivo environment is measured. In this experiment, 800nm excitation light from a Ti:Sapphire laser is dispersion compensated and focused through a SM800 optical fiber and lens system into the tissue phantom. Emission light from the phantom passes through the lens system, reflects off the dichroic and is then collected by a second optical fiber actuated by a micromanipulator. The lateral position of the collection fiber varies, measuring the distribution of emitted light 2000μm on either side of the focal point reimaged to the object plane. This spatial collection measurement is performed at depths up to 200μm from the phantom surface. The tissue phantoms are composed of a 15.8 μM fluorescein solution mixed with microspheres, approximating the scattering properties of human bladder and dermis tissue. Results show that commercially available dual-clad optical fibers collect more than 47% of the total emission returning to the object plane from both phantoms. Based on these results, initial MPM-E devices will image the surface of epithelial tissues.