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M.F. Bird, M.C. W. Campbell; A Rat Model for Two–Photon Excitation in the in vivo Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4066.
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© ARVO (1962-2015); The Authors (2016-present)
Two–photon excitation (TPE) is used in two–photon fluorescence imaging of the in vitro retina and in tissue therapy. It provides a temporally and spatially localized effect. We are developing an animal model to deliver TPE to the in vivo retina and to image its effects. TPE occurs when two photons are simultaneously absorbed and their energies are combined to cause a transition to the excited state of the photosensitizer. Here we determine how monochromatic aberrations and second order dispersion will affect the femtosecond laser pulses required for TPE. We also assess whether adaptive optical (AO) correction of the eye for spatial localization and pulse pre–chirping for temporal control are necessary for targeted TPE.
A schematic model of the rat eye with a gradient refractive index (Campbell, 1982) was re–constructed in the optical design program ZEMAXTM. Using this model, we predict the monochromatic aberrations and the change in the Point Spread Function (PSF) as a function of pupil size and field angle. This information will aid in determining the optimal pupil size for two–photon effects and will allow a comparison with the optimal pupil size for retinal imaging. A simple water model of the nonlinear pulse broadening effect (dispersion) has also been developed as a first approximation for the minimum temporal pulse width that will propagate to the retina.
In the rat eye model, uncorrected for aberration, a 1mm diameter pupil delivers a peak intensity acceptable for a two–photon effect. This focussed spot is diffraction–limited up to a field angle of 20o. A 2mm diameter pupil gives an optimum PSF full width at half height for imaging on the optical axis. The higher order root–mean–square wavefront aberration (H–O RMSA) increases by approximately 50% within a field angle of 5o for a 2mm pupil. For 550nm light, at the largest pupil modeled (3.8mm) the axial H–O RMSA is approximately 1µ on the optical axis, increasing to approximately 2.5µ at a 20o field angle. Therefore, to correct the aberrations at the largest pupil size across the field of view, an AO element with a stroke greater than 6µ would be required. The water model predicts that the minimum temporal pulse width that can be delivered to the retina is approximately 70fs.
The optimum pupil size for a two–photon induction differs from that for optimum imaging in the uncorrected eye. An AO correction of the rat eye requires an AO device with a relatively large stroke. Correction could improve the spatial localization in depth of a two–photon effect by a factor of up to 16 times. Temporal localization of energy delivery could be improved by pre–chirping laser pulses.
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