Abstract
Purpose: :
Together with two-photon fluorescence microscopy, second-harmonic microscopy has become increasingly widespread for multi-dimentional bio-imaging. Collagen-I is a highly efficient generator of second-harmonic radiation and this can be utilized for background-free imaging of the cornea collagen-I fibrils. Such image information may find use for diagnostics of cornea complications including keratoconus but current illumination levels are hazardous for in-vivo applications. The purpose of this study is to use both experiments and theory to clarify the relationship between the illumination conditions and the signal and thereby to facilitate high-resolution imaging with a minimum of light exposure.
Methods: :
A second-harmonic microscope with an ultrafast dispersion-compensated Ti:Sapphire femtosecond laser has been constructed. Second-harmonic signals detected alternatively with lock-in detection at the 80MHz repetition rate of the laser and with photon-counting equipment are compared. Second-harmonic images are generated in transmission through porcine corneas dissected with a trephine and imaged in 3-D using galvo-scanners and a piezoelectric objective scanner. A range of different numerical apertures are tried to optimize the strength of the detected signal. The approach is numerically investigated to clarify the optimal illumination conditions both in terms of signal strength and resolution.
Results: :
The experimental comparison shows clearly that photon-counting equipment provides the higher sensitivity for the detected second-harmonic signal and allows for the faster image acquisition speed. The choice of the optimal numerical aperture of the objective is more dubious. If increased the potential resolution is also increased but the signal decreases as a result of the poorer match between the illumination and the co-linear second-harmonic generated wave. The numerical model shows under which conditions the illumination conditions are optimized for a given speed of image acquisition.
Conclusions: :
We have compared and experimentally confirmed the superiority of the photon-counting approach over lock-in detection for the acquisition of second-harmonic cornea collagen-I fibril images at high speed and at low illumination power. A further optimization is expected to lead to future in-vivo applications if transferred to a reflection geometry the approach may be further complicated.
Keywords: optical properties • cornea: basic science • imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound)