Purchase this article with an account.
JiaJun Li, Denise Valente, Sara Castillo, Brian Vohnsen; Polarization Controlled Second-Harmonic Microscopy For Cornea Collagen-I Fibril Imaging. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2474.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Second-harmonic microscopy has been widely used to image cornea collagen structure in the stroma. Signal efficiency is affected by the angle between incident beam polarization and material polarization. In our experiment two spatial light modulators (SLMs) are used to alter the spatial state of polarization at will from radial to azimuthal. The purpose of this is to examine which polarizations are best at highlighting collagen fibrils at different orientations.
A custom made multiphoton microscope has been combined with two SLMs. An 800nm wavelength femtosecond laser is used as illumination source. The laser beam is scanned by two galvo-scanners and a piezoelectric stage is used to provide 3D scan capability. Signals are collected in both the forward and backward directions to create background-free images. The laser beam polarization is altered prior to each acquisition and images are analyzed to find their specific pros and cons.
The results reveal that the visibility of collagen fibrilar orientation is highly sensitive to the polarization state of the incident beam. For 3D imaging radial polarization beams give the best out-of-plane probing results but the signal is dramatically reduced since less collagen fibrils point perpendicular to the lamellae.
Different laser polarizations have unique imaging potential. It remains a challenge to obtain high quality images for each setting of polarization, but provided that the laser power is kept high, and dispersion in the SLMs is kept at a minimum, the technique holds promise for automated detection of fibrilar orientation at any angle.
This PDF is available to Subscribers Only