Abstract
Volumetric imaging of connective tissue provides insights into the structure of biological tissue. Second harmonic generation (SHG) microscopy has become a standard method to image collagen rich tissue like skin or cornea. Due to the non-centrosymmetric architecture, no additional label is needed and tissue can be visualized noninvasively. Thus, SHG microscopy enables the investigation of collagen associated diseases, providing high resolution images and a field of view of several hundreds of μm. However, the in toto visualization of larger samples is limited to the working distance of the objective and the integration time of the microscope setup, which can sum up to several hours and days. A faster imaging technique for samples in the mesoscopic range is scanning laser optical tomography (SLOT), which provides linear fluorescence, scattering and absorption as intrinsic contrast mechanisms. Due to the advantages of SHG and the reduced measurement time of SLOT, the integration of SHG in SLOT would be a great extension. This way SHG measurements could be performed faster on large samples, providing isotropic resolution and simultaneous acquisition of all other contrast mechanisms available, such as fluorescence and absorption. SLOT is based on the principle of computed tomography, which requires the rotation of the sample. The SHG signal, however, depends strongly on the sample orientation and the polarization of the laser, which results in SHG intensity fluctuation during sample rotation and prevents successful 3D reconstruction. In this paper we investigate the angular dependence of the SHG signal by simulation and experiment and found a way to eliminate reconstruction artifacts caused by this angular dependence in SHG-SLOT data. This way, it is now possible to visualize samples in the mesoscopic range using SHG-SLOT, with isotropic resolution and in correlation to other contrast mechanisms as absorption, fluorescence and scattering.
Highlights
For imaging biological samples, like skin or cornea, second harmonic generation (SHG) has become a standard method [1]
For a complete scanning laser optical tomography (SLOT) measurement, the sample needs to be rotated by 360◦ orthogonal to the optical axis
In this paper have proven for the first time in simulation and in experiment the capability of radon based imaging techniques, as optical projection tomography (OPT) and SLOT, to visualize samples in 3D using SHG as a contrast mechanism
Summary
Like skin or cornea, second harmonic generation (SHG) has become a standard method [1]. The underlying computed tomography reconstruction process (the inverse Radon transform) requires, that the intensity of each microscopic volume element (voxel) is independent on the rotation of the sample This condition is fundamentally violated by SHG. This is because the SHG signal of a scatterer strongly depends on the relative orientation of the polarization of the illuminating light and the scattering objects axis of symmetry During sample rotation this orientation will constantly change and typically result in a fluctuating intensity of each scatterer. We simulate the according resulting artifacts in the reconstructed image using an area of circle as a phantom The extent of these artifacts are measured and displayed for all polarization angles and sample tilts. Light propagates in z-direction and the sample rotation occurs around the x-axis
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