Identifying crossing collagen fibers in human corneal tissues using pSHG images

Abstract

Polarization sensitive second harmonic generation (pSHG) microscopy has been used previously to characterize the structure of collagen fibers in corneal samples. Due to the typical organization of the corneal stroma, the information that pSHG provides may be misleading in points where two different collagen fiber bundles orient along different direction crossings. Here, a simulation that illustrates the problem is presented, along with a novel method that is capable of identifying these crossing points. These results can be used to improve the evaluation of corneal collagen structure, and it has been applied to analyze pSHG data acquired from healthy and keratoconic human corneal samples.

Digital polarimetric second harmonic generation microscopy of partially oriented fiber structures

“…The sign of C ratio changes due to clockwise or counterclockwise tilt of the fiber around X-axis (eq (3)). When the complex values of χ (2) xyz are assumed, some deviations from sinusoidal function appear for both, collagen and TPPS 4 aggregates (Fig. 3 f and g).…”

“…The modeled R ratio value does not change with the displacement in the focal volume when real-valued molecular susceptibility tensor elements are assumed. Interestingly, when complex valued chiral susceptibility χ (2) xyz is assumed, the left side lateral displacement of a right-handed fiber leads to R ratio decrease, while the right side displacement gives higher R ratio. The effect is opposite for left-handed structure (dashed and dash-dotted lines in Fig.…”

“…The polarimetric nonlinear microscopy uses high numerical aperture (NA) objectives for fundamental beam focusing into a tiny voxel. The voxel volume is usually much larger than the diameter of a second harmonic active fibrillar structure, and many fibers with different arrangements can be present in the voxel (2). The hierarchical organization of fibrillar harmonophores in the biological samples can be divided into (i) short range fiber configurations, where phase differences between harmonophores can be neglected, and (ii) configurations separated by a fraction of wavelength of light where phase relations have to be considered.…”

Polarization-Sensitive Second Harmonic Generation Microscopy for Investigations of Diseased Collagenous Tissues

“…In another technique, polarimetry analysis can also be performed using circularly polarized laser excitation, only requiring a single scan [53,69]. Fourier techniques could be used for faster data analysis [16,23,24], while imaging rates can be increased using higher repetition rate pulsed lasers combined with faster scanners such as spinning mirrors, or by increasing the field of view using a wide-field imaging approach [70]. With these developments, it is reasonable that PSHG microscopy may be implemented in a modified hospital pathology slide scanner.…”

“…To avoid delays due to nonlinear data fitting, a fast Fourier transform (FFT) algorithm has been developed to extract ρ values quickly [22]. It was used in the study of normal and keratoconic human corneas [23], human breast carcinoma samples [16,24] and to investigate mechanical load in energy storing versus positional collagen fibrils [25]. The FFT approach has also allowed use of a generic structural model, where rather than assuming a particular symmetry, the second-order FFT coefficients (I 2 and ϕ 2 ), which quantify the modulation depth of the polarization SHG response (I 2 ) and its phase (ϕ 2 ), are reported and compared.…”