High numerical aperture imaging allows chirality measurement in individual collagen fibrils using polarization second harmonic generation microscopy

Abstract

Second harmonic generation (SHG) microscopy is a commonly used technique to study the organization of collagen within tissues. However, individual collagen fibrils, which have diameters much smaller than the resolution of most optical systems, have not been extensively investigated. Here we probe the structure of individual collagen fibrils using polarization-resolved SHG (PSHG) microscopy and atomic force microscopy. We find that longitudinally polarized light occurring at the edge of a focal volume of a high numerical aperture microscope objective illuminated with linearly polarized light creates a measurable variation in PSHG signal along the axis orthogonal to an individual collagen fibril. By comparing numerical simulations to experimental data, we are able to estimate parameters related to the structure and chirality of the collagen fibril without tilting the sample out of the image plane, or cutting tissue at different angles, enabling chirality measurements on individual nanostructures to be performed in standard PSHG microscopes. The results presented here are expected to lead to a better understanding of PSHG results from both collagen fibrils and collagenous tissues. Further, the technique presented can be applied to other chiral nanoscale structures such as microtubules, nanowires, and nanoribbons.

Effect of out of plane orientation on polarization second harmonic generation of single collagen fibrils

“…This variation in was previously investigated in individual unbuckled fibrils and altered values were found to occur when a fibril resided near the edge of the focal volume. The nonuniformity of the polarization within the focal field is a result of high numerical aperture focusing [ 28 ], and the effect is only prominent when no SHG emitters are present in the middle of the focal volume [ 28 ].…”

“…Importantly these results are obtained for individual collagen fibrils rather than collagen in tissue, thus eliminating the possibility of nonuniform fibril orientation leading to experimental errors [ 38 , 39 ]. This is additionally significant since while a very large number of papers have investigated SHG from collagen fibrils in tissues, very little work has been done so far on the SHG response from individual collagen fibrils [ 28 , 40 , 41 ]. These results also open the possibility of using PSHG for the analysis of other nanoscale biological structures such as myofibrils and microtubules [ 42 , 43 ].…”

“…Note that the fibrils used in this study were buckled after being dried. Changes in hydration are known to result in changes in the value of the fibril (see supplement S1 in [ 28 ]), with native collagen typically being in the hydrated state. Therefore, it would be preferable to perform this experiment on hydrated fibrils for increased relevance to PSHG measurements from tissue.…”

“…To reduce the impact of nonuniformities in focal field polarization near the edge of the focal volume [ 28 ] only the most centered (brightest) single pixel across the width of the fibril is included in the profiles of PSHG data.…”

Super-Resolution Second-Harmonic Generation Imaging with Multifocal Structured Illumination Microscopy

“…25,26 Additionally, combining MSIM with nonlinear imaging techniques such as two-photon excitation or SHG can reduce the effects of sample scattering and minimize artifacts in MSIM. 16,27 Polarization-resolved SHG allows for the quantification of collagen fibril orientation, 28,29 even three-dimensional orientation. 30 However, these polarization analyses are mostly based on traditional SHG microscopy with limited resolution, which to some extent impairs the accuracy of quantitative collagen orientation (the average orientation of adjacent collagen fibrils rather than the exact orientation of a single collagen fibril).…”