Schwiedrzik, J. | Search bioRxiv

bioRxiv2026

Residual ellipticity in waveplate-compensated polarization-resolved SHG microscopy may arise from femtosecond laser spectral bandwidth

Polarization-resolved second harmonic generation microscopy provides structural information about non-centrosymmetric biological samples such as collagen. It involves illuminating the sample with a focused laser beam having a variable linear polarization angle and recording the second harmonic signal as a function of this angle. However, accurate linear polarization control is challenging due to ellipticity introduced by reflections from mirrors and dichroic mirrors in the optical path. Waveplate-based compensation has emerged as the standard approach to address these distortions, but its effectiveness for quantitative measurements remains incompletely characterized. Here, we attempt to fill this gap by implementing an established automated waveplate compensation method based on a rotating half-waveplate in combination with a compensating quarter-waveplate. This was done on a commercial Leica TCS SP8 MP multiphoton microscope, making various hardware improvements and carefully documenting important experimental details. Despite significant effort, we consistently observed substantial unwanted residual polarization ellipticity, with amplitudes up to 0.25, persisting under optimal waveplate configurations. Our simulation analysis provides evidence that this limitation may arise from wavelength-dependent dichroic mirror birefringence combined with the broad spectral bandwidth (10nm to 20nm full width at half maximum) of femtosecond laser pulses. While the approach investigated here can compensate a single wavelength, different spectral components within the pulse experience different phase retardations from wavelength-dependent optical elements, potentially resulting in residual ellipticity that cannot be eliminated. Our simulations qualitatively reproduced key features of the experimental observations. These findings have important implications for quantitative polarization-resolved second harmonic generation microscopy and suggest that alternative approaches, including specimen rotation or picosecond laser sources with narrower bandwidth, should be investigated for applications requiring precise polarization control. To facilitate community investigation of these effects, we provide open-source analysis code and simulation files.

bioengineering↗

bioRxiv2022

The elasto-plastic nano- and microscale compressive behaviour of rehydrated mineralised collagen fibres

The multiscale architectural design of bio-based nanostructured materials such as bone enables them to combine unique structure-mechanical properties that surpass classical engineering materials. In biological tissues, water as one of the main components plays an important role in the mechanical interplay, but its influence has not been quantified at the length scale of a mineralised collagen fibre. Here, we combine in situ experiments and a statistical constitutive model to identify the elasto-plastic micro- and nanomechanical fibre behaviour under rehydrated conditions. Micropillar compression and simultaneous synchrotron small angle X-ray scattering (SAXS) and X-ray diffraction (XRD) were used to quantify the interplay between fibre, mineralised collagen fibrils and mineral nanocrystals. Rehydration led to a 65% to 75% decrease of fibre yield stress and compressive strength, and a 70% decrease of stiffness with a 3x higher effect on stress than strain values. While in good agreement with bone extracellular matrix, the decrease is 1.5-3x higher compared to micro-indentation and macro-compression. Hydration has a higher influence on mineral than fibril strain while the highest difference to the macroscale was observed comparing mineral and tissue levels. Results suggest that the effect of hydration is strongly mediated by ultrastructural interfaces while corroborating the previously reported water-mediated structuring of bone apatite providing insights towards the mechanical consequences. Results show that the missing reinforcing capacity of surrounding tissue is more pronounced in wet than dry conditions when testing an excised array of fibrils, mainly related to the swelling of fibrils in the matrix. Differences leading to higher compressive strength between mineralised tissues do not seem to depend on the rehydration state while fibril mobilisation follows a similar regime in wet and dry conditions. The lack of kink bands point towards the role of water as an elastic embedding, thus, adapting the way energy is absorbed. Statement of significanceCharacterising structure-property-function relationships of biomaterials helps us to elucidate the underlying mechanisms that enables the unique properties of these architectured materials. Experimental and computational methods can advance our understanding towards their complex behaviour providing invaluable insights towards bio-inspired material development. In our study, we present a novel method for biomaterials characterisation. We close a gap of knowledge at the micro- and nanometre length scale by combining synchrotron experiments and a statistical model to describe the behaviour of a rehydrated single mineralised collagen fibre. Results suggest a high influence of hydration on structural interfaces, and the role of water as an elastic embedding. Using a statistical model, we are able to deduce the differences in wet and dry elasto-plastic properties of fibrils and fibres close to their natural hydration state.

bioengineering↗