The human middle ear in motion: 3D visualization and quantification using dynamic synchrotron-based X-ray imaging.

The characterization of the vibrations of the middle ear ossicles during sound transmission is a focal point in clinical research. However, the small size of the structures, their micrometer-scale movement, and the deep-seated position of the middle ear within the temporal bone make these types of measurements extremely challenging. In this work, dynamic synchrotron-based X-ray phase-contrast microtomography is used on acoustically stimulated intact human ears, allowing for the three-dimensional visualization of entire human eardrums and ossicular chains in motion. A post-gating algorithm is used to temporally resolve the fast micromotions at 128 Hz, coupled with a high-throughput pipeline to process the large tomographic datasets. Seven ex-vivo fresh-frozen human temporal bones in healthy conditions are studied, and the rigid body motions of the ossicles are quantitatively delineated. Clinically relevant regions of the ossicular chain are tracked in 3D, and the amplitudes of their displacement are computed for two acoustic stimuli.

Quantitative structural imaging of keratoconic corneas using polarization-resolved SHG microscopy.

The human cornea is mainly composed of collagen fibrils aligned together within stacked lamellae. This lamellar structure can be affected in pathologies such as keratoconus, which is characterized by progressive corneal thinning and local steepening. In this study, we use polarization-resolved second harmonic generation (P-SHG) microscopy to characterize 8 control and 6 keratoconic human corneas. Automated processing of P-SHG images of transverse sections provides the collagen orientation in every pixel with sub-micrometer resolution. Series of P-SHG images recorded in the most anterior region of the stroma evidence sutural lamellae inclined at 22° ± 5° to the corneal surface, but show no significant difference between control and keratoconic corneas. In contrast, series of P-SHG images acquired along the full thickness of the stroma show a loss of order in the lamellar structure of keratoconic corneas, in agreement with their defective mechanical properties. This structural difference is analyzed quantitatively by computing the entropy and the orientation index of the collagen orientation distribution and significant differences are obtained along the full thickness of the stroma. This study shows that P-SHG is an effective tool for automatic quantitative analysis of structural defects of human corneas and should be applied to other collagen-rich tissues.

Noninvasive quantitative assessment of collagen degradation in parchments by polarization-resolved SHG microscopy.

Nondestructive and noninvasive investigation techniques are highly sought-after to establish the degradation state of historical parchments, which is up to now assessed by thermal techniques that are invasive and destructive. We show that advanced nonlinear optical (NLO) microscopy enables quantitative in situ mapping of parchment degradation at the micrometer scale. We introduce two parameters that are sensitive to different degradation stages: the ratio of two-photon excited fluorescence to second harmonic generation (SHG) signals probes severe degradation, while the anisotropy parameter extracted from polarization-resolved SHG measurements is sensitive to early degradation. This approach is first validated by comparing NLO quantitative parameters to thermal measurements on artificially altered contemporary parchments. We then analyze invaluable parchments from the Middle Ages and show that we can map their conservation state and assess the impact of a restoration process. NLO quantitative microscopy should therefore help to identify parchments most at risk and optimize restoration methods.

Implementation of artifact-free circular dichroism SHG imaging of collagen.

Second harmonic generation (SHG) enables in situ imaging of fibrillar collagen architecture in connective tissues. Recently, Circular Dichroism SHG (CD-SHG) microscopy has been implemented to take advantage of collagen chirality to improve 3D visualization. It measures the normalized difference in the SHG signal obtained upon excitation by left versus right circular polarizations. However, CD-SHG signal is not well characterized yet, and quite different CD-SHG values are reported in the literature. Here, we identify two major artifacts that may occur in CD-SHG experiments and we demonstrate that thorough optimization and calibration of the experimental setup are required for CD-SHG imaging. Notably it requires a careful calibration of the incident circular polarizations and a perfect mechanical stabilization of the microscope stage. Finally, we successfully record CD-SHG images in human cornea sections and confirm that this technique efficiently reveals collagen fibrils oriented out of the focal plane.

Elimination of imaging artifacts in second harmonic generation microscopy using interferometry.

Conventional second harmonic generation (SHG) microscopy might not clearly reveal the structure of complex samples if the interference between all scatterers in the focal volume results in artefactual patterns. We report here the use of interferometric second harmonic generation (I-SHG) microscopy to efficiently remove these artifacts from SHG images. Interfaces between two regions of opposite polarity are considered because they are known to produce imaging artifacts in muscle for instance. As a model system, such interfaces are first studied in periodically-poled lithium niobate (PPLN), where an artefactual incoherent SH signal is obtained because of irregularities at the interfaces, that overshadow the sought-after coherent contribution. Using I-SHG allows to remove the incoherent part completely without any spatial filtering. Second, I-SHG is also proven to resolve the double-band pattern expected in muscle where standard SHG exhibits in some regions artefactual single-band patterns. In addition to removing the artifacts at the interfaces between antiparallel domains in both structures (PPLN and muscle), I-SHG also increases their visibility by up to a factor of 5. This demonstrates that I-SHG is a powerful technique to image biological samples at enhanced contrast while suppressing artifacts.

Monitoring dynamic collagen reorganization during skin stretching with fast polarization-resolved second harmonic generation imaging.

The mechanical properties of biological tissues are strongly correlated to the specific distribution of their collagen fibers. Monitoring the dynamic reorganization of the collagen network during mechanical stretching is however a technical challenge, because it requires mapping orientation of collagen fibers in a thick and deforming sample. In this work, a fast polarization-resolved second harmonic generation microscope is implemented to map collagen orientation during mechanical assays. This system is based on line-to-line switching of polarization using an electro-optical modulator and works in epi-detection geometry. After proper calibration, it successfully highlights the collagen dynamic alignment along the traction direction in ex vivo murine skin dermis. This microstructure reorganization is quantified by the entropy of the collagen orientation distribution as a function of the stretch ratio. It exhibits a linear behavior, whose slope is measured with a good accuracy. This approach can be generalized to probe a variety of dynamic processes in thick tissues.