Precision of polarization-resolved second harmonic generation microscopy limited by photon noise for samples with cylindrical symmetry

Wasik, Valentine; Réfrégier, Philippe; Roche, Muriel; Brasselet, Sophie

doi:10.1364/josaa.32.001437

Cited by 4 publications

(3 citation statements)

References 16 publications

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“…P-SHG orientation maps are thus reliable up to approximately 2/3 of the cornea thickness and their effective precision is around 4°, considering that the measure uncertainty is similar for OrientationJ and P-SHG and using error propagation. This effective precision is much larger than the one usually claimed for P-SHG data that is assessed from theoretical analysis or provided by fitting or FFT codes 29,30 . It indeed takes into account all experimental uncertainty sources in thick tissues, including the tissue optical heterogeneity that induces wavefront deformation, focal volume deterioration, and ellipticity increase even at moderate depth.…”

Section: Validity Of Collagen Orientation Measurements In Depthmentioning

confidence: 83%

“…We thus use an FFT algorithm to process our data and determine the orientation φ in every pixel (see Supplementary material). The theoretical angular precision is about 1° after the application of a 6 × 6 averaging filter to increase the signal-to-noise ratio 29 , 30 . Our data-processing workflow also provides 21 : (i) the SHG signal averaged over all linear polarizations: ; (ii) a coefficient of determination R 2 that compares the experimental data and the curve obtained from the FFT parameters in every pixel, in order to validate the orientation measure (Fig.…”

Section: Methodsmentioning

confidence: 99%

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Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy

Raoux¹,

Chessel²,

Mahou³

et al. 2023

Light Sci Appl

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A key property of the human cornea is to maintain its curvature and consequently its refraction capability despite daily changes in intraocular pressure. This is closely related to the multiscale structure of the corneal stroma, which consists of 1–3 µm-thick stacked lamellae made of thin collagen fibrils. Nevertheless, the distribution, size, and orientation of these lamellae along the depth of the cornea are poorly characterized up to now. In this study, we use second harmonic generation (SHG) microscopy to visualize the collagen distribution over the full depth of 10 intact and unstained human corneas (500–600 µm thick). We take advantage of the small coherence length in epi-detection to axially resolve the lamellae while maintaining the corneal physiological curvature. Moreover, as raw epi-detected SHG images are spatially homogenous because of the sub-wavelength size of stromal collagen fibrils, we use a polarimetric approach to measure the collagen orientation in every voxel. After a careful validation of this approach, we show that the collagen lamellae (i) are mostly oriented along the inferior–superior axis in the anterior stroma and along the nasal-temporal axis in the posterior stroma, with a gradual shift in between and (ii) exhibit more disorder in the anterior stroma. These results represent the first quantitative characterization of the lamellar structure of the human cornea continuously along its entire thickness with micrometric resolution. It also shows the unique potential of P-SHG microscopy for imaging of collagen distribution in thick dense tissues.

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Section: Validity Of Collagen Orientation Measurements In Depthmentioning

confidence: 83%

Section: Methodsmentioning

confidence: 99%

Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy

Raoux¹,

Chessel²,

Mahou³

et al. 2023

Light Sci Appl

View full text Add to dashboard Cite

show abstract

“…In our case, due to the fact that a sample-based analysis has been followed, the precision of <B> and <f c > is determined as the SD of the corresponding values resulting from all the image pixels. It is worth to be mentioned that, recent studies present an alternative, new method where the precision of the calculated B and f c values can be estimated for each pixel [29,30].…”

Section: Pshg Data Analysismentioning

confidence: 99%

Monitoring aging‐associated structural alterations in Caenorhabditis elegans striated muscles via polarization‐dependent second‐harmonic generation measurements

Tsafas

Giouroukou

Kounakis

et al. 2021

Journal of Biophotonics

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The in‐vivo elucidation of the molecular mechanisms underlying muscles dysfunction due to aging via non‐invasive label free imaging techniques is an important issue with high biological significance. In this study, polarization‐dependent second‐harmonic generation (PSHG) was used to evaluate structural alterations in the striated muscles during Caenorhabditis elegans lifespan. Young and old wild‐type animals were irradiated. The obtained results showed that it was not feasible to detect differences in the structure of myosin that could be correlated with the aging of the worms, via the implementation of the classical, widely used, cylindrical symmetry model and the calculation of the SHG anisotropy values. A trigonal symmetry model improved the extracted results; however, the best outcome was originated from the application of a general model. Myosin structural modifications were monitored via the estimation of the difference in spectral phases derived from discrete Fourier transform analysis. Age classification of the nematodes was achieved by employing both models, proving the usefulness of the usage of PSHG microscopy as a potential diagnostic tool for the investigation of muscle diseases.

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

Raoux

Schmeltz

Bied

et al. 2021

Biomed. Opt. Express

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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.

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Precision of polarization-resolved second harmonic generation microscopy limited by photon noise for samples with cylindrical symmetry

Cited by 4 publications

References 16 publications

Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy

Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy

Monitoring aging‐associated structural alterations in Caenorhabditis elegans striated muscles via polarization‐dependent second‐harmonic generation measurements

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

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