Biology subjects

Barzda, V.

Publications and source records attributed to Barzda, V..

8 recordsLinked to original sources

bioRxiv2025

Collagen fibre organisation revealed with computational scattered light imaging and polarimetric second harmonic generation microscopy

Collagen forms dense fibre networks in the human body, with the organisation directly influencing tissue mechanics and function in health and disease. A good understanding of this relation requires proper imaging techniques for visualising the dense collagen network. Previously, computational scattered light imaging was employed as a fast and easy-to-implement technique to retrieve the orientation of multi-directional collagen fibres, but results were not yet validated quantitatively. In this study, we validate the in-plane orientations of collagen fibres determined with computational scattered light imaging by performing comparative measurements with polarimetric second harmonic generation microscopy on rat tendon and bone sections. For rat tendon, sections with and without hematoxylin-and-eosin staining, folded tendon layers, and obliquely cut sections were investigated. Similar fibre orientations were obtained with both techniques in both tissues, with the highest degree of similarity found for in-plane, unidirectional fibres in the tendon sections. The techniques were able to retrieve the orientations of multi-directional crossing collagen fibres in folded rat tendon layers, and results were found to be unaffected by staining. While polarimetric second harmonic generation microscopy provides high resolution and ultrastructural information on collagen, computational scattered light imaging provides large field of view measurements with micrometre resolution.

bioengineering↗

bioRxiv2025

Polar organization of H&E dyes in histology tissue revealed by polarimetric nonlinear microscopy

Structural organization of harmonophores used in hematoxylin (H) and eosin (E) staining is studied with polarimetric multimodal second-harmonic generation (SHG), third-harmonic generation (THG) and multiphoton excitation fluorescence (MPF) microscopy in rat tail tendon histology sections. The polarimetric microscopy imaging reveals that hemalums (complexes of hematoxylin and aluminum) are well aligned with C6h symmetry along the collagen fibers in H-stained tissue, while eosin Y is partially aligned along the fibers in E-stained tissue and also follows organization of C6h symmetry. When both hemalum and eosin are used for H&E staining, the dye molecules interact and align noncentrosymmetrically with C6 symmetry along the collagen fibers, while the stained nuclei appear isotropically organized. The polar alignment of the hemalum and eosin complexes increases the achiral second order susceptibility tensor component ratio [Formula] in H&E-stained tissue. The alignment of hemalum and eosin molecules, and their complexes in collagenous tissue, must be considered in nonlinear microscopy and polarimetric analysis of H&E-stained histopathology.

biophysics↗

bioRxiv2023

Polarimetric response of second harmonic generation in microscopy of partially oriented chiral fibrillar structures

Polarimetric second harmonic generation (SHG) microscopy is employed to study partially oriented fibrillar structures. The polarimetric SHG parameters are influenced by three-dimensional (3D) configuration of C6 symmetry fibrilar structures in the focal volume (voxel) of a microscope. The achiral and chiral susceptibility tensor components ratios (R and C, respectively) are extracted from the linear polarization-in polarization-out (PIPO) measurements. The analytical derivations along with the polarimetric SHG microscopy results obtained from rat tail tendon, rabbit cornea, pig cartilage and meso-tetra (4-sulfonatophenyl) porphine (TPPS4) cylindrical aggregates demonstrate that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and R and C ratio values change by tilting the fibers out of image plane, as well as by crossing the fibers in 2D and 3D. The polarimetric microscopy results are consistent with the digital microscopy modeling of fibrillar structures. These results facilitate the interpretation of polarimetric SHG microscopy images in terms of 3D organization of fibrilar structures in each voxel of the samples. Statement of SignificancePolarimetric second harmonic generation (SHG) microscopy is used to study partially oriented C6 symmetry chiral fibrillar structures. The linear polarization-in polarization-out (PIPO) SHG imaging is performed on rat tail tendon, rabbit cornea, pig cartilage tissues and meso-tetra (4-sulfonatophenyl) porphine (TPPS4) cylindrical aggregates. The study demonstrates that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and the achiral and chiral susceptibility component ratio values change by tilting the fibers out of image plane, as well as by crossing the fibers in 2D and 3D. The polarimetric microscopy results are consistent with the digital microscopy modeling of fibrillar structures. These results facilitate the interpretation of polarimetric SHG microscopy images in terms of 3D organization of fibrillar structures in each voxel of the samples.

biophysics↗

bioRxiv2022

Digital polarimetric second harmonic generation microscopy of partially oriented fiber structures

Second harmonic generation (SHG) in biological tissue originates predominantly from noncentrosymmetric fibrillar structures partially oriented within the focal volume (voxel) of a multiphoton excitation microscope. The study is aimed to elucidate fibrillar organization factors influencing SHG intensity, as well as achiral, R, and chiral, C, nonlinear susceptibility tensor component ratios. SHG response is calculated for various configurations of fibrils in a voxel using digital nonlinear microscope. The R and C ratios are calculated using linear incident and outgoing polarization states that simulate polarization-in polarization-out (PIPO) polarimetric measurements. The investigation shows strong SHG intensity dependence on parallel/antiparallel fiber organization. The R and C ratio is strongly influenced by the fiber chirality, tilting of the fibers out of image plane and crossing of the fibers. The study facilitates interpretation of polarimetric SHG microscopy images in terms of ultrastructural organization of fibers in the imaged structures. Statement of SignificanceSecond harmonic generation microscopy is widely used for imaging non-centrosymmetric biological structures such as collagen. The ultrastructure of collagen can be determined with polarimetric SHG microscopy. The coherent nonlinear response of biological structures depends on the 3D orientations and positions of the collagen fibers in the focal volume of the microscope. Here, we show how different fiber organizations and 3D orientations in the focal volume can affect the polarimetric SHG responses. The results are important for understanding and interpreting images obtained with polarimetric SHG microscopy.

biophysics↗

bioRxiv2022

Unsupervised determination of lung tumor margin with widefield polarimetric second-harmonic generation microscopy

The extracellular matrix (ECM) is amongst many tissue components affected by cancer, however, morphological changes of the ECM are not well-understood and thus, often omitted from diagnostic considerations. Polarimetric second-harmonic generation (P-SHG) microscopy allows for visualization and characterization of collagen ultrastructure in the ECM, aiding in better understanding of the changes induced by cancer throughout the tissue. In this paper, a large region of hematoxylin and eosin-stained human lung section, encompassing a tumor margin, connecting a significant tumor portion to normal tissue was imaged with P-SHG microscopy. The resulting polarimetric parameters were utilized in principal components analysis and unsupervised k-means clustering to separate normal- and tumor-like tissue. Consequently, a pseudo-color map of the clustered tissue regions is generated to highlight the irregularity of the ECM collagen structure throughout the region of interest and to identify the tumor margin.

pathology↗

bioRxiv2022

Wide-Field Stokes Polarimetric Microscopy for Second Harmonic Generation Imaging

We employ wide-field second harmonic generation (SHG) microscopy together with nonlinear Stokes polarimetry for quick ultrastructural investigation of large sample areas (700 m x 700 m) in thin histology sections. The Stokes vector components for SHG are obtained from the polarimetric measurements with incident and outgoing linear and circular polarization states. The Stokes components are used to construct the images of polarimetric parameters and deduce the sample maps of achiral and chiral nonlinear susceptibility tensor components ratios and cylindrical axis orientation in fibrillar materials. The imaged histology sections with polarimetric wide-field SHG microscopy provide large area maps of ultrastructural information about the collagenous tissue, which can be used for rapid histology investigations.

biophysics↗

bioRxiv2022

Wide-Field Multicontrast Nonlinear Microscopy for Histopathology

A multicontrast polarimetric wide-field second harmonic generation (SHG) and multiphoton excitation fluorescence (MPF) microscope is optimized for large area imaging of hematoxylin and eosin (H&E) stained and unstained histology slides. The bleaching kinetics of MPF and SHG are examined with various laser intensities at different pulse repetition rates to determine the optimal wide-filed imaging conditions for H&E stained histology slides. Several polarimetric parameters are used to investigate the organization of extracellular matrix collagen in the histology samples.

biophysics↗

bioRxiv2021

Machine learning-enabled cancer diagnostics with widefield polarimetric second-harmonic generation microscopy

The extracellular matrix (ECM) collagen undergoes major remodeling during tumorigenesis. However, alterations to the ECM are not widely considered in cancer diagnostics, due to mostly uniform appearance of collagen fibers in white light images of hematoxylin and eosin-stained tissue sections. Polarimetric second-harmonic generation (P-SHG) microscopy enables label-free visualization and ultrastructural investigation of non-centrosymmetric molecules, which, when combined with texture analysis, provides multiparameter characterization of tissue collagen. This paper demonstrates whole slide imaging of breast tissue microarrays using high-throughput widefield P-SHG microscopy. The resulting P-SHG parameters are used in classification to differentiate tumor tissue from normal with 94.2% accuracy and F1-score, and 6.3% false discovery rate. Subsequently, the trained classifier is employed to predict tumor tissue with 91.3% accuracy, 90.7% F1-score, and 13.8% false omission rate. As such, we show that widefield P-SHG microscopy reveals collagen ultrastructure over large tissue regions and can be utilized as a sensitive biomarker for cancer diagnostics and prognostics studies.

pathology↗