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Significance: The spatial organization of collagen fibers has been used as a biomarker for assessing injury and disease progression. However, quantifying this organization for complex structures is challenging. Aim: To quantify and classify complex collagen fiber organizations. Approach: Using quantitative second-harmonic generation (SHG) microscopy, we show that collagen-fiber orientation can be viewed as pseudovector fields. Subsequently, we analyze them using fluid mechanic metrics, such as energy U, enstrophy E, and tortuosity tau. Results: We show that metrics used in fluid mechanics for analyzing fluid flow can be adapted to analyze complex collagen fiber organization. As examples, we consider SHG images of collagenous tissue for straight, wavy, and circular fiber structures. Conclusions: The results of this study show the utility of the chosen metrics to distinguish diverse and complex collagen organizations. We find that the distribution of values for E and U increases with collagen fiber disorganization, where they divide between low and high values corresponding to uniformly aligned fibers and disorganized collagen fibers, respectively. We also confirm that the values of tau cluster around 1 when the fibers are straight, and the range increases up to 1.5 when wavier fibers are present. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Published version

Significance: A non-destructive technique for accurately characterizing the spatial distribution of optical properties of soft tissue membranes may give improved outcomes in many tissue engineering applications. Aim: This study aimed to develop a non-destructive macroscopic imaging technique that is sensitive to optical anisotropy, typical of fibrous components in soft tissue membranes, and can address some of the difficulties caused by the complex turbid nature of these tissues. Approach: A near-infrared Mueller matrix imaging polarimeter employing logarithm decomposition was developed and used to conduct transmission measurements of all the polarization properties across the full thickness of bovine pericardium tissue. Results: The full Mueller matrix was measured across a 70 mm×70 mm sample of calf bovine pericardium and revealed significant retardance (linear and circular) and depolarization in this tissue. Regions with a uniform axis of optical anisotropy were identified. Mueller matrix imaging demonstrated that the exhibited circular retardance was sufficient to lead to possible misinterpretation of apparent fiber orientation when using conventional polarization imaging techniques for such tissues. Conclusions: Mueller matrix imaging can identify regional distributions of optical anisotropy in calf bovine pericardium. This new capability is a promising development in non-destructive imaging for tissue selection.

In this study, the employment of the X-ray irradiation as bioremediation method to treat parchment has been deeply investigated. In particular, the effect of the irradiation doses on the structural stability of collagen, the main constituent of parchment, has been evaluated on a series of modern parchment samples by means of different opto-thermal and spectroscopic techniques in order to obtain the dose-dependent effect of irradiation on collagen. Moreover, the long-term behavior of the irradiated parchment has been considered by analyzing the same series of samples after being hygrothermally artificially aged. Characterizations by light transmission analysis, Fourier transform infrared spectroscopy and the ultraviolet–visible–near infrared (UV–Vis–NIR) reflectance one have been performed for the identification of the radio-induced effect on the parchment structure even in a dose range much wider than the need for sterilization. The obtained results have proved the safeness of the method in the short and long term confirming the applicability of this emerging procedure.

Significance: Collagen is the most abundant protein in vertebrates and is found in tissues that regularly experience tension, compression, and shear forces. However, the underlying mechanism of collagen fibril formation and remodeling is poorly understood. Aim: We explore how a collagen monomer is visualized using fluorescence microscopy and how its spatial orientation is determined. Defining the orientation of collagen monomers is not a trivial problem, as the monomer has a weak contrast and is relatively small. It is possible to attach fluorescence tags for contrast, but the size is still a problem for detecting orientation using fluorescence microscopy. Approach: We present two methods for detecting a monomer and classifying its orientation. A modified Gabor filter set and an automatic classifier trained by convolutional neural network based on a synthetic dataset were used. Results: By evaluating the performance of these two approaches with synthetic and experimental data, our results show that it is possible to determine the location and orientation with an error of ∼37 deg of a single monomer with fluorescence microscopy. Conclusions: These findings can contribute to our understanding of collagen monomers interaction with collagen fibrils surface during fibril formation and remodeling.

Significance: Morphological collagen signatures are important for tissue function, particularly in the tumor microenvironment. A single algorithmic framework with quantitative, multiscale morphological collagen feature extraction may further the use of collagen signatures in understanding fundamental tumor progression. Aim: A modification of the 2D wavelet transform modulus maxima (WTMM) anisotropy method was applied to both digitally simulated collagen fibers and second-harmonic-generation imaged collagen fibers of mouse skin to calculate a multiscale anisotropy factor to detect collagen fiber organization. Approach: The modified 2D WTMM anisotropy method was initially validated on synthetic calibration images to establish the robustness and sensitivity of the multiscale fiber organization tool. Upon validation, the algorithm was applied to collagen fiber organization in normal wild-type skin, melanoma stimulated skin, and integrin α10KO skin. Results: Normal wild-type skin collagen fibers have an increased anisotropy factor at all sizes scales. Interestingly, the multiscale anisotropy differences highlight important dissimilarities between collagen fiber organization in normal wild-type skin, melanoma stimulated, and integrin α10KO skin. At small scales (∼2 to 3 μm), the integrin α10KO skin was vastly different than normal skin (p-value ∼ 10 − 8), whereas the melanoma stimulated skin was vastly different than normal at large scales (∼30 to 40 μm, p-value ∼ 10 − 15). Conclusions: This objective computational collagen fiber organization algorithm is sensitive to collagen fiber organization across multiple scales for effective exploration of collagen morphological alterations associated with melanoma and the lack of α10 integrin binding.

Significance: Idiopathic pulmonary fibrosis (IPF) patients have a poor prognosis with short lifespan following diagnosis as there are limited effective treatment options. Despite matrix stiffening being the hallmark of the disease there remains a lack of knowledge surrounding the underlying collagen alterations in the disease. Specifically, while increased collagen crosslinking has been implicated, the resulting effects on collagen macro/supramolecular changes have not been explored. Aim: We sought to determine if second-harmonic generation (SHG) microscopy could characterize differences in the collagen architecture in 3D spheroid models of IPF grown under different crosslinking modulation conditions (promotion and inhibition). Approach: We used SHG metrics based on the fiber morphology, relative SHG brightness, and macro/supramolecular structure by SHG polarization analyses to compare the structure of the IPF spheroids. Results: Comparison of the fiber morphology of the spheroids showed that the control group had the longest, straightest, and thickest fibers. The spheroids with crosslink enhancement and inhibition had the highest and lowest SHG conversion efficiencies, respectively, consistent with the resulting harmonophore density. SHG polarization analyses showed that the peptide pitch angle, alignment of collagen molecules, and overall chirality were altered upon crosslink modulation and were also consistent with reduced organization relative to the control group. Conclusions: While no single SHG signature is associated with crosslinking, we show that the suite of metrics used here is effective in delineating alterations across the collagen architecture sizescales. The results largely mirror those of human tissues and demonstrate that the combination of 3D spheroid models and SHG analysis is a powerful approach for hypothesis testing the roles of operative cellular and molecular factors in IPF.

In this research, collagen hydrolysates with different average molecular weights ( Mn ¯ ) from leather collagen were chosen as raw materials. Five environmental-friendly sizing agents (SA) were prepared by cross-linking collagen hydrolysates with glycol diglycidyl ether (GDE) and further grafting them with butyl acrylate (BA) and styrene (St). Then the compound sizing agents (SGDESA-x, x = 1, 2, 3 and 4) were obtained by simple physical mixing of GDESA and starch. The surface sizing performance of GDESA and compound sizing agents were studied. The research result shows that both physical, mechanical properties and water resistance of the corrugated paper coated by GDESA were significantly improved when Mn ¯ of collagen hydrolysate was about 10,000, and its emulsion exhibited robust stability in long standing time. Furthermore, when the SGDESA-2 was used as a sizing agent, the coated corrugated paper exhibited strong water resistance, good physical and mechanical properties even after refolded for 20 times.

The piezoelectricity of collagen is purported to be linked to many biological processes including bone formation and wound healing. Although the piezoelectricity of tissue-derived collagen has been documented across the length scales, little work has been undertaken to characterise the local electromechanical properties of processed collagen, which is used as a base for tissue-engineering implants. In this work, three chemically distinct treatments used to form structurally and mechanically stable scaffolds— EDC-NHS, genipin and tissue transglutaminase—are investigated for their effect on collagen piezolectricity. Crosslinking with EDC-NHS is noted to produce a distinct self-assembly of the fibres into bundles roughly 300 nm in width regardless of the collagen origin. These fibre bundles also show a localised piezoelectric response, with enhanced vertical piezoelectricity of collagen. Such topographical features are not observed with the other two chemical treatments, although the shear piezoelectric response is significantly enhanced upon crosslinking. These observations are reconciled by a proposed effect of the crosslinking mechanisms on the molecular and nanostructure of collagen. These results highlight the ability to modify the electromechanical properties of collagen using chemical crosslinking methods.

Significance: The study has confirmed the feasibility of using ultraviolet (UV) excitation to visualize and quantify desmoplasia in fresh tumor tissue of pancreatic adenocarcinoma (PDAC) in an orthotopic xenograft mouse model, which provides a useful imaging platform to evaluate acute therapeutic responses. Aim: Stromal network of collagen prominent in PDAC tumors is examined by imaging fresh tissue samples stained with histological dyes. Fluorescence signals are color-transferred to mimic Masson’s trichrome staining. Approach: Murine tumor samples were stained with Hoechst, eosin, and rhodamine B and excited at 275-nm. Fluorescence signals in the visible spectrum were captured by a CMOS color camera with high contrast and resolution at whole-tumor slice field of view. Results: Fluorescence imaging using UV excitation is capable of visualizing collagen deposition in PDAC tumors. Both fluorescence and histology data showed collagen content of up to 30%. The collagen modulation effect due to photodynamic priming treatment was observed showing 13% of collagen reduction. Necrosis area is visible and perfusion imaging using Texas Red dextran is feasible. Conclusions: The study demonstrates collagen visualization in fresh PDAC tumor samples using UV excitation. This imaging platform also provides quantitative stromal information from fiber analysis and visibility of necrosis and perfusion, suitable for therapeutic response assessment of photodynamic therapy.

Significance: Mueller matrix polarimetry can provide useful information about the function and structure of the extracellular matrix. A portable and low-cost system could facilitate the clinical assessment of cervical anomalies in low-resource settings. Aim: We introduce a low-cost snapshot Mueller matrix polarimeter that does not require external power, has no moving parts, and can acquire a full Mueller matrix in ∼1 s, to conduct a feasibility study for cervical imaging in the low-resource setting. Approach: A snapshot system based on two sets of Savart plates, a ring illuminator with polarizing elements (generating four polarization states), and one camera is introduced. Stokes vectors are formulated to recover the polarization properties of the sample. Then, using Mueller matrix decomposition, the depolarization and retardance information is extracted. Results: We report the results on 16 healthy individuals (out of 22 patients imaged), whose Pap smear showed no malignant findings from mobile clinics in rural region of Mysore, India. The depolarization and retardance information was in agreement with previous reports. Conclusions: We introduce an imaging system and conducted a feasibility study on healthy individuals. This work could futurely translate into diagnostic applications to provide a quantitative platform in the clinical environment (e.g., cervical cancer screening).

PubMed: 31983145 Obesity is associated with a higher risk of developing breast cancer and with worse disease outcomes for women of all ages. The composition, density, and organization of the breast tissue stroma are also known to play an important role in the development and progression of the disease. However, the connections between obesity and stromal remodeling are not well understood. We sought to characterize detailed organization features of the collagen matrix within healthy and cancerous breast tissues acquired from mice exposed to either a normal or high fat (obesity inducing) diet. We performed second-harmonic generation and spectral two-photon excited fluorescence imaging, and we extracted the level of collagen-associated fluorescence (CAF) along with metrics of collagen content, three-dimensional, and two-dimensional organization. There were significant differences in the CAF intensity and overall collagen organization between normal and tumor tissues; however, obesity-enhanced changes in these metrics, especially when three-dimensional organization metrics were considered. Thus, our studies indicate that obesity impacts significantly collagen organization and structure and the related pathways of communication may be important future therapeutic targets..

Melanin is known to provide strong third-harmonic generation (THG) contrast in human skin. With a high concentration in basal cell cytoplasm, THG contrast provided by melanin overshadows other THG sources in human skin studies. For better understanding of the THG signals in keratinocytes without the influence of melanin, an in vivo THG microscopy (THGM) study was first conducted on vitiliginous skin. As a result, the THG-brightness ratio between the melanin-lacking cytoplasm of basal cells and collagen fibers is about 1.106 at the dermal–epidermal junctions of vitiliginous skin, indicating high sensitivity of THGM for the presence of melanin. We further applied the in vivo THGM to assist evaluating the therapeutic outcome from the histopathological point of view for those showed no improvement under narrowband ultraviolet B therapy based on the seven-point Physician Global Assessment score. Our clinical study indicates the high potential of THGM to assist the histopathological assessment of the therapeutic efficacy of vitiligo treatments.

pTwo-photon microscopes have been successfully translated into clinical imaging tools to obtain high-resolution optical biopsies foritalicin vivo/italichistology. We report on clinical multiphoton coherent anti-Stokes Raman spectroscopy (CARS) tomography based on two tunable ultrashort near-infrared laser beams for label-freeitalicin vivo/italicmultimodal skin imaging. The multiphoton biopsies were obtained with the compact tomograph "MPTflex-CARS" using a photonic crystal fiber, an optomechanical articulated arm, and a four-detector-360 deg measurement head. The multiphoton tomograph has been employed to patients in a hospital with diseased skin. The clinical study involved 16 subjects, 8 patients with atopic dermatitis, 4 patients with psoriasis vulgaris, and 4 volunteers served as control. Two-photon cellular autofluorescence lifetime, second harmonic generation (SHG) of collagen, and CARS of intratissue lipids/proteins have been detected with single-photon sensitivity, submicron spatial resolution, and picosecond temporal resolution. The most important signal was the autofluorescence from nicotinamide adenine dinucleotide [NAD(P)H]. The SHG signal from collagen was mainly used to detect the epidermal-dermal junction and to calculate the ratio elastin/collagen. The CARS/Raman signal provided add-on information. Based on this view on the disease-affected skin on a subcellular level, skin areas affected by dermatitis and by psoriasis could be clearly identified. Multimodal multiphoton tomographs may become important label-free clinical high-resolution imaging tools foritalicin vivo/italicskin histology to realize rapid early diagnosis as well as treatment control./p.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis with short lifespan following diagnosis as patients have limited effective treatment options. A fundamental limitation is a lack of knowledge of the underlying collagen alterations in the disease, as this could lead to better diagnostics, prognostics, and measures of treatment efficacy. While the fibroses is the primary presentation of the disease, the collagen architecture has not been well studied beyond standard histology. Here, we used several metrics based on second harmonic generation (SHG) microscopy and optical scattering measurements to characterize the subresolution collagen assembly in human IPF and normal lung tissues. Using SHG directional analysis, we found that while collagen synthesis is increased in IPF, the resulting average fibril architecture is more disordered than in normal tissue. Wavelength-dependent optical scattering measurements lead to the same conclusion, and both optical approaches are consistent with ultrastructural analysis. SHG circular dichroism revealed significant differences in the net chirality between the fibrotic and normal collagen, where the former has a more randomized helical structure. Collectively, the measurements reveal significant changes in the collagen macro/supramolecular structure in the abnormal fibrotic collagen, and we suggest these alterations can serve as biomarkers for IPF diagnosis and progression.

Neoadjuvant chemotherapy (NACT) is routinely administered to subsets of breast cancer patients, including triple negative (TN) or human epidermal growth factor receptor 2-positive (HER2+) cancers. After NACT and subsequent surgical resection, 5% to 30% of patients have no residual invasive carcinoma, termed pathological complete response. Unfortunately, many patients experience little-to-no response after NACT and unnecessarily suffer its side effects. Methods are needed to predict an individual patient’s response to NACT. Core needle biopsies, taken before NACT, consist of tumor cells and the surrounding extracellular matrix. We performed second-harmonic generation (SHG) imaging of fibrillar collagen in core needle biopsy sections as a possible predictor of response to NACT. The ratio of forward-to-backward scattering (F/B) SHG was assessed in the “tumor bulk” and “tumor–host interface” in HER2+ and TN core needle biopsy sections. Patient response was classified post-treatment using the Residual Cancer Burden (RCB) score. In HER2+ biopsies, RCB class was associated with F/B derived from the tumor–stromal interface, but not tumor bulk. F/B was not associated with RCB class in TN biopsies. These findings suggest that F/B from needle biopsy sections may be a useful predictor of which patients will respond favorably to NACT, with the potential to help reduce overtreatment.

The preservation of cultural heritage is one of the major challenges of today’s society. Parchments, a semi-solid matrix of collagen produced from animal skin, are a significant part of the cultural heritage, being used as writing material since ancient times. Due to their animal origin, parchments easily undergo biodeterioration: the most common biological damage is characterized by isolated or coalescent purple spots, that often lead to the detachment of the superficial layer and the consequent loss of written content. Although many parchments with purple spot biodegradative features were studied, no common causative agent had been identified so far. In a previous study a successional model has been proposed, basing on the multidisciplinary analysis of damaged versus undamaged samples from a moderately damaged document. Although no specific sequences were observed, the results pointed to Halobacterium salinarum as the starting actor of the succession. In this study, to further investigate this topic, three dramatically damaged parchments were analysed; belonging to a collection archived as Faldone Patrizi A 19, and dated back XVI-XVII century A.D. With the same multidisciplinary approach, the Next Generation Sequencing (NGS, Illumina platform) revealed DNA sequences belonging to Halobacterium salinarum; the RAMAN spectroscopy identified the pigment within the purple spots as haloarchaeal bacterioruberin and bacteriorhodopsine, and the LTA technique quantified the extremely damaged collagen structures through the entire parchments, due to the biological attack to the parchment frame structures. These results allowed to propose a model of the progressive degradation pattern of the parchment collagen. Overall, these data validate a multi-phase microbial succession model. This demonstration is pivotal to possible new restoration strategies, important for a huge number of ancient documents.

Collagen remodeling occurs in many prostate pathologies; however, the underlying structural architecture in both normal and diseased prostatic tissues is largely unexplored. Here, we use second-harmonic generation (SHG) microscopy to specifically probe the role of the proteoglycan decorin (Dcn) on collagen assembly in a wild type (wt) and Dcn null mouse (Dcn − / − ). Dcn is required for proper organization of collagen fibrils as it regulates size by forming an arch-like structure at the end of the fibril. We have utilized SHG metrics based on emission directionality (forward–backward ratio) and relative conversion efficiency, which are both related to the SHG coherence length, and found more disordered fibril organization in the Dcn − / − . We have also used image analysis readouts based on entropy, multifractal dimension, and wavelet transforms to compare the collagen fibril/fiber architecture in the two models, where all these showed that the Dcn − / − prostate comprised smaller and more disorganized collagen structures. All these SHG metrics are consistent with decreased SHG phase matching in the Dcn − / − and are further consistent with ultrastructural analysis of collagen in this model in other tissues, which show a more random distribution of fibril sizes and their packing into fibers. As Dcn is a known tumor suppressor, this work forms the basis for future studies of collagen remodeling in both malignant and benign prostate disease.

Significance: Collagen is a basic component of many tissues such as tendons, muscles, and skin, and its imaging helps diagnose and monitor treatments in a variety of fields, including orthopedics. However, due to the overlapping peaks of the absorption spectrum with water in the short-wave infrared region (SWIR), it is difficult to select an optimal wavelength and obtain the photoacoustic (PA) image for collagen-based tissues. Therefore, an additional approach to selecting the proper wavelength is needed. Aim: The aim of this study is to derive an effective PA absorption spectrum of collagen to select the optimal wavelength for high-sensitive PA imaging (PAI). Approach: We measure the absorption spectrum by acquiring the PA signal from various collagen-based samples. To derive an effective PA absorption spectrum in the SWIR band, the following two parameters should be considered: (1) the laser excitation for generating the PA signal and (2) the absorption spectrum for water in the SWIR band. This molecular intrinsic property suggests the optimal wavelength for high-sensitive PAI of collagen-based samples. Results: PA absorption spectral peaks of collagen were found at wavelengths of 1200, 1550, and 1700 nm. Thereby, the PA signal increased by up to five times compared with the wavelength commonly used in collagen PAI. We applied a pulsed fiber laser with a center wavelength of 1560 nm, and the three-dimensional PA image of a collagen patch was obtained. Conclusions: The effective PA absorption spectrum contributes to the improvement of the PA image sensitivity by presenting the optimal wavelength of the target samples.

Corneal collagen cross-linking (CXL) strengthens the biomechanical properties of damaged corneas. Quantifying the changes of stiffness due to different CXL protocols is difficult, especially in vivo. A noninvasive elastic wave-based optical coherence elastography system was developed to construct in vivo corneal elasticity maps by excitation of air puff. Biomechanical differences were compared for rabbit corneas given three different CXL protocols while keeping the total energy delivered constant. The Young’s modulus was weaker in corneas treated with higher irradiance levels over shorter durations, and a slight increase of Young’s modulus was present in all groups one week after the recovery process. Due to the noninvasive nature and minimal force to generate corneal elastic waves, this technique has the potential for early detection and treatment of corneal diseases in clinic.

The aim of this study is to evaluate the osteoblastic collagen synthesis under mechanical stimulation using second-harmonic-generation (SHG) microscopy. We apply SHG microscopy to monitor the collagen fibers synthesized by osteoblast-like cells (MC3T3-E1) without the need for fixation and staining. To quantitatively evaluate the influence of mechanical stimulation on osteoblastic collagen synthesis, we compare SHG images of osteoblast-synthesized collagen fibers with and without a cyclic stretch stimulus applied using a lab-made stretching device. We acquire SHG images every 7 days for 3 weeks at different stimulus conditions (5 min/day and 3 h/day with a strain magnitude of 5% and a frequency of 0.5 Hz). Image analysis of the average SHG intensity indicates that the amount of osteoblastic collagen synthesis is significantly enhanced by the cyclic stretch compared with the nonstretched condition, while there is no significant difference between the two mechanical stimulation conditions. Furthermore, the maturity of the collagen fibers in the early stage of bone formation is not affected by the mechanical stimulation. The results can be used in bone regenerative medicine to apply feedback control of collagen synthesis by artificial stimulation.