[This corrects the article DOI: 10.3389/fnagi.2023.1161847.].

Second harmonic generation (SHG) allows for the examination of collagen structure in collagenous tissues. Collagen is a fibrous protein found in abundance in the human body, present in bones, cartilage, the skin, and the cornea, among other areas, providing structure, support, and strength. Its structural arrangement is deeply intertwined with its function. For instance, in the cornea, alterations in collagen organization can result in severe visual impairments. Using SHG imaging, various metrics have demonstrated the potential to study collagen organization. The discrimination between healthy, keratoconus, and crosslinked corneas, assessment of injured tendons, or the characterization of breast and ovarian tumorous tissue have been demonstrated. Nevertheless, these metrics have not yet been objectively evaluated or compared. A total of five metrics were identified and implemented from the literature, and an additional approach adapted from texture analysis was proposed. In this study, we analyzed their effectiveness on a ground-truth set of artificially generated fibrous images. Our investigation provides the first comprehensive assessment of the performance of multiple metrics, identifying both the strengths and weaknesses of each approach and providing valuable insights for future applications of SHG imaging in medical diagnostics and research.

There is a pressing need for novel diagnostic and progression biomarkers of neurodegeneration. However, the inability to determine disease duration and stage in patients with Alzheimer’s disease (AD) hinders their discovery. Because animal models of disease allow us to circumvent some of these limitations, they have proven to be of paramount importance in clinical research. Due to the clear optics of the eye, the retina combined with optical coherence tomography (OCT) offers the perfect opportunity to image neurodegeneration in the retina in vivo, non-invasively, directly, quickly, and inexpensively. Based on these premises, our group has worked towards uncovering neurodegeneration-associated changes in the retina of the triple-transgenic mouse model of familial AD (3×Tg-AD). In this work, we present an overview of our work on this topic. We report on thickness variations of the retina and retinal layers/layer aggregates caused by healthy aging and AD-like conditions and discuss the implications of focusing research efforts solely on retinal thickness. We explore what other information is embedded in the OCT data, extracted based on texture analysis and deep-learning approaches, to further identify biomarkers that could be used for early detection and diagnosis. We were able to detect changes in the retina of the animal model of AD as early as 1 month of age. We also discuss our work to develop an optical coherence elastography system to measure retinal elasticity, which can be used in conjunction with conventional OCT. Finally, we discuss the potential application of these technologies in human patients and the steps needed to make OCT a helpful screening tool for the detection of neurodegeneration.

Second-harmonic generation (SHG) is uniquely capable of imaging collagen non-invasively with high-resolution, making it ideal to evaluate tissue organization in health and disease. For this, quantitative data analysis is essential. Different approaches have been proposed to quantify tissue organization from SHG images. Nevertheless, these methods have never been objectively evaluated or compared. In this study, we performed a comprehensive analysis on the performance of different metrics in computer-generated SHG images with increasing levels of disorganization to evaluate the advantages and limitations of each approach.

Supplementary Figure 3 - Expression of VEGFR-1 and IL-1ß in TAMs in PAN02 tumors.

Supplementary Table 1: Protein quantification of cytokines in tumors and plasma of WT and Flt1TK-/- obese using ELISA; Supplementary Table 2: Quantification of the expression of IL-1ÃŽÃ,² and VEGFR-1 in TAMs from PAN02 tumors by mmunofluorescence.

Animal models of disease are paramount to understand retinal development, the pathophysiology of eye diseases, and to study neurodegeneration using optical coherence tomography (OCT) data. In this study, we present a comprehensive normative database of retinal thickness in C57BL6/129S mice using spectral-domain OCT data. The database covers a longitudinal period of 16 months, from 1 to 16 months of age, and provides valuable insights into retinal development and changes over time. Our findings reveal that total retinal thickness decreases with age, while the thickness of individual retinal layers and layer aggregates changes in different ways. For example, the outer plexiform layer (OPL), photoreceptor inner segments (ILS), and retinal pigment epithelium (RPE) thickened over time, whereas other retinal layers and layer aggregates became thinner. Additionally, we compare the retinal thickness of wild-type (WT) mice with an animal model of Alzheimer's disease (3 × Tg-AD) and show that the transgenic mice exhibit a decrease in total retinal thickness compared to age-matched WT mice, with statistically significant differences observed at all evaluated ages. This normative database of retinal thickness in mice will serve as a reference for future studies on retinal changes in neurodegenerative and eye diseases and will further our understanding of the pathophysiology of these conditions.

The biomechanical properties of the human skin are intrinsically correlated with changes associated with pathological conditions, aging, and hydration. Quantitative measurements can improve diagnostic tools, treatments, and cosmetic product evaluation. Using optical coherence elastography (OCE), an emerging imaging modality combining optical coherence tomography (OCT) with a localized excitation source to induce mechanical disturbances, a quantitative evaluation of tissue biomechanics can be achieved. OCE complements the structural information with elasticity data to attain a complete overview of skin status. In this study, we employed a home-built OCE system, combining a swept-source OCT system with a piezoelectric actuator for tissue displacement, to evaluate changes to the skin biomechanical properties due to the application of an anti-aging cream. Skin elasticity was monitored for a total of five weeks. Anti-aging cream was applied daily for four weeks. OCE measurements continued for one additional week to assess the effect of cream application interruption. Three female volunteers were included in this proof-of-principle investigation. Their counter-arm was used as control. Although no statistical significance was reached, a decrease in skin Young’s modulus was observed with the cream application, indicating an increase in skin elasticity.

Robust methods to compute tissue displacements in optical coherence elastography (OCE) data are paramount, as they play a significant role in the accuracy of tissue elastic properties estimation. In this study, the accuracy of different phase estimators was evaluated on simulated OCE data, where the displacements can be accurately set, and on real data. Displacement (∆d) estimates were computed from (i) the original interferogram data (Δφori) and two phase-invariant mathematical manipulations of the interferogram: (ii) its first-order derivative (Δφd) and (iii) its integral (Δφint). We observed a dependence of the phase difference estimation accuracy on the initial depth location of the scatterer and the magnitude of the tissue displacement. However, by combining the three phase-difference estimates (Δdav), the error in phase difference estimation could be minimized. By using Δdav, the median root-mean-square error associated with displacement prediction in simulated OCE data was reduced by 85% and 70% in data with and without noise, respectively, in relation to the traditional estimate. Furthermore, a modest improvement in the minimum detectable displacement in real OCE data was also observed, particularly in data with low signal-to-noise ratios. The feasibility of using Δdav to estimate agarose phantoms' Young's modulus is illustrated.

We present the initial stages of development of a Finite Element Method-based time-dependent elastic numerical model which seeks to support the employment of our Optical Coherence Elastography system for assessing murine retinal elasticity. The current model is able to reconstruct displacement maps in both homogeneous and heterogeneous domains with errors up to a few hundredths relatively to a known exact displacement map, within 1 millisecond. The results demonstrate the robustness of the numerical algorithm under different elastic domains, and model parametrization with real Optical Coherence Elastography data is already in progress.

Effects of VEGFR-1 signaling ablation on body weight gain, immune cell infiltration and vasculature in adipose tissues during obesity.

Two-photon imaging (TPI) microscopy, namely, two-photon excited fluorescence (TPEF), fluorescence lifetime imaging (FLIM), and second-harmonic generation (SHG) modalities, has emerged in the past years as a powerful tool for the examination of biological tissues. These modalities rely on different contrast mechanisms and are often used simultaneously to provide complementary information on morphology, metabolism, and structural properties of the imaged tissue. The cornea, being a transparent tissue, rich in collagen and with several cellular layers, is well-suited to be imaged by TPI microscopy. In this review, we discuss the physical principles behind TPI as well as its instrumentation. We also provide an overview of the current advances in TPI instrumentation and image analysis. We describe how TPI can be leveraged to retrieve unique information on the cornea and to complement the information provided by current clinical devices. The present state of corneal TPI is outlined. Finally, we discuss the obstacles that must be overcome and offer perspectives and outlooks to make clinical TPI of the human cornea a reality.

In this paper we address the numerical solution of the inverse elastography problem, from the knowledge of the excitation field on the boundary and the displacement field in a grid of points within the domain. We suggest using a representation of the solution by the method of fundamental solutions and using a Newton-type method to iteratively approximate the Lain coefficients of the medium from elastography displacement measurements. We consider a toy model to illustrate the performance of the method.

We present an Optical Coherence Elastography (OCE) system, based on a swept-source Optical Coherence Tomography (OCT) setup, and evaluate its performance in terms of phase stability and minimum detectable displacement. The ability to record sub-pixel movements in samples under dynamic conditions was also assessed. The OCE system has a time stability of 396.9 +/- 46.7 ps. The phase stability, given by the standard deviation of the measured phase difference, was 72.44 mrad, which corresponds to a minimum detectable displacement of 6.11 nm. Tests showed that the OCE system can detect and measure sub-pixel movements in samples under dynamic mechanical excitation.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma (GBM) trials. Here, we show that regulatory T (Treg) cells play a key role in GBM resistance to ICBs in experimental gliomas. Targeting glucocorticoid-induced TNFR-related receptor (GITR) in Treg cells using an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, alleviates Treg cell-mediated suppression of anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. The reprogrammed GBM-infiltrating Treg cells express genes associated with a Th1 response signature, produce IFNγ, and acquire cytotoxic activity against GBM tumor cells while losing their suppressive function. αGITR and αPD1 antibodies increase survival benefit in three experimental GBM models, with a fraction of cohorts exhibiting complete tumor eradication and immune memory upon tumor re-challenge. Moreover, αGITR and αPD1 synergize with the standard of care treatment for newly-diagnosed GBM, enhancing the cure rates in these GBM models.

The human cornea is the tissue of the eye that contributes most to its refractive power. A healthy tissue is highly important to maintain visual acuity. In fact, diseases affecting the cornea are one of the major causes of blindness. Therefore, efficient methods to analyze the status of the healthy and diseased cornea are of outmost importance. Current clinical devices are, however, mostly limited to the morphological analysis of the tissue. The simultaneous analysis of tissue morphology, metabolism, and stromal structural organization using two-photon imaging (TPI) could improve corneal examination. In this study, two systems with TPI capabilities equipped with ultra-short near-infrared Ti:sapphire lasers were optimized for corneal imaging and their advantages for tissue examination demonstrated. Additionally, we show that by using TPI, disease diagnosis, follow-up after medical procedures, and corneal evaluation prior to transplantation could be improved. Thus, a future clinical device based on TPI could enhance the current state corneal examination and improve patient diagnosis and care.

The diagnostic possibilities of multiphoton tomography (MPT) in dermatology have already been demonstrated. Nevertheless, the analysis of MPT data is still time-consuming and operator dependent. We propose a fully automatic approach based on convolutional neural networks (CNNs) to fully realize the potential of MPT. In total, 3,663 MPT images combining both morphological and metabolic information were acquired from atopic dermatitis (AD) patients and healthy volunteers. These were used to train and tune CNNs to detect the presence of living cells, and if so, to diagnose AD, independently of imaged layer or position. The proposed algorithm correctly diagnosed AD in 97.0 ± 0.2% of all images presenting living cells. The diagnosis was obtained with a sensitivity of 0.966 ± 0.003, specificity of 0.977 ± 0.003 and F-score of 0.964 ± 0.002. Relevance propagation by deep Taylor decomposition was used to enhance the algorithm’s interpretability. Obtained heatmaps show what aspects of the images are important for a given classification. We showed that MPT imaging can be combined with artificial intelligence to successfully diagnose AD. The proposed approach serves as a framework for the automatic diagnosis of skin disorders using MPT.

Femtosecond-laser pulses can create transient holes in the membrane of a cell, making it briefly permeable to genetic macromolecules. This is a highly effective method for cell transfection and reprogramming. For sufficiently high irradiance values, the laser radiation leads to plasma formation through multiphoton ionization and a subsequent formation of gas-filled bubbles which are shortly visible after the irradiation. While this bubble formation is well known, the underlying microscopic processes, the optimal bubble size and duration which indicate the transient hole formation are less clear. The correspondence between bubble formation and successful optoporation is further complicated by the fact that the formation greatly depends on the irradiated cell position and laser irradiation parameters (power, exposure time). We have investigated the formation of bubbles resulting from short pulse irradiation with two commercial Ti:sapphire lasers using a high-speed camera. Higher laser powers and longer exposure times yielded bigger bubbles which took longer to collapse. Additionally, a correlation between the bubble characteristics and the cell's metabolism and post-optoporation viability was found. These results can help to optimize the laser parameters for efficient optoporation and high post-treatment cell viability as well as to shine light on the microscopic excitation processes behind the bubble formation.