Lipid content evaluation of Drosophila tumour associated haemocytes through Third Harmonic Generation measurements.

Non-linear microscopy is a powerful imaging tool to examine structural properties and subcellular processes of various biological samples. The competence of Third Harmonic Generation (THG) includes the label free imaging with diffraction-limited resolution and three-dimensional visualization with negligible phototoxicity effects. In this study, THG records and quantifies the lipid content of Drosophila haemocytes, upon encountering normal or tumorigenic neural cells, in correlation with their shape or their state. We show that the lipid accumulations of adult haemocytes are similar before and after encountering normal cells. In contrast, adult haemocytes prior to their interaction with cancer cells have a low lipid index, which increases while they are actively engaged in phagocytosis only to decrease again when haemocytes become exhausted. This dynamic change in the lipid accrual of haemocytes upon encountering tumour cells could potentially be a useful tool to assess the phagocytic capacity or activation state of tumour-associated haemocytes.

Age-dependent nuclear lipid droplet deposition is a cellular hallmark of aging in Caenorhabditis elegans.

Aging is the major risk factor for several life-threatening pathologies and impairs the function of multiple cellular compartments and organelles. Age-dependent deterioration of nuclear morphology is a common feature in evolutionarily divergent organisms. Lipid droplets have been shown to localize in most nuclear compartments, where they impinge on genome architecture and integrity. However, the significance of progressive nuclear lipid accumulation and its impact on organismal homeostasis remain obscure. Here, we implement non-linear imaging modalities to monitor and quantify age-dependent nuclear lipid deposition in Caenorhabditis elegans. We find that lipid droplets increasingly accumulate in the nuclear envelope, during aging. Longevity-promoting interventions, such as low insulin signaling and caloric restriction, abolish the rate of nuclear lipid accrual and decrease the size of lipid droplets. Suppression of lipotoxic lipid accumulation in hypodermal and intestinal nuclei is dependent on the transcription factor HLH-30/TFEB and the triglyceride lipase ATGL-1. HLH-30 regulates the expression of ATGL-1 to reduce nuclear lipid droplet abundance in response to lifespan-extending conditions. Notably, ATGL-1 localizes to the nuclear envelope and moderates lipid content in long-lived mutant nematodes during aging. Our findings indicate that the reduced ATGL-1 activity leads to excessive nuclear lipid accumulation, perturbing nuclear homeostasis and undermining organismal physiology, during aging.

Application of a Deep-Learning Technique to Non-Linear Images From Human Tissue Biopsies for Shedding New Light on Breast Cancer Diagnosis.

The development of label-free non-destructive techniques to be used as diagnostic tools in cancer research is of great importance for improving the quality of life for millions of patients. Previous studies have demonstrated that Third Harmonic Generation (THG) imaging could differentiate malignant from benign unlabeled human breast biopsies and distinguish the different grades of cancer. Towards the application of such technologies to clinic, in the present report, a deep learning technique was applied to THG images recorded from breast cancer tissues of grades 0, I, II, and III. By the implementation of a convolutional neural network (CNN) model, the differentiation of malignant from benign breast tissue samples and the discrimination of the different grades of cancer in a fast and accurate way were achieved. The obtained results provide a step ahead towards the use of optical diagnostic tools in conjunction with the CNN image classifier for the reliable and rapid malignancy diagnosis in clinic.

Application of Multicriteria Decision Analysis to Determine the Value of Prophylaxis Relative to On-Demand Treatment in Hemophilia A and Emicizumab versus Replacement Therapy in the Greek Healthcare Setting.

BACKGROUND AND OBJECTIVE: Hemophilia A (HA) is a rare disease that is characterized by congenital underproduction or dysfunction of the endogenous coagulation factor VIII (FVIII). The aim of the present study was to determine the value of prophylaxis versus on-demand treatment strategies for moderate to severe HA (MtSHA) patients and the value of emicizumab in the prophylaxis of MtSHA in Greece, compared with short half-life (SHL) FVIII and extended half-life (EHL) FVIII through multicriteria decision analysis (MCDA). METHODS: A literature review was performed to identify a set of criteria relevant to the therapeutic approaches and therapies under investigation. A performance matrix was populated by two literature reviews and meta-analyses. The criteria selected were hierarchically classified by allocating weights on a 0-100 scale. The performances of therapies were scored at the 100-point scale. The value judgments utilized for weighing and scoring were sourced via a survey among independent multidisciplinary system stakeholders. A linear additive value function was used for the calculation of total value estimates. RESULTS: The participants ranked 'annual number of bleedings per patient' and 'percentage of target joint bleeds' as the most important criteria, while the least important criterion was the 'annual treatment cost' for both assessments. Based on the weights elicited and the performance in each criterion, the overall value score was higher for prophylaxis treatment (58.27) compared with on-demand treatment (40.13). In the other comparison, the most valued treatment was emicizumab (77.05) followed by EHL FVIII (71.52) and SHL FVIII (19.88). According to the participants, the most important factors for managing MtSHA patients are those related to successful management of bleeding events given their contribution to improved quality of life (QoL) and reduced morbidity. CONCLUSIONS: This MCDA has shown that the prophylaxis strategy was perceived as a more valuable option for managing MtSHA patients when compared with the on-demand strategy. Moreover, emicizumab adds higher value and improves patient QoL compared with replacement therapy for MtSHA in Greece. Emicizumab addresses important unmet needs due to its improved efficacy and mode of administration.

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

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.

Polarization-dependent second-harmonic generation for collagen-based differentiation of breast cancer samples.

Nonlinear optical imaging techniques have been widely used to reveal biological structures for accurate diagnosis at the cellular as well as the tissue level. In the present study, polarization-dependent second-harmonic generation (PSHG) was used to determine collagen orientation in breast cancer biopsy tissues (grades 0, I, II and III). The obtained data were processed using fast Fourier transform (FFT) analysis, while second-harmonic generation (SHG) anisotropy and the "ratio parameter" values were also calculated. Such measurements were shown to be able to distinguish collagen structure modifications in different cancer grades tested. The analysis presented herein suggests that PSHG imaging could provide a quantitative evaluation of the tumor state and the distinction of malignant from benign breast tissues. The obtained results also allowed the development of a biophysical model, which can explain the aforementioned differentiations and is in agreement with the simulations relating the SHG anisotropy values with the mechanical tension applied to the collagen during cancer progression. The current approach could be a step forward for the development of new, nondestructive, label free optical diagnostic tools for cancer reducing the need of recalls and unnecessary biopsies, while potentially improving cancer detection rates.

Third Harmonic Generation microscopy distinguishes malignant cell grade in human breast tissue biopsies.

The ability to distinguish and grade malignant cells during surgical procedures in a fast, non-invasive and staining-free manner is of high importance in tumor management. To this extend, Third Harmonic Generation (THG), Second Harmonic Generation (SHG) and Fourier-Transform Infrared (FTIR) spectroscopy were applied to discriminate malignant from healthy cells in human breast tissue biopsies. Indeed, integration of non-linear processes into a single, unified microscopy platform offered complementary structural information within individual cells at the submicron level. Using a single laser beam, label-free THG imaging techniques provided important morphological information as to the mean nuclear and cytoplasmic area, cell volume and tissue intensity, which upon quantification could not only distinguish cancerous from benign breast tissues but also define disease severity. Simultaneously, collagen fibers that could be detected by SHG imaging showed a well structured continuity in benign tumor tissues, which were gradually disoriented along with disease severity. Combination of THG imaging with FTIR spectroscopy could provide a clearer distinction among the different grades of breast cancer, since FTIR analysis showed increased lipid concentrations in malignant tissues. Thus, the use of non-linear optical microscopy can be considered as powerful and harmless tool for tumor cell diagnostics even during real time surgery procedures.

Combined multiphoton fluorescence microscopy and photoacoustic imaging for stratigraphic analysis of paintings.

We demonstrate the effective combination of multiphoton and photoacoustic (PA) imaging for the high-resolution stratigraphic analysis of multilayered art objects with emphasis on paintings. A novel convolution-based algorithm is additionally applied for the precise discrimination of nonlinear signals, providing valuable information in regard to the thickness and composition of successive varnish and paint layers in the mock-up samples. On the other hand, PA contrast complements the extracted data by revealing well-hidden graphite underdrawings below the paint at high sensitivity levels. The final composite images are directly compared with cross-sectional brightfield observations, validating the capabilities of the bimodal diagnosis in terms of measurement accuracy and contrast specificity. The presented hybrid diagnostic approach has the potential to optimize delicate interventions in works of art such as the selective removal of aged materials, thus promoting a significantly improved restoration outcome.

Detection of the T cell activation state using nonlinear optical microscopy.

The ability to monitor the activation state of T-cells during immunotherapy is of great importance. Although specific activation markers do exist, their abundance and complicated regulation cannot definitely define the activation state of the cells. Previous studies have shown that Third Harmonic Generation (THG) imaging could distinguish between activated versus resting microglia and healthy versus cancerous cells, mainly based on their lipid-body profiles. In the present study, mitogen or antigen-stimulated T-cells were subjected to THG imaging microscopy. Qualitative and quantitative analysis showed statistically significant increase of THG mean area and intensity in activated versus resting T-cells. The connection of THG imaging to chemical information was achieved using Raman spectroscopy, which showed significant differences between the activation processes and controls, correlating of THG signal area with cholesterol and lipid compounds, but not with triglycerides. The obtained results suggested a potential employment of nonlinear microscopy in evaluating of T-cell activation, which is expected to be largely appreciated in the clinical practice.

Differential integrin expression in pre-implantation embryos developing under in vivo and in vitro conditions.

Implantation failure is a major problem in human assisted reproduction, which persists regardless the optimization of endometrial receptivity and selection of genetically and morphologically healthy embryos. Since embryo-endometrium interaction depends on cell junctional, cell adhesion and cell-substratum adhesion molecules, the present study inquired whether in vitro growing murine embryos display similar to the in vivo growing embryos patterns of adhesion molecules. To this extend aVb3 expression and distribution in zygotes and 2-cell stage embryos were studied. The results demonstrated that only the in vivo growing embryos displayed specifically polarized aVb3 distribution, indicating their potential successful interaction with endometrium. Based on previous studies showing that L-carnitine (L-Cn) could affect embryonic development, it was demonstrated that the addition of L-Cn to the culture medium, could lead the in vitro growing embryos to acquire aVb3 expression and distribution similar to the in vivo growing embryos. Visualization of the effect of L-Cn using third harmonic generation imaging showed decreased lipid droplet levels in 2-cell-stage embryos, observation that correlates with an active energetic state of the growing embryos. Thus, the application of L-Cn to the culture medium could assist pre-implantation-state embryos to acquire aVb3 expression and distribution similar to the in vivo developing conditions.

Optical coherence tomography and non-linear microscopy for paintings - a study of the complementary capabilities and laser degradation effects.

This paper examines for the first time the potential complementary imaging capabilities of Optical coherence tomography (OCT) and non-linear microscopy (NLM) for multi-modal 3D examination of paintings following the successful application of OCT to the in situ, non-invasive examination of varnish and paint stratigraphy of historic paintings and the promising initial studies of NLM of varnish samples. OCT provides image contrast through the optical scattering and absorption properties of materials, while NLM provides molecular information through multi-photon fluorescence and higher harmonics generation (second and third harmonic generation). OCT is well-established in the in situ non-invasive imaging of the stratigraphy of varnish and paint layers. While NLM examination of transparent samples such as fresh varnish and some transparent paints showed promising results, the ultimate use of NLM on paintings is limited owing to the laser degradation effects caused by the high peak intensity of the laser source necessary for the generation of non-linear phenomena. The high intensity normally employed in NLM is found to be damaging to all non-transparent painting materials from slightly scattering degraded varnish to slightly absorbing paint at the wavelength of the laser excitation source. The results of this paper are potentially applicable to a wide range of materials given the diversity of the materials encountered in paintings (e.g. minerals, plants, insects, oil, egg, synthetic and natural varnish).

Distinction between breast cancer cell subtypes using third harmonic generation microscopy.

Third Harmonic Generation (THG) microscopy as a non-invasive, label free imaging methodology, allows linkage of lipid profiles with various breast cancer cells. The collected THG signal arise mostly from the lipid droplets and the membrane lipid bilayer. Quantification of THG signal can accurately distinguish HER2-positive cells. Further analysis using Fourier transform infrared (FTIR) spectra reveals cancer-specific profiles, correlating lipid raft-corresponding spectra to THG signal, associating thus THG to chemical information. THG imaging of a cancer cell.

Ectopic fat deposition contributes to age-associated pathology in Caenorhabditis elegans.

Age-dependent collapse of lipid homeostasis results in spillover of lipids and excessive fat deposition in nonadipose tissues. Ectopic fat contributes to lipotoxicity and has been implicated in the development of a metabolic syndrome that increases risk of age-associated diseases. However, the molecular mechanisms coupling ectopic fat accumulation with aging remain obscure. Here, we use nonlinear imaging modalities to visualize and quantify age-dependent ectopic lipid accumulation in Caenorhabditis elegans We find that aging is accompanied by pronounced deposition of lipids in nonadipose tissues, including the nervous system. Importantly, interventions that promote longevity such as low insulin signaling, germ-line loss, and dietary restriction, which effectively delay aging in evolutionary divergent organisms, diminish the rate of ectopic fat accumulation and the size of lipid droplets. Suppression of lipotoxic accumulation of fat in heterologous tissues is dependent on helix-loop-helix (HLH)-30/transcription factor EB (TFEB) and autophagy. Our findings in their totality highlight the pivotal role of HLH-30/TFEB and autophagic processes in the maintenance of lipid homeostasis during aging, in addition to establishing nonlinear imaging as a powerful tool for monitoring ectopic lipid droplet deposition in vivo.

Polarization Second Harmonic Generation Discriminates Between Fresh and Aged Starch-Based Adhesives Used in Cultural Heritage.

In this work, we report that polarization second harmonic generation (PSHG) microscopy, commonly used in biomedical imaging, can quantitatively discriminate naturally aged from fresh starch-based glues used for conservation or restoration of paintings, works of art on paper, and books. Several samples of fresh and aged (7 years) flour and starch pastes were investigated by use of PSHG. In these types of adhesives, widely used in cultural heritage conservation, second harmonic generation (SHG) contrast originates primarily from the starch granules. It was found that in aged glues, the starch SHG effective orientation (SHG angle, θ) shifts to significantly higher values in comparison to the fresh granules. This shift is attributed to the different degree of granule hydration between fresh and aged adhesives. Thus noninvasive high-resolution nonlinear scattering can be employed to detect and quantify the degree of deterioration of restoration adhesives and to provide guidance toward future conservation treatments.

l-Carnitine affects preimplantation embryo development toward infertility in mice.

l-Carnitine (l-Cn), despite the beneficial role as energy-generating substance delivering long-chain fatty acids to the ß-oxidation pathway in mitochondria, has been accused to cause an endometriosis-like state to BALB/c mice manifested by increased inflammatory cytokines in serum and peritoneal fluid, accumulation of immune cells in the peritoneal cavity and uterine walls and most importantly, correlating to infertility. Exploring this type of infertility, the effect of l-Cn on preimplantation embryo development, ovarian integrity and systemic maternal immunity was studied. Using nonlinear microscopy analysis, which was shown to be a powerful tool for determining embryo quality by quantitatively estimating the lipid body (LB) content of the cells, it was shown that in vitro and in vivo administration of l-Cn significantly decreased LB mean area in zygotes. Daily intraperitoneal administration of 2.5mg l-Cn for 3, 4 and 7days to mice significantly decreased the percent of normal zygotes. However, only the 7-day treatment persisted by affecting 2- and 8-cell stage embryos, while almost abolishing blastocyst development. Such effects were accompanied by abnormal ovarian histology, showing increased numbers of corpora luteus and elevated progesterone concentration in the serum. In addition, it was shown that the 7-day l-Cn treatment pushed maternal systemic immunity toward inflammation and immunosuppression by increasing CD11b-, CD25- and CD11bGr1-positive cells in spleen, which opposed the necessity for immunostimulation at these early stages of pregnancy. In conclusion, the results presented here demonstrated that elevated doses of l-Cn affect early stages of embryo development, leading to infertility.

Imaging ectopic fat deposition in Caenorhabditis elegans muscles using nonlinear microscopy.

The elucidation of the molecular mechanisms that lead to the development of metabolic syndrome, a complex of pathological conditions including type-2 diabetes, hypertension, and cardiovascular diseases, is an important issue with high biological significance and requires accurate methods capable of monitoring lipid storage distribution and dynamics in vivo. In this study, the nonlinear phenomena of second and third harmonic generation (SHG, THG) have been employed simultaneously as label-free, nondestructive diagnostic techniques, for the monitoring and the complementary three-dimensional (3D) imaging and analysis of the muscular areas and the lipid content localization. THG microscopy was used as a quantitative tool in order to record the accumulation of lipids in nonadipose tissues in the pharyngeal muscles of 18 Caenorhabditis elegans (C. elegans) specimens, while the SHG imaging provided the detailed anatomical information about the structure of the muscles. The ectopic accumulation of fat on the pharyngeal muscles increases in wild-type (N2) C. elegans between 1 and 9 days of adulthood. This suggests a correlation of ectopic fat accumulation with the process of aging. Our results can contribute to the unraveling of the link between the deposition of ectopic fat and aging, but mainly to the validation of SHG and THG microscopy modalities as new, noninvasive tools to localize and quantify selectively lipid formation and distribution.

Assessment of in-depth degradation of artificially aged triterpenoid paint varnishes using nonlinear microscopy techniques.

The present work investigates the applicability of nonlinear imaging microscopy for the precise assessment of degradation of the outer protective layers of painted artworks as a function of depth due to aging. Two fresh and artificially aged triterpenoid varnishes, dammar and mastic, were tested. Nonlinear imaging techniques have been employed as a new diagnostic tool for determination of the exact thickness of the affected region due to artificial aging of the natural varnishes. The measured thicknesses differ from the calculated mean penetration depths of the samples. These nondestructive, high resolution modalities are valuable analytical tools for aging studies and they have the potential to provide unique in-depth information. Single photon laser induced fluorescence measurements and Raman spectroscopy were used for the integrated investigation and analysis of aging effects in varnishes.

Label-free imaging of lipid depositions in C. elegans using third-harmonic generation microscopy.

Elucidation of the molecular mechanisms regulating lipid storage and metabolism is essential for mitigating excess adiposity and obesity, which has been associated with increased prevalence of severe pathological conditions such as cardiovascular disorders and type II diabetes, worldwide. However, imaging fatty acid distribution and dynamics in vivo, at the cellular or organismal level is challenging. We developed a label-free method for visualizing lipid depositions in vivo, based on third harmonic generation (THG) microscopy. THG imaging requires a single pulsed-laser light source, alleviating the technical challenges of implementing coherent anti-Stokes Raman scattering spectroscopy (CARS) to detect fat stores in living cells. We demonstrate that THG can be used to efficiently and reliably visualize lipid droplets in Caenorhabditis elegans. Thus, THG microscopy offers a versatile alternative to fluorescence and dye-based approaches for lipid biology research.

Nonlinear microscopy techniques for assessing the UV laser polymer interactions.

A new diagnostic approach for assessing the in-depth laser induced modifications upon ultraviolet polymer irradiation is presented. The methodology relies on the observation of morphological alterations in the bulk material (Paraloid B72) by using third harmonic generation. This non destructive methodology allows the detailed and accurate imaging of the structurally laser modified zone extent in the vicinity of the irradiated area. Additionally, for the first time, the visualization and quantitative determination of the contour of the laser-induced swelling/bulk material interface is reported. The observed polymer surface swelling following single-pulse KrF laser irradiation at sub-ablation fluences is interpreted in the context of a model for laser-induced bubble formation due to droplet explosion mechanism.

Femtosecond laser nanosurgery of sub-cellular structures in HeLa cells by employing Third Harmonic Generation imaging modality as diagnostic tool.

Femtosecond laser assisted nanosurgery of microscopic biological specimens is a relatively new technique which allows the selective disruption of sub-cellular structures without causing any undesirable damage to the surrounding regions. The targeted structures have to be stained in order to be clearly visualized for the nanosurgery procedure. However, the validation of the final nanosurgery result is difficult, since the targeted structure could be simply photobleached rather than selectively destroyed. This fact comprises a main drawback of this technique. In our study we employed a multimodal system which integrates non-linear imaging modalities with nanosurgery capabilities, for the selective disruption of sub-cellular structures in HeLa cancer cells. Third Harmonic Generation (THG) imaging modality was used as a tool for the identification of structures that were subjected to nanosurgery experiments. No staining of the biological samples was required, since THG is an intrinsic property of matter. Furthermore, cells' viability after nanosurgery processing was verified via Two Photon Excitation Fluorescence (TPEF) measurements.