Age-dependent collapse of lipid homeostasis results in spillover of lipids and excessive fat deposition in non-adipose 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 ageing remain obscure. Here, we use nonlinear imaging modalities to visualize and quantify age-dependent ectopic lipid accumulation in Caenorhabditis elegans. We find that ageing is accompanied by pronounced deposition of lipids in non-adipose tissues, including the nervous system. Importantly, interventions that promote longevity such as low insulin signalling, germline loss and dietary restriction, which effectively delays ageing 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 HLH-30/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 ageing, in addition to establishing nonlinear imaging as a powerful tool for monitoring ectopic lipid droplet deposition in vivo.

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).

A range of sophisticated imaging techniques have been developed in recent years that can reveal the surface structure of cultural heritage objects with varying precision. In combination with various spectroscopic methods, they allow the study of the chemical composition of the object; thus, conclusions can be drawn about the origin of the object or its initial components, method, or time of creation, authenticity, mechanisms of degradation, and ways of further conservation. At present, different techniques can be applied to a wide range of cultural heritage objects, such as varnishes, paintings, archaeological objects, binding media, paper-based documents, parchments, marbles, frescoes, as well as various objects made of leather, fabric, stone, ceramics and glass, wood, or metal. One of the main needs in the study of cultural heritage (CH) is the transportability/portability of the research equipment, since many pieces under investigation cannot be moved to the laboratory, either because of their size, inseparability (for example, frescoes on walls, mural paintings in caves), or the threat of damage. In this work, we briefly overview the main optical- and laser-based methods used for the study of cultural heritage objects indicating the scope of their application, and we focus on the applications of non-linear microscopic methods for the investigation of a series of artifacts. We also discuss all the requirements for the construction of a prototype transportable non-linear optical system that will be used as a novel diagnostic tool for in situ studies of CH assets. The availability of such a transportable workstation will significantly improve the study and characterization of various types of CH objects and will constitute an extremely useful diagnostic tool for heritage scientists dealing with a variety of investigations.

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.