We present a hybrid microscope combining multiphoton microscopy incorporating second-harmonic generation contrast and optical-resolution optoacoustic (photoacoustic) microscopy. We study the relative performance of the two systems and investigate the complementarity of contrast by demonstrating the label-free imaging capabilities of the hybrid microscope on zebrafish larvae ex vivo, concurrently visualizing the fish musculature and melanocytes. This implementation can prove useful in multiparametric microscopy studies, enabling broader information to be collected from biological specimens.

Biology requires observations at multiple geometrical scales, a feature that is not typically offered by a single imaging modality. We developed a hybrid optical system that not only provides different contrast modes but also offers imaging at different geometrical scales, achieving uniquely broad resolution and a 1000-fold volume sampling increase compared to volumes scanned by optical microscopy. The system combines optoacoustic mesoscopy, optoacoustic microscopy and two-photon microscopy, the latter integrating second and third harmonic generation modes. Label-free imaging of a mouse ear and zebrafish larva ex-vivo demonstrates the contrast and scale complementarity provided by the hybrid system. We showcase the superior anatomical orientation offered by the label-free capacity and hybrid operation, over fluorescence microscopy and the dynamic selection between field of view and resolution achieved, leading to new possibilities in biological visualization.

A novel, non-invasive, imaging methodology, based on the photoacoustic effect, is introduced in the context of artwork diagnostics with emphasis on the uncovering of hidden features such as underdrawings or original sketch lines in paintings. Photoacoustic microscopy, a rapidly growing imaging method widely employed in biomedical research, exploits the ultrasonic acoustic waves, generated by light from a pulsed or intensity modulated source interacting with a medium, to map the spatial distribution of absorbing components. Having over three orders of magnitude higher transmission through strongly scattering media, compared to light in the visible and near infrared, the photoacoustic signal offers substantially improved detection sensitivity and achieves excellent optical absorption contrast at high spatial resolution. Photoacoustic images, collected from miniature oil paintings on canvas, illuminated with a nanosecond pulsed Nd:YAG laser at 1064 nm on their reverse side, reveal clearly the presence of pencil sketch lines coated over by several paint layers, exceeding 0.5 mm in thickness. By adjusting the detection bandwidth of the optically induced ultrasonic waves, photoacoustic imaging can be used for looking into a broad variety of artefacts having diverse optical properties and geometrical profiles, such as manuscripts, glass objects, plastic modern art or even stone sculpture.

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.

Photoacoustic (PA) imaging is a novel, rapidly expanding diagnostic technique, which has been predominately developed in the context of contemporary biomedical research studies. In this review, we demonstrate how PA technologies can break through the barriers of biomedicine to find innovative applications in cultural heritage (CH) diagnostics and laser cleaning monitoring. Having over three orders of magnitude higher transmission through strongly scattering media, compared to light in the visible and near infrared, PA signals offer substantially improved detection sensitivity, providing optical absorption contrast at high spatial resolution. This unique combination of features is employed for establishing novel diagnostic methodologies aiming to uncover well-hidden features and provide structural information in multi-layered CH objects such as paintings and documents. Finally, we demonstrate that the PA effect can be successfully utilized for the reliable monitoring of laser cleaning interventions on stonework, allowing for a safe and well-controlled cleaning procedure which will safeguard CH objects’ original surfaces. Simplicity of implementation, effectiveness and low-cost features provided by the developed diagnostic and monitoring systems, highlight the rich potential of emerging PA technologies in CH studies and offer exciting possibilities for future implementations.