In the context of this work, a prototype hybrid photoacoustic (PA) and optical system for the on-line monitoring of laser cleaning procedures is presented. The developed apparatus has enabled the detection of MHz frequency range acoustic waves generated during the laser ablation process. The intrinsically generated PA signals combined with high resolution optical images provide the opportunity to follow the cleaning process accurately and in real time. Technical mock-ups have been used to demonstrate the potential of this novel technique with emphasis given to applications that refer to the restoration of Cultural Heritage (CH) surfaces. Towards this purpose, the real time monitoring of the laser assisted removal of unwanted encrustation from stonework has been achieved using IR and UV wavelengths. This novel approach has allowed for the precise determination of the critical number of laser pulses required for the elimination of the encrustation layer, while highlighting the dominant ablation mechanisms according to the irradiation wavelength. The promising results obtained using the prototype hybrid PA and optical system can open up new perspectives in the monitoring of laser cleaning interventions, promoting an improved restoration outcome.

We describe a customizable and cost-effective light sheet microscopy (LSM) platform for rapid three-dimensional imaging of protein dynamics in small model organisms. The system is designed for high acquisition speeds and enables extended time-lapse in vivo experiments when using fluorescently labeled specimens. We demonstrate the capability of the setup to monitor gene expression and protein localization during ageing and upon starvation stress in longitudinal studies in individual or small groups of adult Caenorhabditis elegans nematodes. The system is equipped to readily perform fluorescence recovery after photobleaching (FRAP), which allows monitoring protein recovery and distribution under low photobleaching conditions. Our imaging platform is designed to easily switch between light sheet microscopy and optical projection tomography (OPT) modalities. The setup permits monitoring of spatio-temporal expression and localization of ageing biomarkers of subcellular size and can be conveniently adapted to image a wide range of small model organisms and tissue samples.

We investigated the capacity of normalized transillumination methods in imaging fluorescent proteins in visible light, in phantoms and in live mice. We demonstrate significant imaging improvements over epi-illumination imaging, as commonly applied today. Of particular importance is the significant betterment in contrast achieved, due to minimization of the influence of autofluorescence and the enhancement of depth sensitivity compared to epi-illumination imaging. Although normalized transillumination does not match the accuracy and quantification capacity of tomographic imaging, it nevertheless attains key advantages over other planar optical imaging methods while preserving implementation simplicity. Due to the balance achieved between simplicity and accuracy, normalized transillumination approaches could serve as an important alternative molecular imaging method.

Light sheet fluorescence microscopy techniques have revolutionized biological microscopy enabling low-phototoxic long-term 3D imaging of living samples. Although there exist many light sheet microscopy (LSM) implementations relying on fluorescence, just a few works have paid attention to the laser elastic scattering source of contrast available in every light sheet microscope. Interestingly, elastic scattering can potentially disclose valuable information from the structure and composition of the sample at different spatial scales. However, when coherent scattered light is detected with a camera sensor, a speckled intensity is generated on top of the native imaged features, compromising their visibility. In this work, we propose a novel light sheet based optical setup which implements three strategies for dealing with speckles of elastic scattering images: (i) polarization filtering; (ii) reducing the temporal coherence of the excitation laser light; and, (iii) reducing the spatial coherence of the light sheet. Finally, we show how these strategies enable pristine light-sheet elastic-scattering imaging of structural features in challenging biological samples avoiding the deleterious effects of speckle, and without relying on, but complementing, fluorescent labelling.

We demonstrate the development and application of a prototype hybrid microscopy system integrating autofluorescence (AF) and photoacoustic (PA) label-free contrast modes, for the differentiation of ocular tumors in human surgical biopsies. Hybrid imaging was performed in conjunctival nevi and uveal melanomas tissue sections to acquire quantified data for each molecular background. The AF and PA signals were spatially correlated to establish a novel malignancy indicator that could detect melanomas with high accuracy (t-test; p < 0.01 ). The proposed methodology has the potential to simplify relevant diagnostic procedures and paves the way for the development of novel ophthalmoscopes aiming to the early diagnosis of ocular malignancies in a clinical setting.

Simultaneous detection of several biological processes in vivo is a common requirement in biomedical and biological applications, and in order to address this issue the use of multiple fluorophores is usually the method of choice. Existing methodologies however, do not provide quantitative feedback of multiple fluorophore concentrations in small animals in vivo when their spectra overlap, especially when imaging the whole body in 3D. Here we present an approach where a spectroscopic module has been implemented into a custom-built Fluorescence Molecular Tomography (FMT) system. In contrast with other multispectral approaches, this multimodal imaging system is capable of recording the fluorescence spectra from each illumination point during a tomographic measurement. In situ spectral information can thus be extracted and used to improve the separation of overlapping signals associated with different fluorophores. The results of this new approach tested on both in vitro and in vivo experiments are presented, proving that accurate recovery of fluorophore concentrations can be obtained from multispectral tomography data even in the presence of high autofluorescence.