Bimodal optical and optoacoustic multiview microscope in the frequency-domain.

Hybrid fluorescence and optoacoustic microscopy systems have recently emerged as powerful imaging modalities concurrently capturing both radiative and non-radiative molecular relaxations in biological tissues. Nevertheless, such approaches provide limited information as specimens are imaged exclusively from one side, not permitting the acquisition of their full anatomical, structural, or functional features in multiple views of interest. Herein we present a bimodal optical and optoacoustic multiview (BOOM) cost-efficient microscope operating in the frequency-domain for the comprehensive label-free imaging of established and emerging model organisms. Thus, the capabilities of BOOM microscopy have been proven suitable for highly demanding observations in developmental biology and embryology.

Deep learning-assisted frequency-domain photoacoustic microscopy.

Frequency-domain photoacoustic microscopy (FD-PAM) constitutes a powerful cost-efficient imaging method integrating intensity-modulated laser beams for the excitation of single-frequency photoacoustic waves. Nevertheless, FD-PAM provides an extremely small signal-to-noise ratio (SNR), which can be up to two orders of magnitude lower than the conventional time-domain (TD) systems. To overcome this inherent SNR limitation of FD-PAM, we utilize a U-Net neural network aiming at image augmentation without the need for excessive averaging or the application of high optical power. In this context, we improve the accessibility of PAM as the system's cost is dramatically reduced, and we expand its applicability to demanding observations while retaining sufficiently high image quality standards.

High precision photoacoustic interferometer for the determination of the speed of sound in liquid media.

In this work, we introduce the concept and delineate the fundamental principles of photoacoustic interferometry (PAInt), aiming at the development of a novel methodology for the precise assessment of the speed of sound in liquid media. The PAInt apparatus integrates an intensity-modulated continuous wave laser beam at 20 MHz for the efficient generation of monochromatic photoacoustic wavefronts which interfere across the surface of a vertically displaced spherically focused piezoelectric element. In this context, the resulting interference pattern can reveal the acoustic wavelength in the liquid medium with remarkable accuracy, providing thus reliable estimations of the speed of sound in reference liquids (error ∼0.1%) such as distilled and sea water, acetonitrile, and ethanol.

Full image reconstruction in frequency-domain photoacoustic microscopy by means of a low-cost I/Q demodulator.

We present a full image reconstruction methodology in frequency-domain photoacoustic (PA) microscopy using a low-cost I/Q demodulator for the recording of the amplitude and phase of the signals. By modulating the intensity of a continuous-wave diode laser at 10 MHz, we have been able to provide accurate optical absorption images and surface reconstructions of phantom samples, comparing also the extracted results with standard time-domain approaches. The findings of the study in this Letter could be utilized towards the development of inexpensive PA microscopes with multispectral capabilities for a wide range of biomedical studies, requiring the sensitive detection of endogenous or exogenous absorbers in tissues.

A Cost-Efficient Multiwavelength LED-Based System for Quantitative Photoacoustic Measurements.

The unique ability of photoacoustic (PA) sensing to provide optical absorption information of biomolecules deep inside turbid tissues with high sensitivity has recently enabled the development of various novel diagnostic systems for biomedical applications. In many cases, PA setups can be bulky, complex, and costly, as they typically require the integration of expensive Q-switched nanosecond lasers, and also presents limited wavelength availability. This article presents a compact, cost-efficient, multiwavelength PA sensing system for quantitative measurements, by utilizing two high-power LED sources emitting at central wavelengths of 444 and 628 nm, respectively, and a single-element ultrasonic transducer at 3.5 MHz for signal detection. We investigate the performance of LEDs in pulsed mode and explore the dependence of PA responses on absorber's concentration and applied energy fluence using tissue-mimicking phantoms demonstrating both optical absorption and scattering properties. Finally, we apply the developed system on the spectral unmixing of two absorbers contained at various relative concentrations in the phantoms, to provide accurate estimations with absolute deviations ranging between 0.4 and 12.3%. An upgraded version of the PA system may provide valuable in-vivo multiparametric measurements of important biomarkers, such as hemoglobin oxygenation, melanin concentration, local lipid content, and glucose levels.

Hybrid autofluorescence and photoacoustic label-free microscopy for the investigation and identification of malignancies in ocular biopsies.

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.

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.

Combined photoacoustic imaging to delineate the internal structure of paintings.

In this Letter, we present a combined photoacoustic imaging method, based on consecutive excitation using either the fundamental or the second-harmonic wavelength of a pulsed Nd:YAG laser for the stratigraphy of painted artworks. Near-infrared excitation was employed for the imaging of hidden underdrawings in mock-up samples, whereas visible light was used for the thickness mapping of the overlying paint through the detection of photoacoustic signal attenuation. The proposed methodology was proven effective in measuring thick and strongly absorbing layers, which would not be possible by means of other pure optical techniques, while also enabling the visualization of features underneath the painted surface. Such an implementation expands significantly the applicability of the previously presented photoacoustic technique, which was limited to point-measurements, and paves the way for novel application in historical and technical studies, as well as in documenting restoring operations.

Photoacoustic imaging methodology for the optical characterization of contact lenses.

We demonstrate photoacoustic microscopy as a metrology method for the optical characterization and quality control of contact lenses (CLs). Dual-wavelength excitation is applied to CLs tinted on both sides with two thin ink layers, each of them possessing distinctly different optical absorption properties. Thus, the method is capable of measuring the elevation maps of both CL surfaces during two subsequent imaging sessions and extracting the CL thickness, curvatures, and dioptric power. We show that such an easily implementable technique provides robust, high-precision, cost-effective three-dimensional imaging and characterization of both rigid and soft CLs, which renders it highly favorable for a broad range of applications.

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.

Hybrid photoacoustic and optical imaging of pigments in vegetative tissues.

Pigments in vegetative tissues have been a subject of intense research during the previous decades, since they play an active role in several molecular mechanisms regarding plants' physiology and function. Towards this direction, the imaging modality that has been extensively employed and represents the state of the art for mapping pigments' distribution is confocal microscopy. Despite the advantage of a high spatial resolution however, confocal microscopy provides a rather limited imaging depth and requires necessarily strong fluorescence properties from the specimen under observation. To overcome such limitations, we propose a hybrid, photoacoustic and optical imaging methodology for the delineation of various vegetative pigments, such as chlorophylls, anthocyanins and betalains in different plant species. The superior sensitivity and the high contrast complementarity of the hybrid technique, render it a powerful alternative to the conventional fluorescence imaging modalities, significantly expanding the current state of the art.

Hybrid multiphoton and optoacoustic microscope.

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.

Optoacoustic interferometric characterization system (OPTICS) for the evaluation of fuel quality through speed of sound measurements.

Ultrasonic techniques have been applied to assess crucial physical parameters in various types of fuels including the speed of sound (SoS), the bulk modulus and the acoustic attenuation coefficient. Such investigations may have important practical significance as the knowledge of fuel properties is directly related to the analysis of combustion characteristics, engine's overall performance and exhaust emission in the environment. Nevertheless, typical pulse-echo acoustic methods, require the exact determination of both acoustic source - reflector distance and the time of flight of a finite temporal width ultrasonic pulse, setting thus an upper limit as regards the accuracy of the measurements. To encounter these challenges, we present a novel technology implemented through a low-cost and potentially portable optoacoustic interferometric characterization system (OPTICS) for the investigation of SoS variations in common fuels including automotive diesel, hydrous ethanol and gasoline. At 25 °C, diesel/kerosene blends demonstrated a SoS variation ranging from 1322.91 m/s (0.6 diesel volume fraction) to 1349.79 m/s (diesel fuel only), whereas hydrous ethanol samples varied between 1199.92 m/s (0.95 ethanol volume fraction) to 1149.39 m/s (pure ethanol only). Finally, assessments for 95 and 100 research octane number (RON) gasoline blends showed a SoS range from 1134.42 m/s (RON 95) to 1159.86 m/s (RON 100). The high precision and repeatability (relative uncertainty: ∼10-4) of the performed SoS measurements in controlled samples, has demonstrated the promising potential of OPTICS for the evaluation of fuel physical properties as well as the potential detection of contamination with adulterants.

Stratigraphy of Fresco Paintings: A New Approach with Photoacoustic and SORS Imaging.

Photoacoustic (PA) imaging is a novel, powerful diagnostic technique utilized in different research fields. In particular, during recent years it has found several applications in Cultural Heritage (CH) diagnostics. PA imaging can be realized in transmittance or epi-illumination (reflectance) modes, obtaining variable levels of contrast and spatial resolution. In this work, we confirmed the applicability of the PA technique as a powerful tool for the imaging of one of the most challenging artwork objects, namely fresco wall paints, to obtain precise stratigraphic profiles in different layered fresco samples. In this regard, we studied some multi-layered fragments of the vault of San Giuseppe Church in Cagliari (1870 AD) and some mock-ups realized specifically to test the potentiality of this technique. Due to complex structures of the frescoes, we used the Spatially Off-set Raman Spectroscopy (SORS) technique to provide complementary information. The experimental results were in agreement for both techniques, even for the three-layered complex structure, and were confirmed with Scanning Electron Microscopy (SEM) analysis of cross-sections. The combined use of these two techniques proved useful to investigate detailed hidden information on the fresco samples.

Hybrid Autofluorescence and Optoacoustic Microscopy for the Label-Free, Early and Rapid Detection of Pathogenic Infections in Vegetative Tissues.

Agriculture plays a pivotal role in food security and food security is challenged by pests and pathogens. Due to these challenges, the yields and quality of agricultural production are reduced and, in response, restrictions in the trade of plant products are applied. Governments have collaborated to establish robust phytosanitary measures, promote disease surveillance, and invest in research and development to mitigate the impact on food security. Classic as well as modernized tools for disease diagnosis and pathogen surveillance do exist, but most of these are time-consuming, laborious, or are less sensitive. To that end, we propose the innovative application of a hybrid imaging approach through the combination of confocal fluorescence and optoacoustic imaging microscopy. This has allowed us to non-destructively detect the physiological changes that occur in plant tissues as a result of a pathogen-induced interaction well before visual symptoms occur. When broccoli leaves were artificially infected with Xanthomonas campestris pv. campestris (Xcc), eventually causing an economically important bacterial disease, the induced optical absorption alterations could be detected at very early stages of infection. Therefore, this innovative microscopy approach was positively utilized to detect the disease caused by a plant pathogen, showing that it can also be employed to detect quarantine pathogens such as Xylella fastidiosa.

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.

Agar Gel as a Non-Invasive Coupling Medium for Reflectance Photoacoustic (PA) Imaging: Experimental Results on Wall-Painting Mock-Ups.

The new reflectance set-up configuration extended the applicability of the photoacoustic (PA) imaging technique to art objects of any thickness and form. Until now, ultrasound gel or distilled water have been necessary as coupling mediums between the immersion-type transducer and the object's surface. These media can compromise the integrity of real artwork; therefore, known applications of reflectance PA imaging have been limited to only experimental mock-ups. In this paper, we evaluate an alternative non-invasive PA coupling medium, agar gel, applied in two layers of different consistency: first, rigid-for the protection of the object's surface, and second, fluid-for the transducer's immersion and movement. Agar gel is widely used in various conservation treatments on cultural heritage objects, and it has been proven to be safely applicable on delicate surfaces. Here, we quantify and compare the contrast and signal-to-noise ratio (SNR) of PA images, obtained in water and in agar gel on the same areas, at equal experimental conditions. The results demonstrate that the technique's performance in agar is comparable to that in water. The study uncovers the advanced potential of the PA approach for revealing hidden features, and is safely applicable for future real-case studies.

Imaging Parhyale hawaiensis embryogenesis with frequency domain photoacoustic microscopy: A novel tool in developmental biology.

We present the application of a low-cost frequency domain photoacoustic (FDPA) microscope for the label-free imaging of live developing embryos of the crustacean model organism Parhyale hawaiensis. By modulating the intensity of a continuous wave laser source at 9.5 MHz, we achieve the excitation of monochromatic PA waves, which are detected to provide amplitude and phase recordings. The data are subsequently processed to generate accurate maximum amplitude projection and surface reconstructions, delineating the morphological features of the embryos with high resolution and contrast. The findings of this study pave the way for the broader adoption of inexpensive PA diagnostic techniques in developmental biology, shedding light on various fundamental processes in established and emerging model organisms.

Hybrid confocal fluorescence and photoacoustic microscopy for the label-free investigation of melanin accumulation in fish scales.

Lower vertebrates, including fish, can rapidly alter skin lightness through changes in melanin concentration and melanosomes' mobility according to various factors, which include background color, light intensity, ambient temperature, social context, husbandry practices and acute or chronic stressful stimuli. Within this framework, the determination of skin chromaticity parameters in fish species is estimated either in specific areas using colorimeters or at the whole animal level using image processing and analysis software. Nevertheless, the accurate quantification of melanin content or melanophore coverage in fish skin is quite challenging as a result of the laborious chemical analysis and the typical application of simple optical imaging methods, requiring also to euthanize the fish in order to obtain large skin samples for relevant investigations. Here we present the application of a novel hybrid confocal fluorescence and photoacoustic microscopy prototype for the label-free imaging and quantification of melanin in fish scales samples with high spatial resolution, sensitivity and detection specificity. The hybrid images are automatically processed through optimized algorithms, aiming at the accurate and rapid extraction of various melanin accumulation indices in large datasets (i.e., total melanin content, melanophores' area, density and coverage) corresponding to different fish species and groups. Furthermore, convolutional neural network-based algorithms have been trained using the recorded data towards the classification of different scales' samples with high accuracy. In this context, we demonstrate that the proposed methodology may increase substantially the precision, as well as, simplify and expedite the relevant procedures for the quantification of melanin content in marine organisms.

Following the course of pre-implantation embryo patterning by non-linear microscopy.

Embryo patterning is subject to intense investigation. So far only large, microscopically obvious structures like polar body, cleavage furrow, pro-nucleus shape can be evaluated in the intact embryo. Using non-linear microscopic techniques, the present work describes new methodologies to evaluate pre-implantation mouse embryo patterning. Third Harmonic Generation (THG) imaging, by detecting mitochondrial/lipid body structures, could provide valuable and complementary information as to the energetic status of pre-implantation embryos, time evolution of different developmental stages, embryo polarization prior to mitotic division and blastomere equivalence. Quantification of THG imaging detected highest signalling in the 2-cell stage embryos, while evaluating a 12-18% difference between blastomeres at the 8-cell stage embryos. Such a methodology provides novel, non-intrusive imaging assays to follow up intracellular structural patterning associated with the energetic status of a developing embryo, which could be successfully used for embryo selection during the in vitro fertilization process.