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.

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.

The Temozolomide-Doxorubicin paradox in Glioblastoma in vitro-in silico preclinical drug-screening.

Adjuvant Temozolomide is considered the front-line Glioblastoma chemotherapeutic treatment; yet not all patients respond. Latest trends in clinical trials usually refer to Doxorubicin; yet it can lead to severe side-effects if administered in high doses. While Glioblastoma prognosis remains poor, little is known about the combination of the two chemotherapeutics. Patient-derived spheroids were generated and treated with a range of Temozolomide/Doxorubicin concentrations either as monotherapy or in combination. Optical microscopy was used to monitor the growth pattern and cell death. Based on the monotherapy experiments, we developed a probabilistic mathematical framework in order to describe the drug-induced effect at the single-cell level and simulate drug doses in combination assuming probabilistic independence. Doxorubicin was found to be effective in doses even four orders of magnitude less than Temozolomide in monotherapy. The combination therapy doses tested in vitro were able to lead to irreversible growth inhibition at doses where monotherapy resulted in relapse. In our simulations, we assumed both drugs are anti-mitotic; Temozolomide has a growth-arrest effect, while Doxorubicin is able to cumulatively cause necrosis. Interestingly, under no mechanistic synergy assumption, the in silico predictions underestimate the in vitro results. In silico models allow the exploration of a variety of potential underlying hypotheses. The simulated-biological discrepancy at certain doses indicates a supra-additive response when both drugs are combined. Our results suggest a Temozolomide-Doxorubicin dual chemotherapeutic scheme to both disable proliferation and increase cytotoxicity against Glioblastoma.

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.

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.

Development of Erf-Mediated Craniosynostosis and Pharmacological Amelioration.

ETS2 repressor factor (ERF) insufficiency causes craniosynostosis (CRS4) in humans and mice. ERF is an ETS domain transcriptional repressor regulated by Erk1/2 phosphorylation via nucleo-cytoplasmic shuttling. Here, we analyze the onset and development of the craniosynostosis phenotype in an Erf-insufficient mouse model and evaluate the potential of the residual Erf activity augmented by pharmacological compounds to ameliorate the disease. Erf insufficiency appears to cause an initially compromised frontal bone formation and subsequent multisuture synostosis, reflecting distinct roles of Erf on the cells that give rise to skull and facial bones. We treated animals with Mek1/2 and nuclear export inhibitors, U0126 and KPT-330, respectively, to increase Erf activity by two independent pathways. We implemented both a low dosage locally over the calvaria and a systemic drug administration scheme to evaluate the possible indirect effects from other systems and minimize toxicity. The treatment of mice with either the inhibitors or the administration scheme alleviated the synostosis phenotype with minimal adverse effects. Our data suggest that the ERF level is an important regulator of cranial bone development and that pharmacological modulation of its activity may represent a valid intervention approach both in CRS4 and in other syndromic forms of craniosynostosis mediated by the FGFR-RAS-ERK-ERF pathway.

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.

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.

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.

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.

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.

Revealing Underdrawings in Wall Paintings of Complex Stratigraphy with a Novel Reflectance Photoacoustic Imaging Prototype.

Revealing precious hidden features by a completely non-invasive approach is one of the crucial issues in the Heritage Science field. In this regard, concealed fresco paintings still represent an analytical challenge. This paper addresses the specific issue in wall painting diagnostics by the photoacoustic (PA) imaging technique, already proven to be efficient in revealing underdrawings and internal stratigraphy in movable paintings on paper and canvas. A newly set-up reflection PA prototype was applied here for the first time to probe the charcoal, graphite and sinopia hidden sketch drawings in concealed (gypsum, limewash, overpainted) wall paintings. The results presented here push forward the frontiers of the PA imaging technique and point to its potential effectiveness of revealing hidden underdrawings in historical wall paintings with complex stratigraphy.

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.

Revealing Hidden Features in Multilayered Artworks by Means of an Epi-Illumination Photoacoustic Imaging System.

Photoacoustic imaging is a novel, rapidly expanding technique, which has recently found several applications in artwork diagnostics, including the uncovering of hidden layers in paintings and multilayered documents, as well as the thickness measurement of optically turbid paint layers with high accuracy. However, thus far, all the presented photoacoustic-based imaging technologies dedicated to such measurements have been strictly limited to thin objects due to the detection of signals in transmission geometry. Unavoidably, this issue restricts seriously the applicability of the imaging method, hindering investigations over a wide range of cultural heritage objects with diverse geometrical and structural features. Here, we present an epi-illumination photoacoustic apparatus for diagnosis in heritage science, which integrates laser excitation and respective signal detection on one side, aiming to provide universal information in objects of arbitrary thickness and shape. To evaluate the capabilities of the developed system, we imaged thickly painted mock-ups, in an attempt to reveal hidden graphite layers covered by various optically turbid paints, and compared the measurements with standard near-infrared (NIR) imaging. The obtained results prove that photoacoustic signals reveal underlying sketches with up to 8 times improved contrast, thus paving the way for more relevant applications in the field.

PML Differentially Regulates Growth and Invasion in Brain Cancer.

Glioblastoma is the most malignant brain tumor among adults. Despite multimodality treatment, it remains incurable, mainly because of its extensive heterogeneity and infiltration in the brain parenchyma. Recent evidence indicates dysregulation of the expression of the Promyelocytic Leukemia Protein (PML) in primary Glioblastoma samples. PML is implicated in various ways in cancer biology. In the brain, PML participates in the physiological migration of the neural progenitor cells, which have been hypothesized to serve as the cell of origin of Glioblastoma. The role of PML in Glioblastoma progression has recently gained attention due to its controversial effects in overall Glioblastoma evolution. In this work, we studied the role of PML in Glioblastoma pathophysiology using the U87MG cell line. We genetically modified the cells to conditionally overexpress the PML isoform IV and we focused on its dual role in tumor growth and invasive capacity. Furthermore, we targeted a PML action mediator, the Enhancer of Zeste Homolog 2 (EZH2), via the inhibitory drug DZNeP. We present a combined in vitro-in silico approach, that utilizes both 2D and 3D cultures and cancer-predictive computational algorithms, in order to differentiate and interpret the observed biological results. Our overall findings indicate that PML regulates growth and invasion through distinct cellular mechanisms. In particular, PML overexpression suppresses cell proliferation, while it maintains the invasive capacity of the U87MG Glioblastoma cells and, upon inhibition of the PML-EZH2 pathway, the invasion is drastically eliminated. Our in silico simulations suggest that the underlying mechanism of PML-driven Glioblastoma physiology regulates invasion by differential modulation of the cell-to-cell adhesive and diffusive capacity of the cells. Elucidating further the role of PML in Glioblastoma biology could set PML as a potential molecular biomarker of the tumor progression and its mediated pathway as a therapeutic target, aiming at inhibiting cell growth and potentially clonal evolution regarding their proliferative and/or invasive phenotype within the heterogeneous tumor mass.

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.

Hidden phase-retrieved fluorescence tomography.

Fluorescence tomography is a well-established methodology able to provide structural and functional information on the measured object. At optical wavelengths, the unpredictable scattering of light is often considered a problem to overcome, rather than a feature to exploit. Advances in disordered photonics have shed new light on possibilities offered by opaque materials, treating them as autocorrelation lenses able to create images and focus light. In this Letter, we propose tomography through disorder, introducing a modified Fourier-slice theorem, the cornerstone of the computed tomography, aiming to reconstruct a three-dimensional fluorescent sample hidden behind an opaque curtain.

Micro-Computed Tomographic Evaluation of Canal Transportation and Centering Ability of 4 Heat-Treated Nickel-Titanium Systems.

INTRODUCTION: This study aimed to evaluate and compare canal transportation and centering ability of 4 different root canal preparation systems produced with thermal treatments by means of micro-computed tomographic imaging. METHODS: Eighty mesial canals of human extracted mandibular molars were selected based on similar morphologic parameters and were randomly assigned to 4 experimental groups (n = 20) according to the canal instrumentation technique: HyFlex CM (HCM [Coltène-Whaledent, Allstätten, Switzerland]), HyFlex EDM (HEDM [Coltène-Whaledent]), WaveOne Gold (WOG [Dentsply Sirona, Ballaigues, Switzerland]), and OneCurve (OC [Micro-Mega, Besancon, France]). The specimens were scanned before and after root canal preparation using X-ray micro-computed tomographic imaging at a resolution of 19.9 µm. Apical transportation and centering ability were then analyzed at 3 different levels: 3 mm, 5 mm, and 7 mm from the apex, representing the apical, midroot, and coronal thirds of the root, respectively. One-way analysis of variance and Kruskal-Wallis tests were used to statistically compare the groups. The significance level was set at 5%. RESULTS: HCM caused less canal transportation than WOG at the 3-mm level in both the buccal and lingual canals (P < .05). Also, HCM resulted in less canal transportation than WOG and OC at the 7-mm level regarding lingual canals. No statistically significant differences were recorded between the groups when the mean centering ratios were compared. CONCLUSIONS: The 4 evaluated systems safely prepared root canals causing minimal canal transportation and producing relatively centered preparations. In terms of canal transportation, HCM performed better than WOG at the apical level and better than WOG and OC at the coronal level.