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.

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.

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.

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.

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.

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.

Optical projection tomography via phase retrieval algorithms.

We describe a computational method for accurate, quantitative tomographic reconstructions in Optical Projection Tomography, based on phase retrieval algorithms. Our method overcomes limitations imposed by light scattering in opaque tissue samples under the memory effect regime, as well as reduces artifacts due to mechanical movements, misalignments or vibrations. We make use of Gerchberg-Saxton algorithms, calculating first the autocorrelation of the object and then retrieving the associated phase under four numerically simulated measurement conditions. By approaching the task in such a way, we avoid the projection alignment procedure, exploiting the fact that the autocorrelation sinogram is always aligned and centered. We thus propose two new, projection-based, tomographic imaging flowcharts that allow registration-free imaging of opaque biological specimens and unlock three-dimensional tomographic imaging of hidden objects. Two main reconstruction approaches are discussed in the text, focusing on their efficiency in the tomographic retrieval and discussing their applicability under four different numerical experiments.

Hyperuniformity in amorphous speckle patterns.

Hyperuniform structures possess the ability to confine and drive light, although their fabrication is extremely challenging. Here we demonstrate that speckle patterns obtained by a superposition of randomly arranged sources of Bessel beams can be used to generate hyperunifrom scalar fields. By exploiting laser light tailored with a spatial filter, we experimentally produce (without requiring any computational power) a speckle pattern possessing maxima at locations corresponding to a hyperuniform distribution. By properly filtering out intensity fluctuation from the same speckle pattern, it is possible to retrieve an intensity profile satisfying the hyperuniformity requirements. Our findings are supported by extensive numerical simulations.

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.

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.

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.

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.

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.

Tomographic imaging with polarized light.

We report three-dimensional tomographic reconstruction of optical parameters for the mesoscopic light scattering regime from experimentally obtained datasets by using polarized light. We present a numerically inexpensive approximation to the radiative transfer equation governing the polarized light transport. This approximation is employed in the reconstruction algorithm, which computes two optical parameters by using parallel and perpendicular polarizations of transmitted light. Datasets were obtained by imaging a scattering phantom embedding highly absorbing inclusions. Reconstruction results are presented and discussed.