Development of a Deep Learning-Based System for Optic Nerve Characterization in Transorbital Ultrasound Images on a Multicenter Data Set.

OBJECTIVE: Characterization of the optic nerve through measurement of optic nerve diameter (OND) and optic nerve sheath diameter (ONSD) using transorbital sonography (TOS) has proven to be a useful tool for the evaluation of intracranial pressure (ICP) and multiple neurological conditions. We describe a deep learning-based system for automatic characterization of the optic nerve from B-mode TOS images by automatic measurement of the OND and ONSD. In addition, we determine how the signal-to-noise ratio in two different areas of the image influences system performance. METHODS: A UNet was trained as the segmentation model. The training was performed on a multidevice, multicenter data set of 464 TOS images from 110 subjects. Fivefold cross-validation was performed, and the training process was repeated eight times. The final prediction was made as an ensemble of the predictions of the eight single models. Automatic OND and ONSD measurements were compared with the manual measurements taken by an expert with a graphical user interface that mimics a clinical setting. RESULTS: A Dice score of 0.719 ± 0.139 was obtained on the whole data set merging the test folds. Pearson's correlation was 0.69 for both OND and ONSD parameters. The signal-to-noise ratio was found to influence segmentation performance, but no clear correlation with diameter measurement performance was determined. CONCLUSION: The developed system has a good correlation with manual measurements, proving that it is feasible to create a model capable of automatically analyzing TOS images from multiple devices. The promising results encourage further definition of a standard protocol for the automatization of the OND and ONSD measurement process using deep learning-based methods. The image data and the manual measurements used in this work will be available at 10.17632/kw8gvp8m8x.1.

Comparison of the optoacoustic signal generation efficiency of different nanoparticular contrast agents.

Optoacoustic imaging represents a new modality that allows noninvasive in vivo molecular imaging with optical contrast and acoustical resolution. Whereas structural or functional imaging applications such as imaging of vasculature do not require contrast enhancing agents, nanoprobes with defined biochemical binding behavior are needed for molecular imaging tasks. Since the contrast of this modality is based on the local optical absorption coefficient, all particle or molecule types that show significant absorption cross sections in the spectral range of the laser wavelength used for signal generation are suitable contrast agents. Currently, several particle types such as gold nanospheres, nanoshells, nanorods, or polymer particles are used as optoacoustic contrast agents. These particles have specific advantages with respect to their absorption properties, or in terms of biologically relevant features (biodegradability, binding to molecular markers). In the present study, a comparative analysis of the signal generation efficiency of gold nanorods, polymeric particles, and magnetite particles using a 1064 nm Nd:YAG laser for signal generation is described.

Antitumor necrosis factor-α antibody-coupled gold nanorods as nanoprobes for molecular optoacoustic imaging in arthritis.

Optoacoustic molecular imaging can provide spatially resolved information about the presence of molecular markers in vivo. We synthesized elongated gold nanorods having an absorption maximum in the range of 1064 nm modified with the antibodies infliximab and certolizumab for targeting TNF-α to detect inflammation in arthritic mouse knees. We showed an differential enhancement of optoacoustic signal amplitudes after the injection of infliximab-, but not certolizumab-modified and PEGylated control particles on arthritic and healthy control mice by using a fast-scanning optoacoustic imaging platform based on a pulsed Nd:YAG laser and a single focused ultrasound transducer. The excellent photoacoustic properties of the gold nanorods confirmed the overexpression of TNF-α in arthritic knees. Due to the uncomplicated coupling chemistry and the scalability of ultrasound-based imaging approaches, these results potentially allow a transfer to various preclinical and clinical applications. From the Clinical Editor: Gold nanorods were modified with TNF-α targeting antibodies and used to detect inflammation in arthritic mouse knees via optoaoustic imaging. A fast-scanning optoacoustic imaging platform based on a pulsed Nd:YAG laser and a single focused ultrasound transducer was utilized for imaging. The excellent photoacoustic properties of these gold nanorods confirmed the overexpression of TNF-α, paving the way towards further preclinical and future clinical applications.

Targeted cell immobilization by ultrasound microbeam.

Various techniques exerting mechanical stress on cells have been developed to investigate cellular responses to externally controlled stimuli. Fundamental mechanotransduction processes about how applied physical forces are converted into biochemical signals have often been examined by transmitting such forces through cells and probing its pathway at cellular levels. In fact, many cellular biomechanics studies have been performed by trapping (or immobilizing) individual cells, either attached to solid substrates or suspended in liquid media. In that context, we demonstrated two-dimensional acoustic trapping, where a lipid droplet of 125 µm in diameter was directed transversely toward the focus (or the trap center) similar to that of optical tweezers. Under the influence of restoring forces created by a 30 MHz focused ultrasound beam, the trapped droplet behaved as if tethered to the focus by a linear spring. In order to apply this method to cellular manipulation in the Mie regime (cell diameter > wavelength), the availability of sound beams with its beamwidth approaching cell size is crucial. This can only be achieved at a frequency higher than 100 MHz. We define ultrasound beams in the frequency range from 100 MHz to a few GHz as ultrasound microbeams because the lateral beamwidth at the focus would be in the micron range. Hence a zinc oxide (ZnO) transducer that was designed and fabricated to transmit a 200 MHz focused sound beam was employed to immobilize a 10 µm human leukemia cell (K-562) within the trap. The cell was laterally displaced with respect to the trap center by mechanically translating the transducer over the focal plane. Both lateral displacement and position trajectory of the trapped cell were probed in a two-dimensional space, indicating that the retracting motion of these cells was similar to that of the lipid droplets at 30 MHz. The potential of this tool for studying cellular adhesion between white blood cells and endothelial cells was discussed, suggesting its capability as a single cell manipulator.

Preparation and biological evaluation of multifunctional PLGA-nanoparticles designed for photoacoustic imaging.

Nanoparticulate contrast agents for molecular imaging have attracted widespread interest for diagnostic applications with high resolution in medicine. Here we introduce polymer-based multifunctional nanoparticles exhibiting a near-infrared absorption in the range of the Nd:YAG laser wavelength of 1064 nm as a novel resorbable photoacoustic (PA) contrast system and report about their biological evaluation. Submicron-sized spherical nanoparticles with a high encapsulation efficiency (>87%) were created by incorporation of near-infrared dyes (IR5/IR26) in poly[(rac-lactide)-co-glycolide] (PLGA) with 50 mol% glycolide content via a specific spray-drying process in good yield (>75%). Subsequent application of a centrifugation protocol produced two different size fractions with diameters in the ranges 445-540 nm and 253-305 nm; these were further used for investigation of PA properties and cytotoxic effects. The prepared PLGA nanoparticles exhibited PA properties using a Nd:YAG laser-based system. After exposure of particle concentrations up to 10 µg·ml(-1) for 2 days no effects on viability, mitochondrial activity and proliferation, and cell death of human hepatocarcinoma cells and monkey kidney cells were observed. The excellent PA properties in combination with the positive biological results qualify the dye-loaded PLGA particles as promising candidates for a resorbable PA contrast system. FROM THE CLINICAL EDITOR: Photoacoustics (PA), a new modality, in which laser light is shined into tissue and absorbed by inherent proteins or synthetic particles is reflected back and received as ultrasound. This technique was shown to be effective with an erodible polymer particle containing near infrared dyes. In vitro, the PA properties of the PLGA particles persisted for 2 days in cell culture.

Effect of physical stimuli on hair follicle deposition of clobetasol-loaded Lipid Nanocarriers.

Clobetasol propionate (CLO) is a potent glucocorticoid used to treat inflammation-based skin, scalp, and hair disorders. In such conditions, hair follicles (HF) are not only the target site but can also act as drug reservoirs when certain formulations are topically applied. Recently, we have demonstrated nanostructured lipid carriers (NLC) containing CLO presenting epidermal-targeting potential. Here, the focus was evaluating the HF uptake provided by such nanoparticles in comparison to a commercial cream and investigating the influence of different physical stimuli [i.e., infrared (IR) irradiation (with and without metallic nanoparticles-MNP), ultrasound (US) (with and without vibration) and mechanical massage] on their follicular targeting potential. Nanosystems presented sizes around 180 nm (PdI < 0.2) and negative zeta potential. The formulation did not alter skin water loss measurements and was stable for at least 30 days at 5 °C. Nanoparticles released the drug in a sustained fashion for more than 3 days and increased passively about 40 times CLO follicular uptake compared to the commercial cream. Confocal images confirmed the enhanced follicular delivery. On the one hand, NLC application followed by IR for heat generation showed no benefit in terms of HF targeting even at higher temperatures generated by metallic nanoparticle heating. On the other hand, upon US treatment, CLO retention was significantly increased in deeper skin layers. The addition of mechanical vibration to the US treatment led to higher follicular accumulation compared to passive exposure to NLC without stimuli. However, from all evaluated stimuli, manual massage presented the highest follicular targeting potential, driving more than double the amount of CLO into the HF than NLC passive application. In conclusion, NLC showed great potential for delivering CLO to HF, and a simple massage was capable of doubling follicular retention.

Simulation and Experimental Characterization of Lateral Imaging Resolution of Ultrasound Systems and Assessment of System Suitability for Acoustic Optic Nerve Sheath Diameter Measurement.

BACKGROUND AND PURPOSE: Optic nerve sheath diameter (ONSD) is used for the estimation of intracranial pressure (ICP). But there are still doubts about the quality of the images and the lateral resolution. Our aim is to investigate the system suitability and best lateral resolution of different ultrasound systems for acoustic ONSD measurement. METHODS: First, we calculated the theoretically lateral imaging resolution at increasing frequencies: 6.6, 10, and 15 MHz using two different ultrasound systems. Second, we created two phantoms consisting of copper wires or polyvinylchloride (PVC) strips and tested the best lateral resolution at different frequencies with the two ultrasound systems. Using the same ultrasound systems, we evaluated the anatomy of optic nerve at increasing transmission frequencies. Finally, the two probes were tested in two patients with different neurological conditions affected by an increase of ICP. RESULTS: Theoretical resolutions were .63, .43, and .41 mm, respectively, with a frequency of 6.6, 10, and 15 MHz. We found a similar lateral resolution in both phantoms: copper wire; .56 mm at 6.6 MHz, .46 mm at 10 MHz, and .44 mm at 15 MHz; and PVC strips .6 mm at 6.6 MHz, .47 mm at 10 MHz, and .40 mm at 15 MHz in accordance with experimental resolution. The ONSD thickening could be clearly displayed at frequencies higher than 7.5 MHz using the two linear probes and the two patients with an increase of ICP showed thickening of ONSD. CONCLUSION: According to our study, both systems are suitable for ultrasound OSND measurement.

High frequency ultrasound tissue characterization and acoustic microscopy of intracellular changes.

The objective of this work is to investigate changes in the acoustic properties of cells when exposed to chemotherapy for monitoring treatment response. High frequency ultrasound spectroscopy (10-60 MHz) and scanning acoustic microscopy (0.9 GHz) were performed on HeLa cells (Ackermann et al. 1954, Masters 2002) that were exposed to the chemotherapeutic agent cisplatin. Ultrasonic radio-frequency data were acquired from pellets containing HeLa cells after exposure to cisplatin to induce apoptosis. Scanning acoustic and laser fluorescence microscopy images were recorded from single HeLa cells exposed to the same drug. Data acquisition in both cases was performed at several time points throughout the chemotherapeutic treatment for up to 27 h. In the high frequency ultrasound investigation, normalized power spectra were calculated within a region-of-interest. A 20 MHz transducer (f-number 2.35) and a 40 MHz transducer (f-number 3) were used for the data collection in the high frequency ultrasound experiments. The backscatter coefficients, integrated backscatter coefficients, mid-band fit and spectral slope were computed as a function of treatment time to monitor acoustical property changes during apoptosis. Acoustic attenuation was measured using the spectral substitution technique at all time points. Spectral parameter changes were detected after 12 h of exposure and coincided with the initiation of cell damage as assessed by optical microscopy. Integrated backscatter coefficients increased by over 100% between 0 h and 24 h of treatment, with small changes in the associated attenuation ( approximately 0.1 dB/[MHz cm]). Acoustic microscopy was performed at 0.9 GHz frequency. The cell structure was imaged using staining in laser fluorescence microscopy. All cells showed excellent correspondence between the locations of apoptotic nuclear condensation observed in optical imaging and changes in attenuation contrast in acoustic microscopy images. The time after drug exposure at which such changes occurred in the optical images were coincident with the time of changes detected in the acoustic microscopy images and the high frequency ultrasound experiments.

In-vivo observation of cells with a combined high-resolution multiphoton-acoustic scanning microscope.

We present a combined multiphoton-acoustic microscope giving collocated access to the local morphological as well as mechanical properties of living cells. Both methods relay on intrinsic contrast mechanisms and dispense with the need of staining. In the acoustic part of the microscope, a gigahertz ultrasound wave is generated by an acoustic lens and the reflected sound energy is detected by the identical lens in a confocal setup. The achieved lateral resolution is in the range of 1 mum. Contrast in the images arises mainly from the local absorption of sound in the cells related to viscose damping. Additionally, acoustic microscopy can access the sound speed as well as the acoustic impedance of the cell membrane and the cell shape, as it is an intrinsic volume scanning technique. The multiphoton image formation bases on the detection of autofluorescence due to endogenous fluorophores. The nonlinearity of two-photon absorption provides submicron lateral and axial resolution without the need of confocal optical detection. In addition, in the near-IR cell damages are drastically reduced in comparison with direct excitation in the visible or UV. The presented setup was aligned with a dedicated procedure to ensure identical image areas. Combined multiphoton/acoustic images of living myoblast cells are discussed focusing on the reliability of the method.

Imaging of focal contacts of chicken heart muscle cells by high-frequency acoustic microscopy.

A study of the adhesion of embryonic chicken heart muscle cells was conducted with a newly developed time-resolved acoustic microscope, which operates in the GHz-frequency range. The interpretation of the acoustical images of the heart muscle cells was done in combination with the fluorescence optical microscopy. A comparison between the acoustical images of chicken heart muscle cells and optical images of the same cells after staining showed that the actin fibers end inside the dark streaks in the acoustical images and thus represent the focal contacts (FCs). For cell biology applications, this demonstrates (a) the use of SAM as a tool for studying the dynamics of the FCs of living cells without any chemical staining and (b) that the combination of acoustic and optical microscopes allows interpretation of the acoustical images by using the wide variety of techniques available in optical microscopy.

Mechanical properties of single cells by high-frequency time-resolved acoustic microscopy.

In this paper, we describe a new, high-frequency, time-resolved scanning acoustic microscope developed for studying dynamical processes in biological cells. The new acoustic microscope operates in a time-resolved mode. The center frequency is 0.86 GHz, and the pulse duration is 5 ns. With such a short pulse, layers thicker than 3 microm can be resolved. For a cell thicker than 3 microm, the front echo and the echo from the substrate can be distinguished in the signal. Positions of the first and second pulses are used to determine the local impedance of the cell modeled as a thin liquid layer that has spatial variations in its elastic properties. The low signal-to-noise ratio in the acoustical images is increased for image generation by averaging the detected radio frequency signal over 10 measurements at each scanning point. In conducting quantitative measurements of the acoustic parameters of cells, the signal can be averaged over 2000 measurements. This approach enables us to measure acoustical properties of a single HeLa cell in vivo and to derive elastic parameters of subcellular structures. The value of the sound velocity inside the cell (1534.5 +/- 33.6 m/s) appears to be only slightly higher than that of the cell medium (1501 m/s).

Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes.

The present work addresses the question of to what extent a geometrical support acts as a physiological determining template in the setup of artificial cardiac tissue. Surface patterns with alternating concave to convex transitions of cell size dimensions were used to organize and orientate human-induced pluripotent stem cell (hIPSC)-derived cardiac myocytes and mouse neonatal cardiac myocytes. The shape of the cells, as well as the organization of the contractile apparatus recapitulates the anisotropic line pattern geometry being derived from tissue geometry motives. The intracellular organization of the contractile apparatus and the cell coupling via gap junctions of cell assemblies growing in a random or organized pattern were examined. Cell spatial and temporal coordinated excitation and contraction has been compared on plain and patterned substrates. While the α-actinin cytoskeletal organization is comparable to terminally-developed native ventricular tissue, connexin-43 expression does not recapitulate gap junction distribution of heart muscle tissue. However, coordinated contractions could be observed. The results of tissue-like cell ensemble organization open new insights into geometry-dependent cell organization, the cultivation of artificial heart tissue from stem cells and the anisotropy-dependent activity of therapeutic compounds.

Combined ultrasound and optoacoustic system for real-time high-contrast vascular imaging in vivo.

In optoacoustic imaging, short laser pulses irradiate highly scattering human tissue and adiabatically heat embedded absorbing structures, such as blood vessels, to generate ultrasound transients by means of the thermoelastic effect. We present an optoacoustic vascular imaging system that records these transients on the skin surface with an ultrasound transducer array and displays the images online. With a single laser pulse a complete optoacoustic B-mode image can be acquired. The optoacoustic system exploits the high intrinsic optical contrast of blood and provides high-contrast images without the need for contrast agents. The high spatial resolution of the system is determined by the acoustic propagation and is limited to the submillimeter range by our 7.5-MHz linear array transducer. A Q-switched alexandrite laser emitting short near-infrared laser pulses at a wavelength of 760 nm allows an imaging depth of a few centimeters. The system provides real-time images at frame-rates of 7.5 Hz and optionally displays the classically generated ultrasound image alongside the optoacoustic image. The functionality of the system was demonstrated in vivo on human finger, arm and leg. The proposed system combines the merits and most compelling features of optics and ultrasound in a single high-contrast vascular imaging device.

Optoacoustic imaging of subcutaneous microvasculature with a class one laser.

We developed a combined imaging platform allowing optoacoustic and ultrasound imaging based on a low energy laser and a handheld probe. The device is based on a sensitive single element 35-MHz focused transducer, a 2-D piezoscanner and a dual-wavelength switchable Nd:YAG laser. Acoustical detection and optical illumination are confocal for optimization of optoacoustic signal-to-noise ratio. The system allows to scan over a range up to 12 mm ×12 mm in xy-direction with an isotropic lateral resolution of about 90 µm. Although the device is a class 1 laser product having pulse energies in the range, in vivo images of subcutaneous microvasculature could be obtained from human skin with signal-to-noise levels as good as 20 dB.

Fluorescence response of human HER2+ cancer- and MCF-12F normal cells to 200MHz ultrasound microbeam stimulation: a preliminary study of membrane permeability variation.

Targeted mechanical cell stimulation has been extensively studied for a better understanding of its effect on cellular mechanotransduction signaling pathways and structures by utilizing a variety of mechanical sources. In this work, an ultrasound-driven single cell stimulation method is thus proposed, and a preliminary study is carried out by comparing the fluorescence intensities representing a change in cell membrane permeability between MDA-MB-435 human HER2+ cancer cells (∼40-50µm in diameter) and MCF-12F normal cells (∼50-60µm) in the presence of ultrasound. A 200MHz single element zinc oxide (ZnO) transducer is employed to generate ultrasound microbeam (UM) whose beamwidth and depth of focus are 9.5 and 60µm, comparable to typical cell size. The cells in tetramethyl rhodamine methyl ester (TMRM) are interrogated with 200MHz sinusoidal bursts. The number of cycles per burst is 5 and the pulse repetition frequency (PRF) is 1kHz. The temporal variation of fluorescence intensity in each cell is measured as a function of input voltage to the transducer (16, 32, and 47V), and its corresponding fluorescence images are obtained via a confocal microscope. A systematic method for visualizing UM's focus by adding Rhodamine B to the immersion medium is also proposed to enhance the precision in aiming the beam at an individual cell. Both types of cells exhibit a decrease in the intensity upon UM irradiation. In particular, normal cells show more fluorescence reduction (down to 0.7 in normalized intensity) than cancer cells (∼0.9) under the same excitation condition of the transducer. With UM being turned off, the normalized intensity level in normal cells is slowly increased to 1.1. The cell images taken before and after UM exposure indicate that the intensity reduction is more pronounced in those cells after exposure. Hence the results show the potential of UM as a non-invasive in vitro stimulation tool for facilitating targeted drug delivery and gene transfection as well as for studying cellular mechanotransduction.

Near-infrared dye-loaded PLGA nanoparticles prepared by spray drying for photoacoustic applications.

INTRODUCTION: Nanoparticulate contrast agents are of great interest for diagnostic applications with high resolution in medicine. Here we present polymer-based degradable nanoparticles exhibiting a near infrared (NIR) absorption suitable for photoacoustic imaging. METHODS: The nanoparticles were prepared by incorporation of indocyanine green (ICG) as NIR dye in poly[(rac-lactide)-co-glycolide] (PLGA) via an optimized spray drying process. By application of a multi-step centrifugation protocol, two different size fractions were achieved. The biocompatibitilty of the nanoparticles was tested in 2D cell cultures (human hepatocarcinoma cells and monkey kidney cells) using WST-1, BrdU and LDH assay. RESULTS: Spherical particles were obtained with a good yield (>81%), showing a high NIR-dye encapsulation efficiency (>98%). By multi-step centrifugation, two different size fractions with a mean diameter of 640 nm and 390 nm were obtained. Cytotoxicity studies of the synthesized ICG-loaded PLGA particles were performed. No cytotoxic effect on metabolic activity, proliferation, or membrane integrity was observed. CONCLUSION: The high optical absorption at the relevant NIR-wavelength around 800 nm in combination with absence of cytotoxicity qualifies the ICG-loaded PLGA particles as promising candidates for degradable photoacoustic contrast agents.

High frequency optoacoustic microscopy.

Photoacoustic imaging--also called optoacoustic imaging--is a new hybrid modality of high tissue contrast which is based on the varying optical properties of tissue. The acoustic signal generated by pulsed laser absorption reports tissue-specific information with high spatial resolution. To increase the intrinsic contrast in tissue, absorbing particles are of great interest for optical imaging because of their considerable capacity to absorb and scatter light at visible and near-infrared wavelengths. The aim of the work presented here is to establish a scalable photoacoustic technology for volume imaging of biological samples down to diffraction limited microscopy. For this purpose a versatile photoacoustic microscopy platform has been developed with unmatched spatial resolution consisting of a microchip laser and a measurement cell with different transducers attached allowing generation and detection of laser-induced ultrasound signals in a frequency range up to 400 MHz. The performance of a versatile photoacoustic microscopy platform was evaluated via 2D optoacoustic images of light absorbing microparticles (5 microm Fe(3)0(4) and 1 micromblack toner particles) embedded in a polystyrene matrix. High frequency signals in the frequency range of 400 MHz generated by a single 1 microm particle could be recorded with a high signal to noise ratio (SNR) of 34 dB.

Subunits alpha, beta and gamma of the epithelial Na+ channel (ENaC) are functionally related to the hypertonicity-induced cation channel (HICC) in rat hepatocytes.

Specific small interfering RNA (siRNA) constructs were used to test for the functional relation of subunits alpha, beta, and gamma of the epithelial Na(+) channel (ENaC) to the hypertonicity-induced cation channel (HICC) in confluent rat hepatocytes. In current-clamp recordings, hypertonic stress (300 --> 400 mosM) increased membrane conductance from 75.4 +/- 9.4 to 91.1 +/- 11.2 pS (p < 0.001). The effect was completely blocked by 100 microM amiloride and reduced to 46, 30, and 45% of the control value by anti-alpha-, anti-beta-, and anti-gamma-rENaC siRNA, respectively. Scanning acoustic microscopy revealed an initial shrinkage of cells from 6.98 +/- 0.45 to 6.03 +/- 0.43 pl within 2 min. This passive response was then followed by a regulatory volume increase (RVI) by 0.42 +/- 0.05 pl (p < 0.001). With anti-alpha-, anti-beta-, and anti-gamma-rENaC siRNA, the volume response was reduced to 31, 31, and 36% of the reference level, respectively. It is concluded that all three subunits of the ENaC are functionally related to RVI and HICC activation in rat hepatocytes.