Pavlos Anastasiadis

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 mum can be resolved. For a cell thicker than 3 mum, 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 plusmn 33.6 m/s) appears to be only slightly higher than that of the cell medium (1501 m/s).

9D-5 Variation of the Sound Attenuation Inside HeLa Cells During Cell Division Using High-Frequency Time-Resolved Acoustic Microscope

High-frequency, scanning acoustic microscopy (SAM) imaging of the dividing HeLa cells (cell line derived from human cervics carcinoma) has revealed that acoustical images of the HeLa cells become darker during cell division. The dark contrast of the dividing HeLa cells was attributed to increase of sound attenuation in the cytoskeleton. In this report, RF-imaging of adherent HeLa cells at different stages of the cell cycle has been conducted using high-frequency, time-resolved scanning acoustic microscope (SAM). B-Scan images of HeLa cells at different stages of the cell cycle show distinct patterns inside the cell. Velocity of the longitudinal wave, density, thickness and attenuation of the longitudinal waves inside the HeLa cell were determined by measuring the waveform of the echo signals reflected from the top and the bottom of the cell. It was found that attenuation increased after the cell division by 50%. The increase of the attenuation can be attributed to the polymerization of the f-actin.

Targeted ultrasound contrast agents for the imaging of biofilm infections.

Targeted ultrasound contrast agents (UCA) offer a novel, potential method for diagnostic imaging of biofilm infections. Currently, there is no established method for molecular imaging of in vitro biofilm infections with any type of modality. For infective endocarditis, the case of biofilm formation on damaged or diseased heart valves, an early diagnostic method might greatly reduce the associated high mortality rate. Fluorescently labeled lectins were used with targeted ultrasound contrast agents in conjunction with epifluorescence microscopy and time‐resolved scanning acoustic microscopy for visualization and characterization of the extracellular polymeric substances (EPS) of two types of infectious biofilms (Staphylococcus aureus and Pseudomonas aeruginosa). Noninvasive acoustic measurements of the biofilms were conducted at high‐frequency (100 MHz) with a time‐resolved scanning acoustic microscope. Measurements of the reflection and transmission coefficients, over a broad frequency spectrum for a range...

Mechanical properties of HeLa cells at different stages of cell cycle by time‐resolved acoustic microscope

Scanning acoustic microscopy (SAM), particularly time‐resolved acoustic microscopy, is one of the few techniques for study of the mechanical properties of only the cell’s interior, cytosol and nucleus. Unfortunately, time‐resolved acoustic microscopes typically do not provide sufficient resolution to study the elasticity of single cells. We demonstrate that the high‐frequency, time‐resolved acoustic microscope developed at the Fraunhofer Institute for Biomedical Technology (IBMT), Germany, is capable of imaging and characterizing elastic properties of micron size structures in cell’s cytoskeleton with a theoretical resolution limit of 10 m/s for sound speed measurements. Measurements were performed on cells of the HeLa cell line derived from human cervics carcinoma. SAM measurements of the sound speed of adherent HeLa cells at different states of the cell cycle were conducted. They yielded an average value of 1540 m/s. B‐Scan images of HeLa cells at different states of the cell cycle show distinct pattern...

Focus ultrasound for augmenting convection-enhanced delivery of nanoparticles in the brain

We previously demonstrated how ultrasound can enhance the dispersion of locally administrated nanoparticles within the extracellular/perivascular spaces in the ex vivo brain by non-destructively enlarging these regions. The current study aimed to translate these results in vivo , where custom, non-adhering brain-penetrating nanoparticles (BPN: 60, 200, and 500 nm), were administered directly into the brains of Sprague Dawley rats by convection-enhanced delivery. Non-invasive, transcranial focused ultrasound (TCFUS) was carried out using an MRI-guided system (1.5 MHz, 10 ms pulses, 10% duty cycle, and 2.3 MPa). 15 individual exposures in a 3 × 5 matrix (spacing: 1.5 mm) in one hemisphere were given, where the size of the focal zone (-6 dB) was 1 × 1 X 8 mm. At 2hrs post-treatment brains were harvested and sectioned, with digital images captured and processed using a custom MATLAB script. This involved the “Otsu” thresholding method, based on gray level histograms and threshold determinations for maximizing...

A portable transcranial focused ultrasound system for non-invasive applications in small animals

Recent studies using focused ultrasound (FUS) have shown significant progress in the realm of brain applications. These transcranial exposures include localized ablation for the treatment of movement disorders and opening of the blood brain barrier to enhance the delivery of therapeutic agents. Whereas these procedures require expensive, MRI-guided systems, one of the newest and most promising applications of FUS being developed involves more portable, hand-held devices for the purpose of neuromodulation. Here, we describe the development of table-top FUS system comprised of a single element FUS transducer (400—600 kHz) specifically for investigations on the stimulation and suppression of neuronal activity in rodents. The transducer assembly includes a cone and extending flexible bolus filled with degassed water for direct coupling to the head of the animals. A custom 3D stage and two diametrically opposed lasers allow for accurate positioning and targeting. Experiments included simulations of the acousti...

Ultrasound-mediated drug delivery with real-time cell permeability measurements

Ultrasound-mediated drug and gene delivery offers a variety of exciting possibilities for improved localized treatment of vascular- and cancer-related diseases. This therapeutic application benefits from the use of the acoustic radiation force that facilitates the exposure for enhanced binding due to ligand-receptor interactions. The main merits of ultrasound lie in the transient increase of cell permeability without exposing the cell corpus to any detrimental and irreversible side-effects. Nonetheless, the related underlying molecular and cellular pathways of ultrasound-induced permeability and the subsequent recovery of cells have not been answered satisfactorily. Real-time studies of cell behavior during and past-ultrasound exposure have been obstructed by the lack of appropriate techniques. The Electric-Cell Impedance Sensing (ECIS) technique is an attractive way of studying cell permeability changes in real-time. Its nanoscale sensitivity and speedy acquisition of data allows for the accurate and tim...

Shell material parameter measurements of polymer ultrasound contrast agents

Polymer shelled ultrasound contrast agents have been used in ultrasound imaging and tissue perfusion studies. The destruction of the agent produced by the rupture of the shell often through complicated buckling has been quantified with overpressure experiments and optical visualization. Approximate material parameters have been correspondingly estimated with this methodology but additional steps are needed to translate for a viable high throughput technique. Ultra-high frequency acoustic microscopy (1 GHz) provides a non-invasive method to image and measure the shells elastic properties. Scanning acoustic microscopy at 1 GHz is used to determine the shell density and elastic modulus for polymer shelled agents of three different shell thicknesses. The effect bending thickness is estimated for comparison with overpressure experiments.

Ultrasonic biomicroscopy and micro-Raman spectroscopy for the mechanochemical characterization of atheromatous lesions

Atherosclerosis is defined as a focal, inflammatory, and fibro-proliferative response to endothelial injury. The development of atheromatous lesions in the coronary tree is predominantly a quiescent asymptomatic process without any clinical manifestations. The unpredictable and acute nature of cardiovascular complications such as vulnerable plaque rupture makes diagnosis and treatment of this disease an outstanding medical challenge. We investigate non-invasive techniques that facilitate mechanical measurements at the microscopic level, which can then be directly correlated to biomarker localization within lesion sites. To characterize these sites in vitro, we used time-resolved scanning acoustic microscopy (TRSAM). This technique allows for non-invasive interrogation of tissue samples with optical resolution at the micrometer scale. Furthermore, we combined TRSAM with micro-Raman (micro-RS) spectroscopy to investigate plaque morphology with regard to specific biomarkers. We characterized mechanoelastic a...

In vitro quantitation of human femoral artery atherosclerosis using near-infrared Raman spectroscopy

Near-infrared Raman spectroscopy has been used in vitro to identify calcified atherosclerotic plaques in human femoral arteries. Raman techniques allow for the identification of these plaques in a nondestructive manner, which may allow for the diagnosis of coronary artery disease in cardiac patients in the future. As Raman spectroscopy also reveals chemical information about the composition of the arteries, it can also be used as a prognostic tool. The in vivo detection of atherosclerotic plaques at risk for rupture in cardiac patients will enhance treatment methods while improving clinical outcomes for these procedures. Raman spectra were excited by an Invictus 785-nm NIR laser and measured with a fiber-coupled micro-Raman RXN system (Kaiser Optical Systems, Inc., Ann Arbor, MI) equipped with a 785 nm CW laser and CCD detector. Chemical mapping of arteries obtained post mortem allowed for the discrete location of atherosclerotic plaques. Raman peaks at 961 and 1073 cm-1 reveal the presence of calcium hydroxyapatite and carbonate apatite, which are known to be present in calcified plaques. By mapping the locations of these peaks the boundaries of the plaques can be precisely determined. Areas of varying degrees of calcification were also identified. Because this can be useful in determining the degree of plaque calcification and vessel stenosis, this may have a significant impact on the clinical treatment of atherosclerotic plaques in the future.