Showing papers by "Roger J. Zemp published in 2013"

Glancing angle deposited nanostructured film Fabry-Perot etalons for optical detection of ultrasound

Abstract: In this paper a new class of optical Fabry-Perot-based ultrasound detectors using low acoustic impedance glancing angle deposited (GLAD) films is demonstrated. GLAD is a single-step physical vapor-deposition (PVD) technique used to fabricate porous nanostructured thin films. Using titanium dioxide (TiO(2)), a transparent semiconductor with a high refractive index (n = 2.4), the GLAD technique can be employed to fabricate samples with tailored nano-porosity, refractive index periodicities, and high Q-factor reflectance spectra. The average acoustic impedance of the porous films is lower than bulk materials which will improve acoustic coupling, especially for high acoustic frequencies. For this work, two filters with high reflection in the C-band range and high transparency in the visible range (~80%) using GLAD films were fabricated. A 23 µm Parylene C layer was sandwiched between these two GLAD films in order to form a GLAD Fabry Perot Interferometer (GLAD-FPI). A high speed tunable continuous wavelength C-band laser was focused at the FPI and the reflection was measured using a high speed photodiode. The ultrasound pressure modulated the optical thickness of the FPI and hence its reflectivity. The fabricated sensor was tested using a 10 MHz unfocused transducer. The ultrasound transducer was calibrated using a hydrophone. The minimum detectable acoustic pressure was measured as 80 ± 20 Pa and the -3dB bandwidth was measured to be 18 MHz. This ultra-sensitive sensor can be an alternative to piezoelectric ultrasound transducers for any techniques in which ultrasound waves need to be detected including ultrasonic and photoacoustic imaging modalities. We demonstrate our GLAD-FPI for photoacoustic signal detection in optical-resolution photoacoustic microscopy (OR-PAM). To the best of our knowledge, this is the first time that a FPI fabricated using the GLAD method has been used for ultra-sensitive ultrasound detection.

Multifocus optical-resolution photoacoustic microscopy using stimulated Raman scattering and chromatic aberration

Abstract: In this Letter, multifocus optical-resolution photoacoustic microscopy is demonstrated using wavelength tuning and chromatic aberration for depth scanning. Discrete focal zones at several depth locations were created by refocusing light from a polarization-maintaining single-mode fiber pumped by a nanosecond fiber laser. The fiber and laser parameters were chosen to take advantage of stimulated Raman scattering (SRS) in the fiber to create a multiwavelength output that could then be bandpass filtered. The collimator lens and objective lens are chosen to take advantage of chromatic aberration in which each generated SRS wavelength peak focuses at a slightly different depth. The maximum amplitude of photoacoustic signals is mapped to form C-scan images. Additionally, all wavelength peaks fired simultaneously offers improved depth-of-field structural imaging at the cost of slight degradation of mainlobe-to-sidelobe ratios. Wavelength-tuned depth scanning over more than 440 μm is demonstrated, significantly greater than the ∼100 μm depth of field predicted from our focused Gaussian beams. The improved depth of focus could be valuable for structural imaging of microvascular morphology without the need for mechanical scanning in the depth direction.

Label-free in vivo GRIN-lens optical resolution photoacoustic micro-endoscopy

Abstract: In this letter, the feasibility of label-free in vivo GRIN-lens optical resolution photoacoustic micro-endoscopy is demonstrated. An image guide with 100 000 single-mode fibers in a 1.4 mm diameter bundle in conjunction with a 0.29 pitch GRIN lens is used in order to transfer a focused scanning spot through the image guide and refocus it into tissue. A high-repetition-rate (up to 600 kHz) ytterbium fiber laser is used in order to enable near real-time imaging capability. Phantom studies indicate 6 μm resolution. The system, with ~2 mm working distance, overcomes the penetration depth limitation and hence improves the surface laser fluence of previously reported fiber based optical resolution photoacoustic microscopy (OR-PAM). The proposed system with a sub-mm probe footprint is very flexible and now has a significant penetration depth which is another step towards clinical applications.

A least-squares fixed-point iterative algorithm for multiple illumination photoacoustic tomography

Abstract: The optical absorption of tissues provides important information for clinical and pre-clinical studies. The challenge in recovering optical absorption from photoacoustic images is that the measured pressure depends on absorption and local fluence. One reconstruction approach uses a fixed-point iterative technique based on minimizing the mean-squared error combined with modeling of the light source to determine optical absorption. With this technique, convergence is not guaranteed even with an accurate measure of optical scattering. In this work we demonstrate using simulations that a new multiple illumination least squares fixed-point iteration algorithm improves convergence - even with poor estimates of optical scattering.

Human Concentrative Nucleoside Transporter 3 Transfection with Ultrasound and Microbubbles in Nucleoside Transport Deficient HEK293 Cells Greatly Increases Gemcitabine Uptake

Abstract: Gemcitabine is a hydrophilic clinical anticancer drug that requires nucleoside transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of nucleoside transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human concentrative nucleoside transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system - capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors - increased hCNT3 mRNA and 3H-gemcitabine uptake by >2,000– and 3,400–fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative nucleoside transporters and hCNT3 displayed 5% of 3H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative nucleoside transporters caused significant efflux of 3H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of 3H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that of HEK293 cells. Using the MTS proliferation assay, dilazep-treated HEK293 cells demonstrated 13-fold greater resistance to gemcitabine compared to dilazep-untreated HEK293 cells and this resistance could be reversed by transfection of hCNT3 cDNA. We propose that transfection of hCNT3 cDNA using ultrasound and microbubbles may be a method to reverse gemcitabine resistance in pancreatic tumors that have little nucleoside transport activity which are resistant to almost all current anticancer therapies.

In vivo dynamic process imaging using real-time optical-resolution photoacoustic microscopy.

Abstract: The authors demonstrate in vivo dynamic process imaging using a label-free real-time optical-resolution photoacoustic microscope (OR-PAM). This reflection-mode system takes advantage of a 532-nm fiber laser source with a high pulse repetition rate of up to 600 kHz combined with a fast-scanning mirror system. Microvasculature in SCID mouse ears is imaged at near real-time (0.5 fps) for a 1×1 mm 2 field of view (FOV) with micron-scale lateral resolution. We also demonstrate imaging of cardiac-induced microhemodynamics in murine microvasculature at real-time frame-rates (30 fps) over a 250×250 μm 2 FOV using real-time C-scan OR-PAM with ability to provide sustained imaging with near real-time feedback for focusing and positioning.

Multi-frequency CMUT arrays for imaging-therapy applications

Abstract: Novel multi-frequency Capacitive Micromachined Ultrasound Transducer (CMUT) arrays with interlaced low and high-frequency elements are investigated for novel imaging-therapy applications. Our interlaced CMUTs are designed with device dimensions smaller than operating wavelengths. This permits low- and high-frequency CMUT cells to be interlaced monolithically on a scale smaller than the array wavelengths. This dense interlacing scheme thus offers the promise of co-registered dual-frequency-band operation with minimal deleterious grating lobes and with maximal overlap of low- and high-frequency beams.

Microbubble-enhanced ultrasound liberation of mRNA biomarkers in vitro.

Abstract: Blood-borne biomarkers have great potential in diagnostic medicine, but low concentrations, inability to determine their source and lack of a patient baseline have limited their success in both research and clinical medicine. D'Souza et al. previously demonstrated that ultrasound-induced sonoporation can be used to liberate protein biomarkers from a colorectal cancer into the surrounding serum, overcoming many of the limitations of blood-borne biomarkers. In this study we build on D'Souza's work, extending this technique to nucleic acids, specifically mammaglobin mRNA—a potential diagnostic biomarker for breast cancer metastases. Furthermore, we propose to use ultrasound contrast agents, lipid-stabilized microbubbles, to enhance the effects of sonoporation and further amplify the biomarker levels. We demonstrate that microbubbles can enhance mammaglobin mRNA levels by two to three orders of magnitude greater than background levels and one to two orders of magnitude greater than ultrasound alone.

Phase-function corrected diffusion model for diffuse reflectance of a pencil beam obliquely incident on a semi-infinite turbid medium

Abstract: Oblique incidence reflectometry (OIR) is an established technique for the estimation of tissue optical properties. However, a sensing footprint of a few transport mean-free paths is often needed when diffusion-regime-based algorithms are used. Smaller-footprint probes require improved light-propagation models and inversion schemes for diffuse reflectance close to the point of entry but might enable micro-endoscopic form factors for clinical assessments of cancers and precancers. The phase-function corrected diffusion theory presented by Vitkin et al. [Nat. Commun. 2, 587 (2011)] to the case of pencil beams obliquely incident on a semi-infinite turbid medium is extended. The model requires minimal computational resources and offers improved accuracy over more traditional diffusion-theory approximations models when validated against Monte Carlo simulations. The computationally efficient nature of the models lends itself to rapid fitting procedures for inverse problems. The analytical model is used in a nonlinear fitting algorithm to demonstrate the recovery of optical properties using a measurement footprint that is significantly smaller than needed in previous diffusion-regime OIR methods.

S-sequence encoded synthetic aperture B-scan ultrasound imaging

Abstract: Traditional synthetic aperture ultrasound scanning involves firing on one element and receiving on all elements of an array, then firing another transmit element and receiving on all elements until all transmit-receive element pairs have been sampled. It offers excellent resolution but at the expense of signal-to-noise. To remedy this difficulty we propose an aperture encoding scheme that involves firing on multiple elements sampled from a Hadamard S-matrix, then applying matrix inversion methods to decouple signals to recover the effective synthetic aperture imaging set for subsequent image reconstruction. Because more than one elements are fired at a time, signal-to-noise is improved.

Optical resolution photoacoustic microendoscopy with ultrasound-guided insertion and array system detection

Abstract: Using a 0.8-mm-diameter image guide fiber bundle consisting of 30,000 single-mode fibers and an external linear array transducer, we demonstrate a dual-mode photoacoustic system capable of ultrasound-guided microendoscope insertion and photoacoustic imaging. The array optical resolution photoacoustic microendoscopy (AOR-PAME) system is designed to visualize the placement of the distal end of an endoscopy probe several centimeters into tissue, transmit scanning focused laser pulses into tissues via the fiber bundle, and acquire the generated photoacoustic signals. A ytterbium-doped fiber laser is tightly focused and is scanned across the proximal tip of the image guide fiber bundle using a two-dimensional galvanometer scanning mirror system. The end of the fiber bundle is used in contact mode with the object. The capabilities of AOR-PAME are demonstrated by imaging carbon fiber networks embedded in tissue-mimicking phantoms and the ears of a 60-g rat. The lateral resolution and signal-to-noise ratio are measured as 9 μm and 40 dB, respectively.

Synthetic aperture 3D ultrasound imaging schemes with S-sequence bias-encoded top-orthogonal-to-bottom-electrode 2D CMUT arrays

Abstract: Crossed electrode arrays, wherein electrodes are connected in individually addressable rows and columns, have been previously investigated as a means to reduce the number of channels in two-dimensional ultrasound arrays for three-dimensional imaging Previously envisioned imaging schemes have been limited in their resolution and focusing ability due to their inability to perform active focusing in both the lateral and elevational direction for both transmit and receive As well, signal-to-noise ratio (SNR) can be compromised when transmitting or receiving on only one row or column at a time A new imaging method is proposed for this array architecture using capacitive micromachined ultrasound transducers (CMUTs) which can provide two-way focusing in both lateral and elevational directions while providing optimal SNR This method is a variation of synthetic aperture imaging wherein the columns active upon transmit and receive events are determined by applying a bias to specific columns upon transmission and reception in an S-sequence encoded pattern Mathematical derivations of this method are presented and simulations are done to compare this imaging method to previously published crossed-electrode array imaging schemes, as well as to fully-populated 2-D phased array imaging

Pre-charged CMUTs with efficient low-bias voltage operation for medical applications

Abstract: Traditionally dielectric charging has been viewed as an undesirable feature of Capacitive Micromachined Ultrasound Transducers (CMUTs) because charging can alter performance of devices and lead to permanent collapse. We introduce a beneficial aspect of dielectric charging in CMUTs. We demonstrate CMUTs that may be pre-charged with a shift in collapse voltage so that at zero applied bias voltage they experience an electrostatic deflection due to trapped dielectric charge. We demonstrate efficient pre-collapse operation at zero applied bias voltage with exceptional long-term reliability. Similar existing strategies for pre-charging CMUTs were not implemented for diagnostic frequencies for immersion or tissue-coupled applications.

S-sequence bias-encoded photoacoustic imaging with top Orthogonal to Bottom Electrode (TOBE) CMUT arrays

Abstract: Recently we introduced Top Orthogonal to Bottom Electrode (TOBE) Capacitive Micromachined Ultrasound Transducers (CMUTs) as novel 2D arrays capable of 3D ultrasound imaging using only N transmit/receive and N bias channels for an NxN array. Here we adapt these devices for photoacoustic imaging by biasing one column and receiving across rows in parallel, then biasing another column and receiving along rows, and repeating this procedure until all columns have been biased in turn. Only elements in rows which are biased are dominantly sensitive to receive signals. Additionally, we propose a method to bias more than one column at a time using a biasing pattern sampled from rows or columns of a Hadamard S-matrix to improve signal-to-noise ratio, and demonstrate simulation results validating the proposed theory.

Phase-function corrected diffusion model for diffuse reflectance of a pencil beam obliquely incident on a semi-infinite turbid medium

Abstract: Oblique incidence reflectometry (OIR) is an established technique for estimation of tissue optical properties, however, a sensing footprint of a few transport mean-free paths is often needed when diffusion-regime-based algorithms are used. Smaller-footprint probes require improved light-propagation models and inversion schemes for diffuse reflectance close to the point-of-entry but might enable micro-endoscopic form factors for clinical assessments of cancers and pre-cancers. In this paper we extend the phase-function corrected diffusion-theory presented by Vitkin et al. (Nat. Comm 2011) to the case of pencil beams obliquely incident on a semi-infinite turbid medium. The model requires minimal computational resources and offers improved accuracy over more traditional diffusion-theory approximations models when validated against Monte Carlo simulations. The computationally efficient nature of the models may lend themselves to rapid fitting procedures for inverse problems.

In vivo multi-wavelength optical-resolution photoacoustic microscopy with stimulated Raman scattering fiber-laser source

Abstract: In this paper we demonstrate a multi-wavelength optical resolution photoacoustic microscopy system for both phantom and in vivo imaging. Using a 1ns pulse width, 40-kHz repetition-rate ytterbium-doped fiber laser and a 3m single-mode polarization maintaining fiber, we produced numerous Raman-shifted wavelength peaks at with pulse energies between 100 and 400nJ per peak. Peak wavelengths were selected by using 10-nm linewidth bandpass filters. The capabilities of this system is shown by creating C-scan photoacoustic images of carbon fiber networks, 200μm dye-filled tubes, and Swiss Webster mouse ears at several wavelengths. Functional imaging potential was confirmed by assessing tubes filled with varying concentrations of two different dyes.

Optical-resolution photoacoustic micro-endoscopy with ultrasound array system detection

Abstract: Recently we demonstrated the feasibility of Optical-Resolution Photoacoustic Micro-Endoscopy (OR-PAME) using an image guide fiber. However, the use of an ultrasound transducer for signal collection limited useful applications. We demonstrate detection of OR-PAM signals using an external array transducer in order to make endoscopic imaging practical for clinical use for the first time. The array system is able to visualize the placement of the image-guide fiber using pulse-echo ultrasound then switch to an OR-PAME acquisition mode. Photoacoustic signals are captured by a Verasonics ultrasound system using an L7-4 linear array transducer. A high-repetition-rate 532-nm fiber laser was used as the excitation source. This light was focused and raster­ scanned into a 800m-diameter image-guide fiber bundle consisting of 30,000 individual fiber elements. The operator finds the end of the endoscope using a flash ultrasound imaging mode, then captures endoscopic data by clicking a button. This activates the motion of scanning mirrors into the end of the image guide, and engages an endoscopic capture sequence. Endoscopic data are used to form a maximum amplitude image by simply taking the maximum of the absolute value of the signal across the 64 center channel lines used for capture. Using this technique, we have captured images of carbon fibers with a resolution of 6 microns at an SNR of greater than 30dB. Electronic focusing is expected to improve the SNR. The use of an ultrasound array transducer for both endoscope guidance and data collection allows for a much smaller endoscope footprint while opening up clinical possibilities.

Comparing nanodroplets and microbubbles for enhancing ultrasound-mediated gene transfection

Abstract: Ultrasound is capable of transfecting cells with DNA through the process of sonoporation. Cell transfection by ultrasound alone is a relatively inefficient process but sonicating cells in the presence of microbubbles can significantly increase transfection efficiency. Unfortunately, microbubbles are relatively large (~3 μm diameter) and cannot leave the vasculature, preventing their direct interaction with cancer cells in solid tumors. Perfluorobutane microbubbles can be condensed into submicron nanodroplets that may be able to extravasate out of the tumor vasculature, potentially allowing these nanodroplets to accumulate in tumors. The current study compares microbubbles and nanodroplets for their abilities to enhance ultrasound-mediated transfection of cultured HEK293 cells. The nanodroplets in the current study were almost 6-fold smaller than their corresponding microbubbles and could be phase changed back into microbubbles with 400 cycles of 1.7 mechanical index ultrasound. Given enough cycles of ultrasound, low concentrations of nanodroplets ( 2 % v/v) were 2- to 3-fold more effective than microbubbles for transfecting HEK293 cells. The current study supports the use of nanodroplets to enhance ultrasound-mediated transfection due to their equivalent or greater transfection enhancing abilities as well as their smaller size which may allow nanodroplets to accumulate in tumors.

Investigation of photoacoustic signal strength as a function of scan-speed and laser-repetition-rate

Abstract: Optical-resolution photoacoustic microscopy (OR-PAM) can produce micron-scale, high-resolution images of optically-absorbing chromophores. The pressure rise of photoacoustic signals is proportional to the Grueneisen parameter, which is temperature dependent. High laser repetition-rates may cause overlapping of adjacent laser pulses on targets in laser-scanning OR-PAM. When laser-pulse-repetition intervals are shorter than thermal relaxation times, the zone of laser-spot overlap between pulses can generate higher photoacoustic signal than cases where beam-spots do not overlap or in cases where laser pulse-intervals are longer than the thermal relaxation time. This is because subsequent laser pulses experience higher Grueneisen parameters than previous pulses due to temperature rises induced by the previous pulses. Here, we present our recent studies on photoacoustic signals strength varying with scanning speed and laser-repetition-rate on black tape, human hair and rat blood respectively.