This study explores a novel approach to detect virus-laden droplets in the ambient air. An air-coupled photoacoustic (PA) technique is considered for this purpose. The free space PA system is developed using an air-coupled transducer with a center frequency of 350 kHz and a nanosecond pulsed laser operating at wavelength 533 nm. Water droplets containing 80 nm gold (Au) nanoparticles were aerosolized using a custom-built spraying system. The size of the droplets generated was in the range of a few hundred nanometers to 100 μm. Au nanoparticles of four concentrations (0, 8x10-12, 16x10-12, and 32x10-12 mol/L) were sprayed into the investigation domain interrogated by a laser beam, where the average PA signal from the droplets was 3.11±2.35, 1.28±1.26, 0.99±0.97, and 0.92±1.11 mV/mJ, respectively. The study showed, surprisingly, that water droplets without Au nanoparticles had a higher PA signal than those containing Au nanoparticles. A numerical analysis using a finite difference time domain method was used to explore the reasons for this unexpected finding. Results suggested that the undoped droplets could potentially focus the light, significantly increasing the fluence at the focus. When Au nanoparticles were present, the fluence within the droplet decreased, resulting in a lower PA signal.
In this work, speckle in acoustic-resolution photoacoustic (PA) imaging systems is discussed. Simulations and experiments were used to demonstrate that PA speckle carries structural information related to sub-resolution absorbers.
Numerical simulations of phantoms containing spherical absorbers were performed using Green’s function solutions to the PA wave equation. A 21 MHz linear array was simulated (256 elements, 75×165 µm resolution, bandwidth 9-33 MHz) and used to record, bandlimit and beamform the generated PA signals. The effects of absorber size (10-270 µm) and concentration (10-1000/mm3) on PA speckle were examined using envelope statistics and radiofrequency spectroscopy techniques. To examine PA speckle experimentally, a VevoLAZR system was used to image gelatin phantoms containing 3 and 15µm polystyrene beads, a tissue mimicking radial artery phantom, and murine tumour vasculature in vivo.
Fully developed speckle, as assessed by Rayleigh distribution fits to PA signal envelopes, was present in all images (simulated and experimental) containing at least 10 absorbers per resolution volume, irrespective of absorber size. Changes in absorber size could be detected using the spectral slope of the normalized power spectrum (4.5x decrease for an 80 µm increase in size). PA images of flowing blood in the radial artery phantom also revealed the presence of speckle with intensity that fluctuated periodically with beat rate (4 dB per cycle). Speckle was ubiquitous to all murine tumor vasculature images. During treatment-induced vascular hemorrhaging, the spectral slope decreases by 80% compared to untreated mice. These results demonstrate that photoacoustic speckle encodes information about the underlying absorber distribution.
We have developed a flow cytometer based on simultaneous detection of ultrasound and photoacoustic waves from individual particles/cells flowing in a microfluidic channel. Our polydimethylsiloxane (PDMS) based hydrodynamic 3-dimensional (3D) flow-focusing microfluidic device contains a cross-junction channel, a micro-needle (ID 100 μm and OD 200 μm) insert, and a 3D printed frame to hold and align a high frequency (center frequency 375 MHz) ultrasound transducer. The focused flow passes through a narrow focal zone with lateral and axial focal lengths of 6-8 μm and 15-20 μm, respectively. Both the lateral and axial alignments are achieved by screwing the transducer to the frame onto the PDMS device. Individual particles pass through an interrogation zone in the microfluidic channel with a collinearly aligned ultrasound transducer and a focused 532 nm wavelength laser beam. The particles are simultaneously insonified by high-frequency ultrasound and irradiated by a laser beam. The ultrasound backscatter and laser generated photoacoustic waves are detected for each passing particle. The backscattered ultrasound and photoacoustic signal are strongly dependent on the size, morphology, mechanical properties, and material properties of the flowing particles; these parameters can be extracted by analyzing unique features in the power spectrum of the signals. Frequencies less than 100 MHz do not have these unique spectral signatures. We show that we can reliably distinguish between different particles in a sample using the acoustic-based flow cytometer. This technique, when extended to biomedical applications, allows us to rapidly analyze the spectral signatures from individual single cells of a large cell population, with applications towards label-free detection and characterization of healthy and diseased cells.
An inexpensive noncontact photoacoustic (PA) imaging system using a low-power continuous wave laser and a kilohertz-range microphone has been developed. The system operates in both optical and PA imaging modes and is designed to be compatible with conventional optical microscopes. Aqueous coupling fluids are not required for the detection of the PA signals; air is used as the coupling medium. The main component of the PA system is a custom designed PA imaging sensor that consists of an air-filled sample chamber and a resonator chamber that isolates a standard kilohertz frequency microphone from the input laser. A sample to be examined is placed on the glass substrate inside the chamber. A laser focused to a small spot by a 40× objective onto the substrate enables generation of PA signals from the sample. Raster scanning the laser over the sample with micrometer-sized steps enables high-resolution PA images to be generated. A lateral resolution of 1.37 μm was achieved in this proof of concept study, which can be further improved using a higher numerical aperture objective. The application of the system was investigated on a red blood cell, with a noise-equivalent detection sensitivity of 43,887 hemoglobin molecules (72.88×10−21 mol or 72.88 zeptomol). The minimum pressure detectable limit of the system was 19.1 μPa. This inexpensive, compact noncontact PA sensor is easily integrated with existing commercial optical microscopes, enabling optical and PA imaging of the same sample. Applications include forensic measurements, blood coagulation tests, and monitoring the penetration of drugs into human membrane.
We have developed a low-cost, non-contact, multispectral photoacoustic microscope system to study the functional parameters of cellular choromophores. The system uses low power continuous wave lasers and a photoacoustic sensor made of a kHz microphone coupled to a resonant chamber. Methemoglobin has relatively high optical absorption at 500 nm and 630 nm. Moreover, it has an almost the same optical absorption as hemoglobin at the isosbestic point of 525 nm. Photoacoustic data collected from methemoglobin using our system at wavelengths of 473 nm, 533 nm, and 633 nm show the similar trends as the methemoglobin optical absorption spectrum. The PA amplitude at 473 nm is about 1.03 times greater than at 533 nm and about 2.4 times greater than at 633 nm. Similarly, it possesses optical absorption of about 1.08 greater than at 533 nm and 1.34 times greater than at 633 nm. The developed system can be used as a differential photoacoustic microscope.
Single sensor (pixel) signals require scanning of the sample in order to obtain spatial information. In this paper we show that using interference, optically induced signals can be reconstructed when recorded using interference pattern excitation, rather than a point illumination. This method reduces the need for dense scanning and requires a small number of scans, or can eliminate the need for scanning in some cases. It is shown that this method can be used in particular in photo-acoustic imaging.
In this study, we report photoacoustic (PA) measurements of gold-covered polystyrene nanoparticles (Au nanoshells). Two types of Au nanoshells were examined: 1) polystyrene core with sparsely covered Au nanoparticles, and 2) polystyrene core which were fully covered by Au nanoparticles. The fully covered Au nanoshell exhibited a broad extinction cross section (500 nm – 850 nm), which is in the first infrared optical window where light transmission is optimal for optical based studies in tissues. The optical properties were compared to numerical simulations using Mie scattering theory. Using a photoacoustic microscope, the PA signal measured from fully covered Au nanoshells was 1.27 ± 0.18 mV per fluence (mJ/cm2), which was 10x greater than the PA signal from sparsely covered Au nanoshells (0.12 ± 0.14 mV). These novel Au nanoshell nanoparticles can be used for multispectral optical and PA imaging.
Hydrodynamic 3D flow-focusing techniques in microfluidics are categorized as (a) sheathless techniques which require high flow rates and long channels, resulting in high operating cost and high flow rates which are inappropriate for applications with flow rate limitations, and (b) sheath-flow based techniques which usually require excessive sheath flow rate to achieve hydrodynamic 3D flow-focusing. Many devices based on these principles use complicated fabrication methods to create multi-layer microchannels. We have developed a sheath-flow based microfluidic device that is capable of hydrodynamic 3D self-flow-focusing. In this device the main flow (black ink) in a low speed, and a sheath flow, enter through two inlets and enter a 180 degree curved channel (300 × 300 μm cross-section). Main flow migrates outwards into the sheath-flow due to centrifugal effects and consequently, vertical focusing is achieved at the end of the curved channel. Then, two other sheath flows horizontally confine the main flow to achieve horizontal focusing. Thus, the core flow is three-dimensionally focused at the center of the channel at the downstream. Using centrifugal force for 3D flow-focusing in a single-layer fabricated microchannel has been previously investigated by few groups. However, their demonstrated designs required high flow speed (>1 m/s) which is not suitable for many applications that live biomedical specie are involved. Here, we introduce a new design which is operational in low flow speed (<0.05 m/s) and is suitable for applications involving live cells. This microfluidic device can be used in detecting, counting and isolating cells in many biomedical applications.
A new technique for simultaneously acquiring photoacoustic images as well as images based on the optical attenuation of single cells in a human blood smear was developed. An ultra-high frequency photoacoustic microscope equipped with a 1 GHz transducer and a pulsed 532 nm laser was used to generate the images. The transducer and 20X optical objective used for laser focusing were aligned coaxially on opposing sides of the sample. Absorption of laser photons by the sample yielded conventional photoacoustic (PA) signals, while incident photons which were not attenuated by the sample were absorbed by the transducer, resulting in the formation of a photoacoustic signal (tPA) within the transducer itself. Both PA and tPA signals, which are separated in time, were recorded by the system in a single RF-line. Areas of strong signal in the PA images corresponded to dark regions in the tPA images. Additional details, including the clear delineation of the cell cytoplasm and features in red blood cells, were visible in the tPA image but not the corresponding PA image. This imaging method has applications in probing the optical absorption and attenuation characteristics of biological cells with sub-cellular resolution.
An acoustic/photoacoustic microscope was used to create micrometer resolution images of stained cells from a blood smear. Pulse echo ultrasound images were made using a 1000 MHz transducer with 1 μm resolution. Photoacoustic images were made using a fiber coupled 532 nm laser, where energy losses through stimulated Raman scattering enabled output wavelengths from 532 nm to 620 nm. The laser was focused onto the sample using a 20x objective, and the laser spot co-aligned with the 1000 MHz transducer opposite the laser. The blood smear was stained with Wright-Giemsa, a common metachromatic dye that differentially stains the cellular components for visual identification. A neutrophil, lymphocyte and a monocyte were imaged using acoustic and photoacoustic microscopy at two different wavelengths, 532 nm and 600 nm. Unique features in each imaging modality enabled identification of the different cell types. This imaging method provides a new way of imaging stained leukocytes, with applications towards identifying and differentiating cell types, and detecting disease at the single cell level.
A flow cytometer that uses sound waves to determine the size of biological cells is presented. In this system, a microfluidic device made of polydimethylsiloxane (PDMS) was developed to hydrodynamically flow focus cells in a single file through a target area. Integrated into the microfluidic device was an ultrasound transducer with a 375 MHz center frequency, aligned opposite the transducer was a pulsed 532 nm laser focused into the device by a 10x objective. Each passing cell was insonfied with a high frequency ultrasound pulse, and irradiated with the laser. The resulting ultrasound and photoacoustic waves from each cell were analyzed using signal processing methods, where features in the power spectra were compared to theoretical models to calculate the cell size. Two cell lines with different size distributions were used to test the system: acute myeloid leukemia cells (AML) and melanoma cells. Over 200 cells were measured using this system. The average calculated diameter of the AML cells was 10.4 ± 2.5 μm using ultrasound, and 11.4 ± 2.3 μm using photoacoustics. The average diameter of the melanoma cells was 16.2 ± 2.9 μm using ultrasound, and 18.9 ± 3.5 μm using photoacoustics. The cell sizes calculated using ultrasound and photoacoustic methods agreed with measurements using a Coulter Counter, where the AML cells were 9.8 ± 1.8 μm and the melanoma cells were 16.0 ± 2.5 μm. These results demonstrate a high speed method of assessing cell size using sound waves, which is an alternative method to traditional flow cytometry techniques.
In this study, multifunctional theranostic agents for photoacoustic (PA), ultrasound (US), fluorescent imaging, and for therapeutic drug delivery were developed and tested. These agents consisted of a shell made from a biodegradable Poly(lactide-co-glycolic acid) (PLGA) polymer, loaded with perfluorohexane (PFH) liquid and gold nanoparticles (GNPs) in the core, and lipophilic carbocyanines fluorescent dye DiD and therapeutic drug Paclitaxel (PAC) in the shell. Their multifunctional capacity was investigated in an in vitro study. The PLGA/PFH/DiD-GNPs particles were synthesized by a double emulsion technique. The average PLGA particle diameter was 560 nm, with 50 nm diameter silica-coated gold nano-spheres in the shell. MCF7 human breast cancer cells were incubated with PLGA/PFH/DiDGNPs for 24 hours. Fluorescent and PA images were recorded using a fluorescent/PA microscope using a 1000 MHz transducer and a 532 nm pulsed laser. For the particle vaporization and drug delivery test, MCF7 cells were incubated with the PLGA/PFH-GNPs-PAC or PLGA/PFH-GNPs particles for 6, 12 and 24 hours. The effects of particle vaporization and drug delivery inside the cells were examined by irradiating the cells with a laser fluence of 100 mJ/cm2, and cell viability quantified using the MTT assay. The PA images of MCF7 cells containing PLGA/PFH/DiD-GNPs were spatially coincident with the fluorescent images, and confirmed particle uptake. After exposure to the PLGA/PFHGNP- PAC for 6, 12 and 24 hours, the cell survival rate was 43%, 38%, and 36% respectively compared with the control group, confirming drug delivery and release inside the cells. Upon vaporization, cell viability decreased to 20%. The particles show potential as imaging agents and drug delivery vehicles.
A method to detect and differentiate circulating melanoma tumor cells (CTCs) from blood cells using ultrasound and photoacoustic signals with frequencies over 100 MHz is presented. At these frequencies, the acoustic wavelength is similar to the dimensions of a cell, which results in unique features in the signal; periodically varying minima and maxima occur throughout the power spectrum. The spacing between minima depends on the ratio of the size to sound speed of the cell. Using a 532 nm pulsed laser and a 375 MHz center frequency wide-bandwidth transducer, the ultrasound and photoacoustic signals were measured from single cells. A total of 80 cells were measured, 20 melanoma cells, 20 white blood cells (WBCs) and 40 red blood cells (RBCs). The photoacoustic spectral spacing Δf between minima was 95 ± 15 MHz for melanoma cells and greater than 230 MHz for RBCs. No photoacoustic signal was detected from WBCs. The ultrasonic spectral spacing between minima was 46 ± 9 MHz for melanoma cells and 98 ± 11 for WBCs. Both photoacoustic and ultrasound signals were detected from melanoma cells, while only ultrasound signals were detected from WBCs. RBCs showed distinct photoacoustic spectral variations in comparison to any other type of cell. Using the spectral spacing and signal amplitudes, each cell type could be grouped together to aid in cell identification. This method could be used for label-free counting and classifying cells in a sample.
Phase-change contrast agents consisting of a perfluorocarbon (PFC) liquid core stabilized by a lipid, protein, or polymer shell have been proposed for a variety of clinical applications. Previous work has demonstrated that vaporization can be induced by laser irradiation through optical absorbers incorporated inside the droplet. In this study, Poly-lactide-coglycolic acid (PLGA) particles loaded with PFC liquid and silica-coated gold nanoparticles (GNPs) were developed and characterized using photoacoustic (PA) methods. Microsized PLGA particles were loaded with PFC liquid and GNPs (14, 35, 55nm each with a 20nm silica shell) using a double emulsion method. The PA signal intensity and optical vaporization threshold were investigated using a 375 MHz transducer and a focused 532-nm laser (up to 450-nJ per pulse). The laser-induced vaporization threshold energy decreased with increasing GNP size. The vaporization threshold was 850, 690 and 420 mJ/cm2 for 5μm-sized PLGA particles loaded with 14, 35 and 55 nm GNPs, respectively. The PA signal intensity increased as the laser fluence increased prior to the vaporization event. This trend was observed for all particles sizes. PLGA particles were then incubated with MDA-MB-231 breast cancer cells for 6 hours to investigate passive targeting, and the vaporization of the PLGA particles that were internalized within cells. The PLGA particles passively internalized by MDA cells were visualized via confocal fluorescence imaging. Upon PLGA particle vaporization, bubbles formed inside the cells resulting in cell destruction. This work demonstrates that GNPs-loaded PLGA/PFC particles have potential as PA theranostic agents in PA imaging and optically-triggered drug delivery systems.
Red blood cell (RBC) rouleaux formation is a reversible phenomenon that occurs during low blood flow and small shearing forces in circulation. Certain pathological conditions can alter the molecular constituents of blood and properties of the RBCs leading to enhanced rouleaux formation, which results in impaired perfusion and tissue oxygenation. In this study rouleaux were artificially generated using Dextran-70 and examined using a photoacoustic (PA) microscope. Individual rouleau were irradiated with a 532 nm pulsed laser focused to a 10 μm spot size, and the resulting PA signals recorded with a 200 MHz transducer. The laser and transducer were co-aligned, with the sample positioned between them. The frequency-domain PA ultrasound spectra were calculated for rouleaux with lengths ranging from 10 to 20 μm. For the rouleaux, a single spectral minimum at 269±4 MHz was observed. The spectral minima were in good agreement with a theoretical thermoelastic expansion model using an infinite length cylindrical absorber, bearing a diameter equivalent to an average human RBC (7.8 μm). These results suggest that PA ultrasound spectroscopy can be potentially used as a tool for monitoring blood samples for the presence of rouleaux.
We demonstrate a new technique to non-invasively determine the diameter and sound speed of single cells using a
combined ultrasonic and photoacoustic technique. Two cell lines, B16-F1 melanoma cells and MCF7 breast cancer cells
were examined using this technique. Using a 200 MHz transducer, the ultrasound backscatter from a single cell in
suspension was recorded. Immediately following, the cell was irradiated with a 532 nm laser and the resulting
photoacoustic wave recorded by the same transducer. The melanoma cells contain optically absorbing melanin particles,
which facilitated photoacoustic wave generation. MCF7 cells have negligible optical absorption at 532 nm; the cells
were permeabilized and stained with trypan blue prior to measurements. The measured ultrasound and photoacoustic
power spectra were compared to theoretical equations with the cell diameter and sound speed as variables (Anderson
scattering model for ultrasound, and a thermoelastic expansion model for photoacoustics). The diameter and sound speed
were extracted from the models where the spectral shape matched the measured signals. However the photoacoustic
spectrum for the melanoma cell did not match theory, which is likely because melanin particles are located around the
cytoplasm, and not within the nucleus. Therefore a photoacoustic finite element model of a cell was developed where the
central region was not used to generate a photoacoustic wave. The resulting power spectrum was in better agreement
with the measured signal than the thermoelastic expansion model. The MCF7 cell diameter obtained using the spectral
matching method was 17.5 μm, similar to the optical measurement of 16 μm, while the melanoma cell diameter obtained
was 22 μm, similar to the optical measurement of 21 μm. The sound speed measured from the MCF7 and melanoma cell
was 1573 and 1560 m/s, respectively, which is within acceptable values that have been published in literature.
Perfluorocarbon droplets containing nanoparticles (NPs) have recently been investigated as theranostic and dual-mode contrast agents. These droplets can be vaporized via laser irradiation or used as photoacoustic contrast agents below the vaporization threshold. This study investigates the photoacoustic mechanism of NP-loaded droplets using photoacoustic frequencies between 100 and 1000 MHz, where distinct spectral features are observed that are related to the droplet composition. The measured photoacoustic spectrum from NP-loaded perfluorocarbon droplets was compared to a theoretical model that assumes a homogenous liquid. Good agreement in the location of the spectral features was observed, which suggests the NPs act primarily as optical absorbers to induce thermal expansion of the droplet as a single homogenous object. The NP size and composition do not affect the photoacoustic spectrum; therefore, the photoacoustic signal can be maximized by optimizing the NP optical absorbing properties. To confirm the theoretical parameters in the model, photoacoustic, ultrasonic, and optical methods were used to estimate the droplet diameter. Photoacoustic and ultrasonic methods agreed to within 1.4%, while the optical measurement was 8.5% higher; this difference decreased with increasing droplet size. The small discrepancy may be attributed to the difficulty in observing the small droplets through the partially translucent phantom.
Perfluorocarbon droplets containing optical absorbing nanoparticles have been developed for use as theranostic agents
(for both imaging and therapy) and as dual-mode contrast agents. Droplets can be used as photoacoustic contrast agents,
vaporized via optical irradiation, then the resulting bubbles can be used as ultrasound imaging and therapeutic agents.
The photoacoustic signals from micron-sized droplets containing silica coated gold nanospheres were measured using
ultra-high frequencies (100-1000 MHz). The spectra of droplets embedded in a gelatin phantom were compared to a
theoretical model which calculates the pressure wave from a spherical homogenous liquid undergoing thermoelastic
expansion resulting from laser absorption. The location of the spectral features of the theoretical model and experimental
spectra were in agreement after accounting for increases in the droplet sound speed with frequency. The agreement
between experiment and model indicate that droplets (which have negligible optical absorption in the visible and
infrared spectra by themselves) emitted pressure waves related to the droplet composition and size, and was independent
of the physical characteristics of the optical absorbing nanoparticles. The diameter of individual droplets was calculated
using three independent methods: the time domain photoacoustic signal, the time domain pulse echo ultrasound signal,
and a fit to the photoacoustic model, then compared to the diameter as measured by optical microscopy. It was found the
photoacoustic and ultrasound methods calculated diameters an average of 2.6% of each other, and 8.8% lower than that
measured using optical microscopy. The discrepancy between the calculated diameters and the optical measurements
may be due to the difficulty in resolving the droplet edges after being embedded in the translucent gelatin medium.
An acoustic and photoacoustic characterization of micron-sized perfluorocarbon (PFC) droplets is presented. PFC
droplets are currently being investigated as acoustic and photoacoustic contrast agents and as cancer therapy agents.
Pulse echo measurements at 375 MHz were used to determine the diameter, ranging from 3.2 to 6.5 μm, and the sound
velocity, ranging from 311 to 406 m/s of nine droplets. An average sound velocity of 379 ± 18 m/s was calculated for
droplets larger than the ultrasound beam width of 4.0 μm. Optical droplet vaporization, where vaporization of a single
droplet occurred upon laser irradiation of sufficient intensity, was verified using pulse echo acoustic methods. The
ultrasonic backscatter amplitude, acoustic impedance and attenuation increased after vaporization, consistent with a
phase change from a liquid to gas core. Photoacoustic measurements were used to compare the spectra of three droplets
ranging in diameter from 3.0 to 6.2 μm to a theoretical model. Good agreement in the spectral features was observed
over the bandwidth of the 375 MHz transducer.
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