Eric M. Strohm

Probing red blood cell morphology using high-frequency photoacoustics.

A method that can rapidly quantify variations in the morphology of single red blood cells (RBCs) using light and sound is presented. When irradiated with a laser pulse, an RBC absorbs the optical energy and emits an ultrasonic pressure wave called a photoacoustic wave. The power spectrum of the resulting photoacoustic wave contains distinctive features that can be used to identify the RBC size and morphology. When particles 5-10 μm in diameter (such as RBCs) are probed with high-frequency photoacoustics, unique periodically varying minima and maxima occur throughout the photoacoustic signal power spectrum at frequencies >100 MHz. The location and distance between spectral minima scale with the size and morphology of the RBC; these shifts can be used to quantify small changes in the morphology of RBCs. Morphological deviations from the normal biconcave RBC shape are commonly associated with disease or infection. Using a single wide-bandwidth transducer sensitive to frequencies between 100 and 500 MHz, we were able to differentiate healthy RBCs from irregularly shaped RBCs (such as echinocytes, spherocytes, and swollen RBCs) with high confidence using a sample size of just 21 RBCs. As each measurement takes only seconds, these methods could eventually be translated to an automated device for rapid characterization of RBC morphology and deployed in a clinical setting to help diagnose RBC pathology.

Vaporization of perfluorocarbon droplets using optical irradiation.

Micron-sized liquid perfluorocarbon (PFC) droplets are currently being investigated as activatable agents for medical imaging and cancer therapy. After injection into the bloodstream, superheated PFC droplets can be vaporized to a gas phase for ultrasound imaging, or for cancer therapy via targeted drug delivery and vessel occlusion. Droplet vaporization has been previously demonstrated using acoustic methods. We propose using laser irradiation as a means to induce PFC droplet vaporization using a method we term optical droplet vaporization (ODV). In order to facilitate ODV of PFC droplets which have negligible absorption in the infrared spectrum, optical absorbing nanoparticles were incorporated into the droplet. In this study, micron-sized PFC droplets loaded with silica-coated lead sulfide (PbS) nanoparticles were evaluated using a 1064 nm laser and ultra-high frequency photoacoustic ultrasound (at 200 and 375 MHz). The photoacoustic response was proportional to nanoparticle loading and successful optical droplet vaporization of individual PFC droplets was confirmed using photoacoustic, acoustic, and optical measurements. A minimum laser fluence of 1.4 J/cm(2) was required to vaporize the droplets. The vaporization of PFC droplets via laser irradiation can lead to the activation of PFC agents in tissues previously not accessible using standard ultrasound-based techniques.

High frequency label-free photoacoustic microscopy of single cells

Photoacoustic measurements of melanoma cells and red blood cells (RBCs) using ultra-high frequency (UHF) wide-bandwidth transducers are reported. In this detection system, the resolution typically depends on the parameters of the receiving transducer, and not the focus of the laser. A single melanoma cell was imaged with 200, 375 and 1200 MHz transducers. As the frequency increased, the resolution increased, resulting in greater detail observed. A single RBC was imaged at 1200 MHz, showing the contours of the cell. While lateral and axial resolutions approaching 1 μm are possible with this microscope, the key advantage is the ability to perform a wide-bandwidth quantitative signal analysis of the photoacoustic signals. The power spectrum of the signals measured from RBCs showed distinct spectral minima around 800 and 1500 MHz which are directly related to the RBC geometry. This study reports on the high-resolution imaging capabilities and quantitative analyses using UHF photoacoustic microscopy.

Quantitative measurements of apoptotic cell properties using acoustic microscopy

Time-resolved acoustic microscopy was used to measure properties of cells such as the thickness, sound velocity, acoustic impedance, density, bulk modulus, and attenuation, before and after apoptosis. A total of 12 cells were measured, 5 apoptotic and 7 non-apoptotic. Measurements made at 375 MHz showed a statistically significant increase in the cell thickness from 13.6 ± 3.1 μm to 17.3 ± 1.6 μm, and in the attenuation from 1.08 ± 0.21 dB/cm/MHz to 1.74 ± 0.36 dB/cm/MHz. The other parameters, such as the sound velocity, density, acoustic impedance, and bulk modulus remained similar within experimental error. Acoustic images obtained at 1.0 GHz showed increased RF-signal backscatter and a clear delineation of the nucleus and cytoplasm from apoptotic cells compared with non-apoptotic cells. Extensive activity was observed optically and acoustically within apoptotic cells. Acoustic measurements made one minute apart showed variations in the ultrasonic backscatter but not attenuation in the cells, which indicated rapid structural changes were occurring but not changes in bulk composition. The normalized crosscorrelation coefficient was used to quantify the variations in the backscatter RF-signal during apoptosis by comparing the first RF signal measured to each successive RF signal every 10 s. A coefficient of 1 indicates strong correlation, whereas a coefficient of 0 indicates no correlation. An average correlation coefficient of 0.93 ± 0.05 was measured for non-apoptotic cells, compared with 0.68 ± 0.17 for apoptotic cells, indicating that the RF signal as a function of time varied rapidly during apoptosis.

Single Cell Photoacoustic Microscopy: A Review

Photoacoustic imaging has experienced exponential growth over the past decade, with many applications in biomedicine. One application ideally suited to the analysis of single cells is photoacoustic microscopy (PAM). Using PAM, detailed submicrometer resolution images of single cells can be produced, with contrast dependent primarily on the optical absorption properties of the cell. A multiwavelength approach for targeting specific endogenous or exogenous chromophores can enhance cellular detail and resolve single organelles with contrast not possible with traditional optical microscopy. A quantitative analysis of the photoacoustic signals acquired from single cells can provide insight into their anatomical, biomechanical, and functional properties. This information can be used to identify specific cells, or to enhance the understanding of biological processes at the single cell level. This comprehensive review on PAM covers recent advances in high-resolution PAM, signal processing methods, and potential clinical applications targeting single cells in vitro and in vivo.

Acoustic and photoacoustic characterization of micron-sized perfluorocarbon emulsions

Abstract. 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.

High resolution ultrasound and photoacoustic imaging of single cells

Graphical abstract

Modeling photoacoustic spectral features of micron-sized particles

The photoacoustic signal generated from particles when irradiated by light is determined by attributes of the particle such as the size, speed of sound, morphology and the optical absorption coefficient. Unique features such as periodically varying minima and maxima are observed throughout the photoacoustic signal power spectrum, where the periodicity depends on these physical attributes. The frequency content of the photoacoustic signals can be used to obtain the physical attributes of unknown particles by comparison to analytical solutions of homogeneous symmetric geometric structures, such as spheres. However, analytical solutions do not exist for irregularly shaped particles, inhomogeneous particles or particles near structures. A finite element model (FEM) was used to simulate photoacoustic wave propagation from four different particle configurations: a homogeneous particle suspended in water, a homogeneous particle on a reflecting boundary, an inhomogeneous particle with an absorbing shell and non-absorbing core, and an irregularly shaped particle such as a red blood cell. Biocompatible perfluorocarbon droplets, 3-5 μm in diameter containing optically absorbing nanoparticles were used as the representative ideal particles, as they are spherical, homogeneous, optically translucent, and have known physical properties. The photoacoustic spectrum of micron-sized single droplets in suspension and on a reflecting boundary were measured over the frequency range of 100-500 MHz and compared directly to analytical models and the FEM. Good agreement between the analytical model, FEM and measured values were observed for a droplet in suspension, where the spectral minima agreed to within a 3.3 MHz standard deviation. For a droplet on a reflecting boundary, spectral features were correctly reproduced using the FEM but not the analytical model. The photoacoustic spectra from other common particle configurations such as particle with an absorbing shell and a biconcave-shaped red blood cell were also investigated, where unique features in the power spectrum could be used to identify them.

Sound velocity and attenuation measurements of perfluorocarbon liquids using photoacoustic methods

A method to measure the phase velocity and attenuation of liquids over a frequency range of 100-1000 MHz using a photoacoustic method is presented. A pulsed laser directed at a thin gold or ink layer was used to create a broadband ultrasonic wave and was used as the source. An ultrasound transducer (375 or 750 MHz center frequency) positioned above the source was used as the receiver. The phase and amplitude spectra of signals transmitted through a liquid between the photoacoustic source and ultrasound receiver were used to determine the phase velocity and attenuation as a function of frequency. The method was first validated using water, ethanol and castor oil. The phase velocity and attenuation were similar to published values. Water and ethanol showed no dispersion with frequency, while castor oil increased from 1508 m/s at 200 MHz to 1561 m/s at 700 MHz. Three perfluorocarbon (PFC) liquids (perfluorohexane, perfluoroheptane and FC-77) were then measured. The phase velocity was 480, 516 and 557 m/s at 200 MHz for perfluorohexane, perfluoroheptane and FC-77, respectively, and increased by approximately 1.5% at 700 MHz. The attenuation was similar for all three PFC liquids at 0.352f1.56 dB/cm/MHzn.

Optical droplet vaporization (ODV): Photoacoustic characterization of perfluorocarbon droplets

Optical droplet vaporization (ODV) of nanoscale and micron-sized liquid perfluorocarbon (PFC) droplets via a 1064 nm laser is presented. The stability and laser fluence threshold were investigated for PFC compounds with varying boiling points. Using an external optical absorber to facilitate droplet vaporization, it was found that droplets with boiling points at 29°C and 56°C were consistently vaporized upon laser irradiation using a fluence of 0.7 J/cm2 or greater, while those with higher boiling points did not, up to a maximum laser fluence of 3.8 J/cm2. Upon vaporization, the droplet rapidly expanded to approximately 10–20× the original diameter, then slowly and continuously expanded at a rate of up to 1 µm/s. Lead sulphide (PbS) nanoparticles were incorporated into perfluoropentane (PFP) droplets to facilitate vaporization. The fluence threshold to induce vaporization ranged from 0.8 to 1.6 J/cm2, the wide range likely due to variances of the PbS concentration within the droplets. Prior to vaporization, the photoacoustic spectral features of individual droplets 2–8 µm in diameter measured at 375 MHz agreed very well with the theoretical prediction using a liquid sphere model. In summary, the use of liquid droplets for photoacoustic imaging and cancer therapy has been demonstrated.