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.

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.

Current methods to evaluate the status of a cell are largely focused on fluorescent identification of molecular biomarkers. The invasive nature of these methods -requiring either fixation, chemical dyes, genetic alteration, or a combination of these -prevents subsequent analysis of samples. In light of this limitation, studies have considered the use of physical markers to differentiate cell stages. Acoustic microscopy is an ultrahigh frequency (>100 MHz) ultrasound technology that can be used to calculate the mechanical and physical properties of biological cells in realtime, thereby evaluating cell stage in live cells without invasive biomarker evaluation. Using acoustic microscopy, MCF-7 human breast adenocarcinoma cells within the G1, G2, and metaphase phases of the proliferative cell cycle, in addition to early and late programmed cell death, were examined. Physical properties calculated include the cell height, sound speed, acoustic impedance, cell density, adiabatic bulk modulus, and the ultrasonic attenuation. A total of 290 cells were measured, 58 from each cell phase, assessed using fluorescent and phase contrast microscopy. Cells actively progressing from G1 to metaphase were marked by a 28% decrease in attenuation, in contrast to the induction of apoptosis from G1, which was marked by a significant 81% increase in attenuation. Furthermore late apoptotic cells separated into 2 distinct groups based on ultrasound attenuation, suggesting that presently-unidentified sub-stages may exist within late apoptosis. A methodology has been implemented for the identification of cell stages without the use of chemical dyes, fixation, or genetic manipulation.

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