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.

The size, shape, and intrinsic optical absorption properties of RBCs make them ideal candidates for high-frequency quantitative photoacoustic methods. The photoacoustic signal can be used to infer the RBC size and shape, and thus give further insight into the specific abnormality observed than do current clinical methods. These methods could be translated into an automated device capable of measuring a large sample size for a rapid determination of RBC morphology and thus pathology.

Our photoacoustic microscope uses transducers with frequencies >100 MHz, with bandwidths in the hundreds of MHz (). Like other photoacoustic microscopes, it produces images with micrometer resolution (). However, the distinct advantage of this microscope is the broad ultrasound/photoacoustic frequency bandwidth, which can be used for a quantitative analysis of micron-sized particles (). For any particle in the 1–50 μm size range (such as RBCs), unique features in the photoacoustic spectrum over 100 MHz are observed. These periodically varying spectral minima and maxima depend strongly on the size, morphology, orientation, and composition of the particle (). Quantitative analysis of the photoacoustic spectra can be used to help identify these parameters and extract information from the particle examined.

RBCs contain large amounts of hemoglobin, a molecule capable of binding oxygen. Hemoglobin significantly absorbs visible light, whereas other tissues do not, thus making blood an ideal contrast agent for photoacoustic imaging in vivo (). After absorbing energy, the particles rapidly increase in temperature and pressure, resulting in a thermoelastic expansion and emission of a photoacoustic wave. These waves can be detected using conventional ultrasound transducers with frequency sensitivities typically between 10 and 40 MHz (). This effect has been used in vivo for functional imaging (), photoacoustic tomography (), the detection of tumors (), and imaging of vasculature with millimiter-scale resolution (). The photoacoustic effect can be exploited on the microscale using a photoacoustic microscope (). In this system, conventional ultrasound transducers are combined with a highly focused laser to create in vitro and in vivo micrometer-resolution images of vasculature (), and even of single cells (). Current photoacoustic microscopes use inexpensive conventional transducers to produce stunning microsized images with excellent contrast; however, they have a limited photoacoustic frequency range of operation.

Clinical diagnoses of blood disorders use a set of indices to provide information about individual RBC physical characteristics. The average RBC is ∼7.8 μm in diameter and 1–2 μm in height and has a 94 μmvolume (). Current automated diagnostic methods use electrical impedance or light-scattering methods to determine the RBC concentration and mean corpuscular volume. In healthy RBCs, the diameter can be inferred from the volume, as these parameters are directly related (), but this relation may break down for abnormally shaped RBCs. These tools cannot determine the dimensions or shape directly, indicating only whether a sample is outside the accepted guidelines. Because of their limited ability to diagnose pathology, additional testing is required to identify abnormal RBC morphologies such as spherocytes (spherical RBCs) or echinocytes (spherical shape with crenations). Methods to determine the individual RBC shape through blood smears or optical interference methods () are laborious and time intensive, prohibiting analysis of large sample sizes.

Red blood cells (RBCs) have a flexible biconcave disk shape that enables efficient transport of oxygen to the peripheral cells of the body through the narrow and tortuously winding capillary system (). Disease, infection, genetic disorders, and variations in blood chemistry can alter the RBC shape, reducing its ability to bend and deform (). Abnormal RBC morphologies can impede or even obstruct the circulation, causing tissue necrosis in severe cases.

Materials and Methods

RBCs were extracted from a healthy male volunteer in accordance with the guidelines of the Ryerson Ethics Board (REB #2012-210). A drop of blood was drawn from the subject’s finger using a lancet and immediately deposited in 1 mL Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum to maintain viability (Sigma Aldrich, St. Louis, MO). All measurements were made on 35 mm glass-bottom dishes (Mattek Corporation, Ashland, MA). The dishes were coated with a thin layer of 0.5% agar ∼200 μm thick to reduce photoacoustic back reflections from the glass substrate. Approximately 30 μL of the DMEM-RBC solution was deposited into 2 mL of coupling fluid inside the glass-bottom dish to dilute the sample.

For the orientation and echinocyte measurements, DMEM was used as the coupling fluid. For the osmolality measurements, phosphate-buffered saline (PBS) was used as the coupling fluid. For both the PBS and DMEM liquids, osmolality was measured three times using a Vapro 5520 vapor pressure osmometer (Wescor, Logan, UT) and was found to be, on average, 294 and 332 mmol/kg, respectively. The standard deviation of three measurements was ±10 mmol/kg. The osmolality of PBS solutions was adjusted by adding water. For the ATP-depletion procedure, the extracted blood was stored in PBS. A small sample of the PBS-RBC solution was deposited on the glass-bottom dish containing 2 mL DMEM, and 21 RBCs were measured. The PBS-RBC solution was stored at 4°C for 24 h to induce echinocyte formation, and a small sample was then added to a glass-bottom dish containing 2 mL PBS. Again, 21 RBCs were measured. Another PBS-RBC sample was added to a glass-bottom dish containing 2 mL DMEM and an additional 21 RBCs were measured. All measurements were performed at 36°C.

2 at the sample. For the orientation measurements, photoacoustic signals were recorded approximately once per second. For all other measurements, the RBC was centered over the laser spot and the photoacoustic signal was recorded. A Hamming window was applied to the measured signals, and the normalized power spectrum, P(f), was then calculated using the formula P ( f ) = 20 log 10 | P m ( f ) P n ( f ) | ,

where P m (f) is the Fourier transform of the measured signal p m (t), and P n (f) is the Fourier transform of p n (t), which is the spectral response of the transducer system and electronics. This normalization signal is used to remove any artifacts due to the transducer/electronics of the system; it is generated by a 200 nm thick gold film that is spectrally flat over the transducer bandwidth, with variations of <3 dB from 100–1000 MHz. The normalization signal is typically recorded using pulse-echo ultrasound from an ultrasonically flat reflective surface ( 22 Baddour R.E.

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Kolios M.C. Quantitative measurements of apoptotic cell properties using acoustic microscopy. Figure 1 The photoacoustic microscope. (a) The photoacoustic microscope. The sample is positioned between the transducer and the optical objective. (b) RBCs after deposition into the sample holder. Most RBCs align horizontally on the substrate, but a small number align vertically (arrow). Scale bar, 20 μm. (c) A schematic showing the sample positioning relative to the transducer and focused laser. Figure 1 (a) used with permission from Robert Lemor at Kibero GmbH. All measurements were completed using a photoacoustic microscope developed by Kibero (Saarbrücken, Germany). This is an Olympus IX81 inverted optical microscope fitted with a transducer above the sample stage ( Fig. 1 A). The sample was positioned between the optical objective and the transducer. The system optics allows for viewing the sample and aligning the transducer; it also focuses the laser onto the sample. A dilute suspension of freshly extracted human RBCs was deposited into the liquid in the sample holder. Most RBCs would fall to the substrate and align horizontally, but some would initially come to rest in a vertical orientation ( Fig. 1 B); these would eventually tip over to a horizontal orientation. The laser was focused onto single RBCs and the transducer recorded the resulting photoacoustic wave ( Fig. 1 C). The photoacoustic signals of RBCs were measured in both a vertical and horizontal orientation. The diameter was obtained from the optical images for comparison to theoretical simulations. A transducer with a center frequency of 375 MHz and f number of 1 (Kibero) was used for all measurements. A 532 nm laser (Teem Photonics, Meylan, France) was focused by a 10× optical objective to an ∼10 μm spot size on the sample, sufficient to irradiate a single RBC. The laser had a pulse width of 330 ps and a pulse repetition frequency of 4 kHz. Signals were amplified by a 40 dB amplifier (Miteq, Hauppage, NY), digitized at 8 GS/s using a DC252 digitizer (Agilent, Santa Clara, CA), and averaged 100–200 times to increase the signal/noise ratio. The laser power was measured at the objective using a Nova II power meter and PD10 low energy sensor (Ophir Optronics, Jerusalem, Israel). The laser power was adjusted so that the laser fluence was 20–150 mJ/cmat the sample. For the orientation measurements, photoacoustic signals were recorded approximately once per second. For all other measurements, the RBC was centered over the laser spot and the photoacoustic signal was recorded. A Hamming window was applied to the measured signals, and the normalized power spectrum, P(f), was then calculated using the formulawhere P(f) is the Fourier transform of the measured signal p(t), and P(f) is the Fourier transform of p(t), which is the spectral response of the transducer system and electronics. This normalization signal is used to remove any artifacts due to the transducer/electronics of the system; it is generated by a 200 nm thick gold film that is spectrally flat over the transducer bandwidth, with variations of <3 dB from 100–1000 MHz. The normalization signal is typically recorded using pulse-echo ultrasound from an ultrasonically flat reflective surface (), but the transducer bandwidth for photoacoustic measurements is larger than that for pulse-echo ultrasound. Pulse-echo ultrasound waves also travel twice the distance of photoacoustic waves emitted from the sample, and they are therefore attenuated more strongly than photoacoustic waves. More specific details about the equipment, signal processing methods, and normalization procedure can be found elsewhere ().