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.