Ultrasonic Characterization of Whole Cells and Isolated Nuclei

Abstract

High frequency ultrasound imaging (20 to 60 MHz) is increasingly being used in small animal imaging, molecular imaging and for the detection of structural changes during cell and tissue death. Ultrasonic tissue characterization techniques were used to measure the speed of sound, attenuation coefficient and integrated backscatter coefficient for (a) acute myeloid leukemia cells and corresponding isolated nuclei, (b) human epithelial kidney cells and corresponding isolated nuclei, (c) multinucleated human epithelial kidney cells and d) human breast cancer cells. The speed of sound for cells varied from 1522 to 1535 m/s, while values for nuclei were lower, ranging from 1493 to 1514 m/s. The attenuation coefficient slopes ranged from 0.0798 to 0.1073 dB mm⁻¹ MHz⁻¹ for cells and 0.0408 to 0.0530 dB mm⁻¹ MHz⁻¹ for nuclei. Integrated backscatter coefficient values for cells and isolated nuclei showed much greater variation and increased from 1.71 × 10⁻⁴ Sr⁻¹ mm⁻¹ for the smallest nuclei to 26.47 × 10⁻⁴ Sr⁻¹ mm⁻¹ for the cells with the largest nuclei. The findings suggest that integrated backscatter coefficient values, but not attenuation or speed of sound, are correlated with the size of the nuclei. (E-mail: [email protected])

Introduction

Ultrasound imaging is the most frequently used clinical imaging modality, accounting for almost 25% of all imaging procedures (Forsberg 2003). Recent advances in transducer technology and electronics have increased ultrasonic frequencies to 20 to 60 MHz, providing better image resolution at the expense of reduced ultrasound penetration depth (Foster et al. 2000). The associated ultrasound wavelengths are of the order of 25 to 75 μm, the same order of magnitude as the size of cells. Imaging cell ensembles results in a speckle pattern because, even at these high frequencies, individual cells cannot be resolved. Recent applications of high-frequency, ultrasound imaging (often referred to as ultrasound biomicroscopy) are in the fields of developmental and tumor biology (Foster et al. 2000), molecular imaging (Liang et al. 2003), ophthalmology (Pavlin et al. 1991), tumor characterization (Oelze et al. 2004) and monitoring anticancer treatment effects (Czarnota et al. 1999). Tissue scattering and attenuation at these frequencies are not well understood, necessitating ultrasonic characterization experiments.

Our group has shown that high-frequency ultrasound can detect changes in cell morphology during various forms of cell death (Czarnota et al 2002, Kolios et al 2002, Kolios et al 2003, Tunis et al 2005). One such process is apoptosis, a significant process in normal prenatal development and potentially in the response of tumors to anticancer agents (Hengartner 2000). The most striking morphologic features of apoptosis are the condensation and fragmentation of the nucleus, as well as blebbing of the cell membrane (Hengartner 2000). We have shown that high-frequency ultrasound is sensitive to apoptosis in vitro and in vivo (Czarnota et al. 1999). Apoptotic cells and tissues can exhibit up to a sixteen-fold increase in backscatter intensity in comparison with viable cells, as well as subtle changes in the power spectrum. Moreover, similar changes in ultrasound backscatter have been detected in cells (Kolios et al. 2003) and tissues (Vlad et al. 2005) exposed to lethal ischemic insults. These backscatter changes are not well understood because the nature of the interaction between high-frequency ultrasound waves and cellular or nuclear scatterers is not yet fully known. Many studies have been performed on ultrasonic tissue characterization at lower frequencies (for a review see Shung and Thieme 1993) and recent work has been done at higher frequencies for tissue such as skin (Raju and Srinivasan 2001), eye (Ursea et al. 1998) and blood (Cloutier et al 2004, Lupotti et al 2004). Work using tumors and cell systems has shown the potential for tissue characterization, especially at higher frequencies (Kolios et al 2002, Kolios et al 2004, Oelze et al 2004, Tunis et al 2004). An examination of ultrasonic parameters from cells with different sizes and different properties may yield valuable insights into the backscattering process.

In this study, we ultrasonically characterized various types of cells and their isolated nuclei that differ in size and, most likely, mechanical properties. Measurement of the speed of sound, attenuation and integrated backscatter coefficient of these samples provides information regarding the physical characteristics of the cells and the nature of the scatterers. These results may also play an important role in the development of quantitative models of the scattering process (Baddour et al. 2005), which in turn could be utilized to generate parametric images of diagnostic relevance to clinicians and also could aid in the design of appropriate pulsing sequences to enhance tissue contrast.