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Scanning acoustic microscopy (SAM) is a useful observational tool in cellular study as non-invasive living observation is feasible, unlike in conventional optical microscopy. In a previous study, cell morphological changes were successfully visualized using acoustic impedance measurements. These acoustic impedance changes correspond to cell elasticity, mainly reflecting changes in the cytoskeleton. In this study, we evaluate the elastic changes in murine breast cancer cell C127I during mitosis. C127I cells were cultured to ∼75% confluency before measurement, using a transducer with a central frequency of 320 MHz. Dynamic changes during mitosis were successfully mapped using SAM and confirmed by laser confocal microscopy. Cells in prometaphase, anaphase, and telophase, which could previously only be confirmed through immunostaining, were successfully visualized using SAM. This suggests that SAM is capable of distinguishing cells in different mitotic phases based on the changes in acoustic impedance.

Herein, we propose a method to estimate the reflection coefficient of the ultrasonic wave transmitted onto an object and to display this with acoustic impedance distribution. The observation targets were glial cells, which have a rigid cytoskeleton and spread out well on a culture substrate. A reflection coefficient derived only from the cells was then obtained using a deconvolution process. In the conventional method, the deconvolution process that was performed only in the frequency domain would cause an error in the reconstructed signal, and it formed an artifact when the result was converted into the acoustic impedance image. To solve this problem, two types of deconvolution techniques were applied in either the full frequency or time–frequency domain. The results of both methods were then compared. Since the characteristic acoustic impedance is a physical property substantially equivalent to the bulk modulus, it can be considered that the internal elastic parameter is thus estimated. An analysis of the nucleus based on its position in the acoustic impedance image was then performed. The results indicated that the proposed time–frequency domain deconvolution method is able to maintain the structure of the cell, while the cell itself is free from unwanted artifacts. The nucleus was also estimated to be located toward the center of the cell, with lower acoustic impedance value than the cytoskeleton. The results of this study could contribute to establishing a method for monitoring the internal condition of cultured cells in regenerative medicine and drug discovery.

Ultrasound microscope is a useful tool for living organ and tissues observation as it works quickly and nondestructively without chemical staining. Previously, the cytoskeleton was successfully mapped by utilizing the cell elasticity, which corresponds to changes in acoustic impedance. In this research, we switched the focus to the intracellular dynamics to illustrate the biological phenomenon under cellular level. 0.05 μg/ml of demecolcine were applied to C1271 breast cancer cells to synchronize the cell cycle in mitotic phase. The result revealed that phenomena in the mitosis which until now is only observable through optical microscopy is achievable in ultrasound microscope. This implies that non-invasive intracellular observation of cancer cells is feasible, and this technique can improve the current anticancer drug development.

The mechanical properties of living cells are known to be associated with disease states and cell function. In this study, acoustic impedance microscopy using a sapphire lens transducer with a center frequency of 320 MHz was employed to characterize the elasticity of the C127I cell line against an anticancer drug, nimustine hydrochloride (ACNU), and an anticancer agent, betulinic acid (BA). Confocal laser scanning microscopy was also used to investigate the drug affecting actin filaments, the nucleus, and mitochondria structures. Breast cancer cells were found to have significantly lower acoustic impedance after treatment with ACNU and BA than intact cells. Confocal images showed a significant difference in the localization of actin filaments and mitochondria structures, which suggested a difference in cell elasticity. An important insight emerging from this work is that the acoustic impedance of cells may potentially serve as a useful biomarker for anticancer drug efficacy tests, as diseases such as cancer have their own particular mechanical properties.

An ultrasonic microscope is a useful tool for observing living tissue without chemical fixation or histochemical processing. Two-dimensional (2D) acoustic impedance microscopy developed in our previous study for living cell observation was employed to visualize intracellular changes. We proposed a brain tumor model by cocultivating rat glial cells and C6 gliomas to quantitatively analyze the effects of two types of anticancer drugs, cytochalasin B (CyB) and temozolomide (TMZ), when they were applied. We reported that CyB treatment (25 µg/ml, T = 90 min) significantly reduced the acoustic impedance of gliomas and has little effect on glial cells. Meanwhile, TMZ treatment (2 mg/ml, T = 90 min) impacted both cells equally, in which both cells' acoustic impedances were decreased. As CyB targets the actin filament polymerization of the cells, we have concluded that the decrease in acoustic impedance was in fact due to actin filament depolymerization and the data can be quantitatively assessed for future studies in novel drug development.