Noninvasive imaging of rat-derived microglia and its reactivity to inflammatory molecules via acoustic impedance microscopy.

PURPOSE: Microglia, the brain's immune cells, play important roles in neuronal differentiation, survival, and death. The function of microglia is deeply related to the morphologies; however, it is too complex to observe conventionally and identify the condition of living microglia using optical microscopes. Herein, we proposed a new method to observe living cultured microglia and their reactivity to inflammation via the acoustic impedance mode of a scanning acoustic microscope. METHODS: Primary cultured microglia collected from rat pups exposed to acetamiprid, an insecticide, in utero were observed with both acoustic interface impedance mode (C-mode) and transparent three-dimensional impedance mode (B-mode). RESULTS: We characterized microglia into four types based on the results obtained from acoustic impedance, cytoskeletal information, and laser confocal imaging. Biphasic acoustic observation using B-mode and C-mode gave us information regarding the dynamic morphologies of living microglia treated with adenosine triphosphate (ATP) (600 µmol/L), which reflects distress signals from inflamed neurons. Acetamiprid exposure induced microglia response even in the neonatal period. ATP stimulus altered the shape and thickness of microglia with a change in the bulk modulus of the cell. Three-dimensional alteration with ATP stimulus could be observed only after biphasic acoustic observation using B-mode and C-mode. This acoustic observation was consistent with confocal observation using anti-Iba-1 and P2Y12 immunocytochemistry. CONCLUSION: This study demonstrated the adequacy of using a scanning acoustic microscope in analyzing microglia's shape, motility, and response to inflammation.

Time and frequency domain deconvolution for cross-sectional cultured cell observation using an acoustic impedance microscope.

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.

Suppression of reflected signals from substrate as clutters for cell measurements using acoustic impedance microscopy.

Recently, a method for estimating three-dimensional acoustic impedance profiles in cultured cells and human dermal organs was proposed by interpreting the reflected ultrasonic signal based on a 1-D transmission line model for acoustic impedance microscopy (AIM). However, AIM has a disadvantage that reflected signals from cells overlap with that from a reference substrate. Additionally, the amplitudes of the reflected signals from the specimens are significantly weaker than that from the substrate. In this paper, we proposed a new method for separation of those signals based on a concept of clutter filter, which had been developed for a color Doppler method in medical ultrasonic imaging. The proposed filter using singular value decomposition (SVD) could separate original signals into desired signals such as those from the substrate and cells. Additionally, an effect from a tilt of the substrate was investigated in this study. Separability of the proposed filter was evaluated by two investigations. First one was to evaluate the separability by estimating a correlation coefficient between the filtered signal and signal reflected from a position only with the substrate. Second one was to compare a slope of the substrate estimated from the original signal with that estimated from the filtered signals from the substrate. The experimental results showed that the proposed filter could separate signals from the substrate, and the compensation of the tilt of the substrate could improve the performance of the proposed filter.

Three-dimensional acoustic impedance mapping of cultured biological cells.

The acoustic microscope is a powerful tool for the observation of biological matters. Non-invasive in-situ observation can be performed without any staining process. Acoustic microscopy is contrasted by elastic parameters like sound speed and acoustic impedance. We have proposed an acoustic microscope that can acquire three-dimensional acoustic impedance profile. The technique was applied to cell-size observation. Glial cells were cultured on a 70 µm-thick polypropylene film substrate. A highly focused ultrasound beam was transmitted from the rear side of the substrate, and the reflection was received by the same transducer. An acoustic pulse, its spectrum spreading briefly 100 through 450 MHz, was transmitted. By analyzing the internal reflections in the cell, the distribution of characteristic acoustic impedance along the beam direction was determined. Three-dimensional acoustic impedance mapping was realized by scanning the transducer, exhibiting the intra-cellular structure including nucleus and cytoskeleton.

Numerical analysis of acoustic impedance microscope utilizing acoustic lens transducer to examine cultured cells.

A new technique is proposed for non-contact quantitative cell observation using focused ultrasonic waves. This technique interprets acoustic reflection intensity into the characteristic acoustic impedance of the biological cell. The cells are cultured on a plastic film substrate. A focused acoustic beam is transmitted through the substrate to its interface with the cell. A two-dimensional (2-D) reflection intensity profile is obtained by scanning the focal point along the interface. A reference substance is observed under the same conditions. These two reflections are compared and interpreted into the characteristic acoustic impedance of the cell based on a calibration curve that was created prior to the observation. To create the calibration curve, a numerical analysis of the sound field is performed using Fourier Transforms and is verified using several saline solutions. Because the cells are suspended by two plastic films, no contamination is introduced during the observation. In a practical observation, a sapphire lens transducer with a center frequency of 300 MHz was employed using ZnO thin film. The objects studied were co-cultured rat-derived glial (astrocyte) cells and glioma cells. The result was the clear observation of the internal structure of the cells. The acoustic impedance of the cells was spreading between 1.62 and 1.72 MNs/m(3). Cytoskeleton was indicated by high acoustic impedance. The introduction of cytochalasin-B led to a significant reduction in the acoustic impedance of the glioma cells; its effect on the glial cells was less significant. It is believed that this non-contact observation method will be useful for continuous cell inspections.

Numerical analysis of ultrasound propagation and reflection intensity for biological acoustic impedance microscope.

This paper proposes a new method for microscopic acoustic imaging that utilizes the cross sectional acoustic impedance of biological soft tissues. In the system, a focused acoustic beam with a wide band frequency of 30-100 MHz is transmitted across a plastic substrate on the rear side of which a soft tissue object is placed. By scanning the focal point along the surface, a 2-D reflection intensity profile is obtained. In the paper, interpretation of the signal intensity into a characteristic acoustic impedance is discussed. Because the acoustic beam is strongly focused, interpretation assuming vertical incidence may lead to significant error. To determine an accurate calibration curve, a numerical sound field analysis was performed. In these calculations, the reflection intensity from a target with an assumed acoustic impedance was compared with that from water, which was used as a reference material. The calibration curve was determined by changing the assumed acoustic impedance of the target material. The calibration curve was verified experimentally using saline solution, of which the acoustic impedance was known, as the target material. Finally, the cerebellar tissue of a rat was observed to create an acoustic impedance micro profile. In the paper, details of the numerical analysis and verification of the observation results will be described.

Acoustic impedance microscopy for biological tissue characterization.

A new method for two-dimensional acoustic impedance imaging for biological tissue characterization with micro-scale resolution was proposed. A biological tissue was placed on a plastic substrate with a thickness of 0.5mm. A focused acoustic pulse with a wide frequency band was irradiated from the "rear side" of the substrate. In order to generate the acoustic wave, an electric pulse with two nanoseconds in width was applied to a PVDF-TrFE type transducer. The component of echo intensity at an appropriate frequency was extracted from the signal received at the same transducer, by performing a time-frequency domain analysis. The spectrum intensity was interpreted into local acoustic impedance of the target tissue. The acoustic impedance of the substrate was carefully assessed prior to the measurement, since it strongly affects the echo intensity. In addition, a calibration was performed using a reference material of which acoustic impedance was known. The reference material was attached on the same substrate at different position in the field of view. An acoustic impedance microscopy with 200×200 pixels, its typical field of view being 2×2 mm, was obtained by scanning the transducer. The development of parallel fiber in cerebella cultures was clearly observed as the contrast in acoustic impedance, without staining the specimen. The technique is believed to be a powerful tool for biological tissue characterization, as no staining nor slicing is required.

High frequency ultrasound imaging of surface and subsurface structures of fingerprints.

High frequency ultrasound is suitable for non-invasive evaluation of skin because it can obtain both morphological and biomechanical information. A specially developed acoustic microscope system with the central frequency of 100 MHz was developed. The system was capable of (1) conventional C-mode acoustic microscope imaging of thinly sliced tissue, (2) ultrasound impedance imaging of the surface of in vivo thick tissue and (3) 3D ultrasound imaging of inside of the in vivo tissue. In the present study, ultrasound impedance imaging and 3D ultrasound imaging of in vivo fingerprints were obtained. The impedance image showed pores of the sweat glands in the surface of fingerprint and 3D ultrasound imaging showed glands of the rear surface of fingerprint. Both findings were not visualized by normal optical imaging, thus the system can be applied to pathological diagnosis of skin lesions and assessment of aging of the skin in cosmetic point of view.

High frequency ultrasound characterization of artificial skin.

Regenerated skin with 3-dimensional structure is desired for the treatment of large burn and for the plastic surgery. High frequency ultrasound is suitable for non-destructive testing of the skin model because it provides information on morphology and mechanical properties. In this paper, spectral parameters of ultrasound radio-frequency signal from a specially developed high-frequency ultrasound imaging system were evaluated for tissue characterization of artificial skin. Results suggest that spectral parameters are useful for classification of epidermis and dermis in the artificial skin model. The system is also a useful tool for the noninvasive and nondestructive evaluation of skin.

Ultrasound speed and impedance microscopy for in vivo imaging.

Ultrasound speed and impedance microscopy was developed in order to develop in vivo imaging system. The sound speed mode realized non-contact high resolution imaging of cultured cells. This mode can be applied for assessment of biomechanics of the cells and thinly sliced tissues. The impedance mode visualized fine structures of the surface of the rat's brain. This mode can be applied for intra-operative pathological examination because it does not require slicing or staining.

Ultrasonic tissue characterization of atherosclerosis by a speed-of-sound microscanning system.

We have been developing a scanning acoustic microscope (SAM) system for medicine and biology featuring quantitative measurement of ultrasonic parameters of soft tissues. In the present study, we propose a new concept sound speed microscopy that can measure the thickness and speed of sound in the tissue using fast Fourier transform of a single pulsed wave instead of burst waves used in conventional SAM systems. Two coronary arteries were frozen and sectioned approximately 10 microm in thickness. They were mounted on glass slides without cover slips. The scanning time of a frame with 300 x 300 pixels was 90 s and two-dimensional distribution of speed of sound was obtained. The speed of sound was 1680 +/- 30 m/s in the thickened intima with collagen fiber, 1520 +/- 8 m/s in the lipid deposition underlying the fibrous cap, and 1810 +/- 25 m/s in a calcified lesion in the intima. These basic measurements will help in the understanding of echo intensity and pattern in intravascular ultrasound images.

Ultrasonic speed microscopy for imaging of coronary artery.

We have been developing a scanning acoustic microscope (SAM) system for medicine and biology featuring quantitative measurement of ultrasonic speed and attenuation of soft tissues. In the present study, we will propose a new concept ultrasonic speed microscopy that can measure the thickness and ultrasonic speed using fast Fourier transform of a single pulsed wave instead of continuous waves used in conventional SAM systems. Six coronary arteries were frozen and sectioned approximately 10 microm in thickness. They were mounted on glass slides without cover slips. The scanning time of a frame with 300 x 300 pixels was 121 s and two-dimensional distribution of ultrasonic speed was obtained. The ultrasonic speed was 1720 m/s in the thickened intima with collagen fiber, 1520 m/s in lipid deposition underlying fibrous cap and 1830 m/s in calcified lesion in the intima. These basic measurements will help understanding echogenecity in intravascular ultrasound (IVUS) images. Imaging of coronary artery with the ultrasonic speed microscopy provides important information for study of IVUS coronary imaging.

Sound speed scanning acoustic microscopy for biomedical applications.

Since 1985, we have been developing a scanning acoustic microscope (SAM) system for biomedical use and have been investigating the acoustic properties of various organs and disease states by using this SAM system. In biomedicine, SAM is useful for intraoperative pathological examination, study of low-frequency ultrasonic images, and assessment of biomechanics at a microscopic level. Recently, we have proposed a new concept -- acoustic microscopy -- using a single pulsed wave instead of continuous waves used in conventional SAM systems. In the present study, we compared two systems by measuring the same biological material. The sound speed image obtained by sound speed microscopy corresponded well to that obtained using a conventional SAM system. Lesions with hyaline degeneration showed a lower sound speed when compared with that of normal myocardium. Frequency domain analysis of amplitude and phase by both methods also showed similar characteristics. Although the data acquisition time of one frame was greater than that in conventional SAM, the total time required for calculation was significantly shorter. The SAM system can be applied to intraoperative pathological examination.

Ultrasonic tissue characterization of photodamaged skin by scanning acoustic microscopy.

The aim of this study was to ultrasonically characterize photodamaged skin of the elderly at the microscopic level using scanning acoustic microscopy which showed two-dimentional distribution of sound speed in the skin section. We confirmed that the expression level of the elastin gene was increased in the preauricular skin (photodamaged area), compared with postauricular skin (photo-protected area). The expression level of the procollagen gene was also increased in the preauricular skin compared with postauricular skin. The preauricular skin showed higher sound speed in the papillary dermis (Grenz zone). The site of progressive solar elastosis showed a somewhat sound speed velocity than that of the Grenz zone. Immunohistochemical staining showed conserved deposition of collagen in the Grenz zone even in the more photodamaged preauricular skin. These results suggest that fibrosis in the Grenz zone compensates tissue strength with the progress of solar elastosis. The sound speed analysis of skin will provide important information on heterogeneous mechanical changes in the skin during the process of photoaging.