A534. IN VITRO IMAGING OF LIVING CELLS WITH ULTRASOUND INTENSITY MICROSCOPE

Abstract

Introduction: Acoustic microscopy for medicine and biology has been developed for more than twenty years at Tohoku University [1–8]. Application of acoustic microscopy in medicine and biology has three major features and objectives. First, it is useful for intra-operative pathological examination because staining is not required. Second, it provides basic acoustic properties to assess the origin of lower frequency ultrasonic images. Third, it provides information on biomechanical properties at a microscopic level because ultrasound has close correlation with mechanical properties of the tissues. This paper describes the preliminary results obtained using 300 MHz ultrasound intensity microscopy for in vitro characterization of rat synovial cell cultures. The novelty of the approach lies in the fact that it allows remote, non-contact and disturbance-free imaging of cultured synovial cells and the changes in the cells’ properties due to external stimulants such as transforming growth factor beta-1 (TGFbeta1).

Materials and Methods: Ultrasound intensity microscope: An electric impulse was generated by a high speed switching semiconductor. The electric pulse was input to a transducer with sapphire rod as an acoustic lens and with the central frequency of 300 MHz. The reflections from the tissue was received by the transducer and were introduced into a Windows-based PC (Pentium D, 3.0 GHz, 2GB RAM, 250GB HDD) via a digital oscilloscope (Tektronix TDS7154B, Beaverton, USA). The frequency range was 1GHz, and the sampling rate was 20 GS/s. Four values of the time taken for a pulse response at the same point were averaged in order to reduce random noise. The transducer was mounted on an X-Y stage with a microcomputer board that was driven by the PC through RS232C. The Both X-scan and Y-scan were driven by linear servo motors. The ultrasound propagates through the thin specimen such as cultured cells and reflects at the interface between the specimen and substrate. A two-dimensional distribution of the ultrasound intensity, which is closely related to the mechanical properties, was visualized with 200 by 200 pixels.

Tissue preparation: The synovial membrane was obtained from non-operated male rats weighing from 380 to 400 g through medial parapatellar incision. The tissue was diluted and loosened 0.15% DispaseII (Boehringer, Mannheim) in DMEM for 2 hours at 37 C°. Then centrifuged at 400 g for 5 min and discard the supernatant. The cells were plated in 75 mm2 dish (Falcon) with Dulbecco’s modified Eagle’s medium (DMEM, GIBCO Laboratories) containing 10% fetal bovine serum (SIGMA Chemical Co.) at 37 C° in a CO2 incubator. To determine changes of intensity, the cells were treated with 1 ng/ml of human recombinant TGF-β1 (hTGF-β1, R& D Systems, Inc.) for 1 and 3 days after reaching confluent. The non-treated cells was harvested at 3 days after reaching confluent and defined as control. Randomized four points at each dish were measured and averaged data was defined as the representative value of each dish. The cells used for experiments were at the third passage.

Signal processing: The reflection from the tissue area contains two components. One is from the tissue surface and another from the interface between the tissue and the substrate (phosphate buffered saline). Frequency domain analysis of the reflection enables the separation of these two components and the calculation of the tissue thickness and intensity by Fourier-transforming the waveform [9].

Image analysis: Randomized point regions were determined using ultrasound intensity microscopic images. This was done by employing commercially available image analysis software (PhotoShop CS2, Adobe Systems Inc.). Ultrasound intensity microscopic images with a gradation color scale were also produced for clear visualization of the ultrasound intensity variations.

Statistics: Statistical analysis among groups was performed using one factor analysis of variance. Data were expressed as mean ± standard deviation. A value of P < 0.05 was accepted as statistically significant.

Results: The ultrasound intensity microscope can clearly visualize cells. The high intensity variations area of the reflected ultrasound energy at the central part of the cell corresponded to the nucleus and the high intensity area at the peripheral zone corresponded to the cytoskeleton mainly consisting of actin filaments. The intensity of the reflected ultrasound energy at the peripheral zone was significantly increased after stimulation with hTGF-b1.