PNNL-13501 - Pacific Northwest National Laboratory

Results and Accomplishments
Two experiments were performed; one imaged living
cells and the second evaluated acoustic attenuation in
water as a function of temperature.
Xenopus oocytes were selected for imaging living cells
since the National Institute of Health recognizes them as a
reference system, their large size makes them attractive
for imaging internal features, and their opaqueness
hinders imaging by optical confocal techniques. Images
acquired from stage 6 Xenopus oocytes in media at 20°C
are illustrated in Figure 1. An amplitude-color bar was
placed in the far right-hand side of the images. The top of
the bar denoted amplitude saturation while the bottom of
the bar denoted no signal and a linear relation between
them. The wave passed through the oocyte, reflected off
the slide, and passed a second time through the oocyte as
it returned to the transducer for Figure 1A. The false
color was signal amplitude modulated by an integrated
attenuation (scattering and absorption). Signal saturation
was set to black to provide a dark background. Red
denoted a high but unsaturated signal level (minimal
attenuation). Yellow, green, and blue were decreasing
values of amplitude (increasing attenuation). Gray was
the lowest amplitude values (highest attenuation in a cell).
The nucleus was clearly observed, and the animal pole
was the circumferential position of the cell membrane
closest to the nucleus. The nucleus was less attenuative
than other regions within the oocyte. If we employ a
simple model and assume that lipids are more attenuative
than water, data indicated that the nucleus has a higher
concentration of water, as expected. The animal pole also
B) Signal Gate
Positioned at Top
A) Signal Passes Through Cell
and is Reflected off a Glass Plate
C) Signal Gate
Positioned at Middle
D) Signal Gate
Positioned at Bottom
Figure 1. Integrated through-transmission image and pulseecho
images at selected depths
416 FY 2000 Laboratory Directed Research and Development Annual Report
was higher amplitude (less attenuation), as evident from
the reds and yellows about the periphery.
Because acoustic velocity was relatively constant, a time
gate was shifted to successively later values to image
features at a constant depth within the oocyte. Four
planes were digitized simultaneously. The images of the
lower portion of Figure 1 were the top, middle, and
bottom of the oocytes. These were digitized by gates 1
through 3 and were placed side-by-side from left to right,
respectively. Each gate had a duration that roughly
corresponded to one quarter of the oocyte. Note the
color-amplitude bar to the far right-hand side. A
reflection or backscatter occurred at each localized
change in density and stiffness—the greater the change,
the higher the signal amplitude. Organelle membranes
were expected to be reasonably strong signal sources
since cytoplasm existed to either side of a stretched
membrane between the two regions. The top and bottom
of the nucleus were of high amplitude since the
membrane was specular and an efficient reflector of
ultrasound. The middle image gives a well-rounded
image of the oocyte since it cut through the central axis,
and internal features within the nucleus and within the
oocyte were observed. Examination of the acoustic signal
traversing through the nucleus revealed a complex series
of internal responses. We concluded that more work is
needed to identify these responses. Gate 4 was set to
digitize the surface that the oocytes rested against and was
described above (Figure 1A). All four gates were
digitized simultaneously from a train of responses with
the transducer at one location. The transducer was moved
to the next adjacent (x, y) coordinate, and the process was
repeated in milliseconds, assigning an amplitude value to
each of the four gates. Thus, the picture was painted in as
the transducer was moved back and forth along the
scanning axis (x-axis) and incremented along the y-axis at
each pass. The entire scan took 5 minutes, although it
was not optimized for speed. The result was the four
images shown. Two false-color relations were assigned,
as indicated by amplitude-color bars to the right side of
the images, Figure 1A and Figure 1B-1D, respectively.
Both were selected to highlight the internal structures of
the oocyte.
Acoustic attenuation in water was evaluated as a function
of temperature. Amplitude and peak frequency were
monitored for a pulse-echo response from a 50-MHz
transducer oriented normal to a glass plate that was placed
at the focal length, i.e., 13 mm. A heater-chiller permitted
a temperature swing from 20°C to 40°C. As temperature
increased, a 6 dB increase (factor or 2) was observed in