Supplementary Data
Theoretical considerations
Sound waves travel at 1520 m/s in water at 36 °C. The sound waves that reflect from the cell surface and the
substrate directly underneath the cell are separated in time due to the high frequencies used; the amplitude and time
difference of these echoes can be used to determine the cell height, sound speed, acoustic impedance, density, bulk
modulus and acoustic attenuation.1,2,3 The experimental setup of the acoustic microscopic system is shown in Figure
S1.
Figure S1. An overview of the acoustic microscope scanning setup. For every cell, two measurements were made.
The first measurement was made over the cell while ensuring the ultrasound focus would pass through the nuclear
area (blue-purple mix oval) and provided time-of-flight backscatter t1 and t2 from the apical cell membrane surface
and cell-glass substrate interface. The second measurement was performed on the glass substratum near the cell
(<20 m away from plasma membrane) and provided a reference time-of-flight backscatter t0 from the glass
substrate.
First, sound waves produced from the transducer are focused onto the cell center. Ultrasound backscatter echoes
from the cell membrane (t1) and cell-substrate interface (t2) are recorded. For a highly focused transducer (numerical
aperture = 1.0 in our work) the signal amplitude will change depending on the ultrasound focal position axially through
the cell. The signals are recorded as the transducer moves axially through the cell. Recording the signal amplitude as
a function of axial position through the cell is called a V(z) curve.4,5,6
Amplitude (mV)
A0
A2
A1
t1
Time (ns)
t2
t0
Figure S2. A schematic of the resulting envelope backscatter signal after application of a Hilbert transform. The first
measurement results in the recording of two echoes, with the first coming from the apical side of the cell membrane
(red), and the second coming from the cell-glass substrate interface (blue). The second measurement over the glass
substratum without any cell coverage records a single echo coming from the glass substrate itself (black). The
maximum amplitudes A0, A1, and A2 of the glass substrate, the cell membrane, and the cell-glass interface,
respectively, were used along with the times associated with those amplitudes in the Briggs equations.
A V(z) measurement is then made on the glass substrate beside the cell to record a reference signal, t0 (Figure S1).
The signal amplitude will vary depending on the ultrasound focus through the cell. From these V(z) measurements,
the maximum amplitude from each surface (cell, substrate under the cell and the reference from glass) is calculated.
The time of the echoes from each surface are calculated when the transducer focus is directly on the substrate.
Figure S2 shows representative measurements of the V(z) curves recorded when the ultrasound transducer is
positioned above the cell and the reference measurement beside the cell. Both t0 and t2 are related to signals
reflected from the glass substrate, however, t2 is the measurement from the reflection of the substrate under the cell.
The slight shift (~4-6 nanoseconds) of the signal under the cell results from the change in sound speed within the cell
compared to the surrounding coupling fluid. From the time and amplitude of each measured signal, the cellular height
(d), speed of sound through the cell (c), acoustic impedance (Z), cell density (), bulk modulus (K), and attenuation
() can be derived.
The cell height and speed of sound are calculated using the time of the echoes from the cell membrane (t1), the cellglass substrate interface (t2), and the reference glass measurement (t0) as denoted in figure S1 in the following
equations
c0
t 0  t1 
2
,
(1)
t t 
c  c0  0 1 
 t 2  t1  ,
(2)
d
where c0 is the speed of sound in the medium surrounding the cell.
When a sound wave is reflected from a surface, some is reflected and some is transmitted. The acoustic impedance
describes the magnitude of sound pressure reflected and transmitted due to variations within the medium. The
acoustic impedance of the cell can be calculated using the signal amplitude from the glass of the reference signal A0
and from the cell membrane A1 using:
 A  A1 

Z  Z 0  0
 A0  A1  ,
(3)
The cell density,, and bulk modulus, K, are found using the acoustic impedance (calculated using equation 3) and
the sound speed (using equation 2) using the following relations

Z
c
,
(4)
K  cZ .
(5)
Ultrasound attenuation is due to energy absorption and wave scattering by particles in heterogeneous media. Within
this study, the attenuation for a cell was computed from
  c 
1  A0  Z s  Z  4ZZ 0  Z s  Z 0 


 ,
ln  
2d  A2  Z s  Z  ( Z  Z 0 ) 2  Z s  Z 0 
(6)
where c is the attenuation of the medium the cell is immersed in, through a distance of 2d.1 The acoustic
impedances Zs, Z and Z0 account for the reflection and transmission of the sound waves from various interfaces.
References
1
Briggs A, Kolosov O. Acoustic microscopy. 2nd rev. ed. New York: Oxford University Press; 2009. 380 p.
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Trans Ultrason Ferroelectr Freq Control 2010; 57:2293–304.
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4 Weglein RD, Wilson RG. Characteristic material signatures by acoustic microscopy. Electron Lett 1978; 14:352-4.
5 Atalar A. A physical model for acoustic signatures. J Appl Phys 1979; 50:8237.
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2