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ACOUSTIC CHARACTERIZATION OF TISSUE AT THE MICROSCOPIC LEVEL - SONOHISTOLOGY
673 CHAPTER XIII ACOUSTIC CHARACTERIZATION OF TISSUE AT THE MICROSCOPIC LEVEL SONOHISTOLOGY Lawrence W. Kessler 1.
Introduction The application of acoustic microscopy to biological tissues is a
new field which deals with the problem of delineating the physical characteristics of tissue at a microscopic level.
Knowledge of the physi-
cal characteristic of tissues in their viable state instead of histologically fixed and stained, can be rendered directly with acoustic microscopy. This is especially important when trying to identify subtle changes in tissue architecture which may exist with pathological states. The technique of acoustic microscopy is analogous to optical microscopy in that high resolution, magnified images are produced, however, ultrasonic energy, rather than an "optical frequency" of electromagnetic energy, is transmitted through the sample.
The very high ultrasonic frequencies em-
ployed to produce high resolution are new to the medical ultrasonics arena. The high frequencies are associated with large acoustic attenuation values in tissue, thus making small samples necessary.
On the other hand, the
obtainable resolution is several orders of magnitude better than conventional diagnostic ultrasound.
The applications of acoustic microscopy are
somewhat different than conventional medical ultrasound.
In particular,
at frequencies of a few megahertz, i.e., the "macro-range", ultrasonic diagnosis profits from being able to penetrate optically opaque bodies and differentiate soft tissue interfaces without ionizing radiation.
On the
other hand, at frequencies two orders of magnitude greater, the "microrange" is defined.
Here, in addition to providing the same capabilities
on a much finer scale, acoustic microscopy provides direct access to elastic properties of tissue at the microscopic level. At this level, localized
674 variations in mass density, modulus of elasticity and acoustic attenuation, are much more prevalent than for bulk tissue. Although there may be many possible techniques for producing acoustic micrographs of tissue, for example, see the review article reference 17 t this paper concentrates on the various aspects of tissue characterization, per se, rather than on instrumentation.
A brief discussion of the instru-
ment employed to produce the results contained herein is presented for general review. 2.
TECHNIQUE The following brief description of the technique employed for acoustic
microscopy is presented for reference. listed references
Interested readers may review the
for more detailed accounts.
In general terms, pro-
ducing an acoustic micrograph involves placing a sample between sending and receiving ultrasonic transducers, and displaying the point by point pattern of sonic transmission on a CRT.
Figure 1 schematically illustrates
the operation of a commercially available acoustic microscope at 100 MHz and produces resolution of 20 microns.
which operates
Biological specimens are
placed on a sonically activated glass stage and are covered with a mirrored coverslip which also allows a small amount of light transmission.
The
acoustic energy transmitted through the sample imparts oscillatory mechanical pertubations to the coverslip surface.
These disturbances occur at the
acoustic frequency and are proportional to the acoustic amplitude in each region.
These are detected by a focused, scanning laser beam probe which
drives an opto-acoustic receiver.
The laser beam acts as a non-contacting
high resolution microphone. An optical image of the specimen is produced simultaneously with the acoustic by means of the through-transmitted laser light, i.e., through the mirror and sample.
By virtue of this point by point scanning method, the
optical and acoustic signals are in perfect register electronically and corresponding images may be either presented on separate side-by-side TV monitors or superimposed on separate color channels of a single color TV
675
Laser
Beam Scanners
V Mirrors/
Imaging Optics ι
î a
Ultrasonic Transducer
Φ
Φ
Acoustic Signal Processor
**
Mirrored Coverstipj, ^ Acoustic Frequency Generator
Demodulator and Photodetector
Specimen Stage
*
Photodetector
▼
Optical Signal Processor
Acoustic Image Display
j Fig. la
Schematic diagram of a 100 MHz acoustic microscope which produces optical and acoustic micrographs of a specimen simultaneously. A focused scanning laser beam is employed as a high resolution acoustic receiver.
monitor.
The latter display method has been especially useful for sorting
through and precisely correlating the abundant acoustic and optical image details. In addition to displaying the sound field intensity or amplitude throughout the field of view, i.e., the acoustic micrograph, the S0N0MICROSCOPE provides an acoustic interference mode of operation. Here, the phase of the acoustic beam is measured as it propagates through various structures within the field of view.
Thus, velocity of sound measure-
ments can be made in each region of the sample and, under the appropriate conditions, fluctuations of mass density and elasticity can be observed on a microscopic basis. The procedures for mounting specimens in the acoustic microscope are very similar to those normally encountered in optical microscopy.
The
acoustic energy is coupled directly to the specimen without need for an
676
677 acoustically lossy water bath, although the specimen is usually moistened with a thin layer of liquid (saline, culture medium, etc.) to insure good acoustic transmission. 3.
SONOMICROSCOPIC CHARACTERIZATION OF TISSUES The characterization of tissue microstructure is traditionally ac-
complished by optical, X-ray or electron microscopy methods. However, these methods are intrinsically capable of revealing only the physical "electronic properties" of structure as defined by the dielectric constant. (The magnetic properties are usually unimportant).
A great number of use-
ful histological methods have been developed which clarify, fix, and selectively stain tissues in order to render visible, otherwise unobservable, aspects of structure. tissues.
However, these methods alter the natural state of
Classically speaking, the size, shape, position, and staining
characteristics of tissue components provide the body of well documented information which is diagnostically available at present. The important aspect of acoustic microscopy is that it intrinsically differentiates the elastic and density features of structure. parameters are not directly accessible by other techniques.
These
Elasticity
and density can be determined through the acoustic impedance value Z and the velocity of sound, C.
The familiar relationships which define these
quantities can be rearranged slightly to show that k = ZC
(1)
P = Z/C
(2)
where k is the elastic modulus (the reciprocal of the compressibility), and p is the mass density. The essence of the characterization procedure is to experimentally de-
Fig. lb
Photograph of a commercially available acoustic microscope apparatus, the SONOMICROSCOPE™ 100, employing the principles outlined in Figure la. 6
678 termine Z and C, with acoustic microscopy. city can be determined.
From this, the density and elasti-
A more detailed description of the methods for de-
termining acoustic attenuation, acoustic impedance and velocity of sound is presented below. 4.
ACOUSTIC ATTENUATION AND IMPEDANCE DISTRIBUTION In general, the attenuation suffered by an acoustic beam propagating
through tissue results from several possible mechanisms.
As an acoustic
beam propagates through an inhomogeneous medium, reflection and refraction at boundaries divert energy away from the primary beam.
However, as the
spatial extent of the interfaces becomes comparable to or smaller than the dimension of the acoustic wavelength, the energy diversion is referred to as scattering.
It should be recognized that structures, which at clinical
diagnostic frequencies may be considered as scatterers, at the higher frequencies employed in microscopy, may be specular reflectors. Frictional losses in tissue cause a fraction of the acoustic energy to change into heat.
Furthermore, since the physical state of the environ-
ment is altered with sound beam propagation, shifts may occur in chemical or structural equilibria at a molecular level.
The frictional and relax-
ational losses are termed absorption. Acoustic attenuation measurements are made by means of a comparison 8 technique.
With the specimen in place and the acoustic image being
presented on the CRT, the appropriate region of interest is selected. Positive identification of the region is made with the assistance of the corresponding optical image as well.
The relative image brightness in the
region of interest, compared to that when no specimen is in place, is a measure of the attenuation.
Experimentally, an image brightness level is
established with an optically apertured light meter to confine the field of view of the sample.
With the specimen removed, the microscope is re-
focused on the illuminating sound field alone.
Known values of electrical
attenuation are then inserted into the signal path of the insonification transducer in order to restore the brightness to the previous level.
The
679 inserted electrical attenuation is equal to the acoustic attenuation provided by the specimen.
Confidence in the method, established by measuring
the absorption coefficient in water, indicates that an overall accuracy of + 10% can be achieved. In order to separate transmission losses due to impedance mismatch from absorption and scattering losses, a measurement procedure may be performed on several samples of the same tissue.
The sample can be made suf-
ficiently thin so as to insure that absorption and scattering losses are an insignificant fraction of the total observed attenuation.
By way of
example, if the absorption of sound within tissue at 100 MHz is of the order of 1 db/cm/MHz, then for sample thickness of 10, 100 and 1000 microns, the anticipated absorption losses would be 0.1, 1.0 and 10 db, respectively. Since typical sections prepared for optical microscopy are sliced 5-10 microns thick to produce some measure of optical transparency, obtaining such thin samples is no problem. In order to evaluate the loss in tissue due to the localized level of acoustic impedance, consider the simple case of an acoustic beam emerging from the acoustic microscope stage, impedance Z , propagating through the sample, impedance Z , and then entering the coverslip, impedance Z . The general equation for the intensity transmission coefficient a is given by
9
4 Z Z (Z3 + Ζ χ ) 2 cos 2 k2l +
Z
+ Z3 Zl ] Z
2
2
sin2 k2l
J
In this expression 1 is the sample thickness and k the sample, i.e., 2 π/wavelength.
(3)
is the wave number of
Although expression (3) appears to be
rather cumbersome, reasonable simplifications can be made based on certain assumptions on the tissue, stage and coverslip without loss of accuracy. 5.
VELOCITY OF SOUND DISTRIBUTION The velocity of sound has been measured in many soft tissues over the
frequency range of 1-10 MHz and the values observed don't seem to vary by more than 100 m/sec or less, even though there may be substantial differences
680 10 in the tissue types.
It may be reasoned that at the level of "bulk tissue"
the velocity of sound characteristics present in small components are masked by the predominant aqueous constituents.
Microscopically, however, the
velocity of sound may be measured in the small components of the tissue by 3 4 means of the acoustic interferometry mode. ' The principle of measuring changes in velocity of sound can be explained as follows.
Consider the intersection of two plane, coherent sound beams
at a detector plane, viz., the coverslip.
Depending upon the angle between
the two beams a series of regularly spaced fringe lines, caused by mutual interference, will appear.
If an object, whose velocity of sound charac-
teristics differ from that of the surrounding medium, interrupts one of the beams, localized shifting of the fringe lines will result.
The displace-
ment of a fringe, measured graphically, is related to the localized disturbance in transmission time of one sound beam compared with the unperturbed beam. A simple formula has been derived in an "unknown" sample.
to calculate the velocity of sound
In the acoustic microscopy apparatus described,
the geometry required by the high acoustic attenuation precludes two acoustic beams. Instead, the reference beam is simulated electronically at normal incidence, and the acoustic beam is angled slightly with respect to the normal.
According to Snell's Law, an acoustic beam, incident upon the
specimen at angle Θ
from the normal will be refracted to angle Θ
within
the sample according to this relationship sin Θ
sin Θ (4)
C x
where C and C are the velocities of sound in the initial medium and the o x sample, respectively.
If the thickness of the sample, or of the region
of interest is denoted by Δτ, then the lateral shift of the fringes, normalized by the unperturbed spacing of the fringes, N, is given by Δ T sin Θ N<
L o
tan Θ o
VI
tan
(5)
681 where L is the wavelength of sound in the initial material, viz., the o microscope stage.
Note that N is a dimensionless number, corresponding to
the number of fringes of shift in the interferogram. Θ
and then using the Snell's Law relationship, C
X
By solving Eq. 5 for
is readily calculated.
2C
Typically Θ
is 10°, however, it depends upon the particular configuration
of the stage employed. The capability of this technique for differentiating velocity of sound regions within the matrix of a tissue, is dependent upon the region thickness.
This is consistent with other methods of velocity measurements such
as "time of flight" techniques.
For example, if fringe line displacements
can be measured to the nearest 1/10 fringe, then in a section of tissue, say, 300 microns thick, i.e., 20 wavelengths at 100 MHz, 7.5 m/sec variations in C
may be discriminated.
X
6.
RESULTS AND DISCUSSION During the development of the very young field of acoustic microscopy
a variety of biomédical investigations have been conducted.
A few of the
highlights are discussed below. Isolated mouse embryo hearts have been maintained alive for several days on the stage of the SONOMICROSCOPE 100 using an organ culture tech12 13 nique. '
Simultaneous optical and acoustical viewing in real time
permitted more complete observation of the contractile event than heretofore possible.
An example of micrographs produced is shown in
Figure 2. Internal and surface structures of the heart such as valves, chambers, and coronary arteries are imaged on a TV monitor and precise measurements of atrial, ventricular and sinus originated beats are measured and recorded on video tape. tinuous perfusion.
Drugs are introduced either by pulsing or by con-
Various states of contractility, e.g., synchrony,
arrhythmia or fibrillation, is observed either spontaneously or through the introduction of drugs.
Effectiveness of cardioactive drugs such as
lidocaine, epinephrine, aconitine, etc. in modifying such induced or in-
682
Fig. 2
Still frame photographs taken from video tapes of a live mouse embryo heart maintained in organ culture on the stage of the acoustic microscope.-'-3 The cross marks represent spacings of 1 millimeter. a)
Optical through transmission image demonstrating the opacity of the organ.
b)
Corresponding acoustic micrograph at 100 MHz. The functional anatomy of the cardiac cycle can be viewed clearly in the acoustic micrograph. The instantaneous positions of the muscle fibers and valves in diastole are shown here.
trinsic contractile states is measured by this non-invasive, non-destructive technique. The embryological development of CFI mice has been studied with an acoustic microscope.
14
The embryos were removed from time-mated mice and
placed on the microscope stage where they remained alive for substantial periods of time (hours) without the assistance of culture techniques. fetal mice were at gestational ages ranging from 10 to 12 days.
The
Major
morphological differentiation of tissues has occurred immediately prior to this period.
By the tenth day, the heart and its functional aspects
are completely discernible. embryos were produced.
Video tape recordings and movies of live
These illustrate the high degree of tissue differen-
683 tiation at this stage of development, as well as the great amount of structural detail that can be observed acoustically compared with that observed optically.
Figure 3 shows acoustic and optical micrographs of
a 10^ day specimen. Optical visualization of whole-mount embryos is usually very difficult since the specimens are thick and optically opaque.
Therefore, it is
necessary to employ fixing, clearing and staining techniques which alter the natural state of the tissue.
However, the microanatomy of a fixed but
unstained, optically opaque 72-hr. chick embryo is nearly completely revealed with an acoustic microscope.
Soft tissue structures have been
differentiated and information on elasticity and density at the microscopic level have been obtained.
Comparison of the acoustic micrograph with the
known, optically revealed, microanatomy of the chick embryo indicates that a rather complete visualization of organ primordia has been achieved without clearing and staining the specimen.
It was further suggested that
the effects of the fixative may be selective in the manner in which the elastic properties of the tissues are altered.
This can, perhaps, be used
to advantage. Acoustic micrographs of unstained specimens of mouse kidney have been produced.
The acoustically revealed structure has been examined in re-
lation to established microanatomy.
High contrast details corresponding
to connective tissue boundaries of supporting elements of the nephron are exhibited.
In addition, various regions of the kidney such as the cortex
and the three medullary regions can be differentiated.
The acoustically
exhibited structures result from wave scattering at connective tissue interfaces.
The scattering is caused by localized elasticity and density dis-
continuities . Acoustic attenuation measurements have been made in kidney tissue. It has been well established that for mammalian tissues such as kidney and brain, the attenuation coefficient exhibits a linear dependence over the frequency range of investigation, i.e., 0.3-10 MHz, and the magnitude is
684
685 of order of 1 db/cm/MHz.
The absorption coefficient for water is signifi-
cantly less than for tissue, and exhibits a square-law frequency dependence. Projecting the linear dependence for tissue to higher frequencies, and comparing this to the known absorption in water, leads to speculation that beyond about 500 MHz, the attenuation for water might exceed that for tissue.
Since tissue is composed mainly of water, this would not ordinarily
be expected. The attenuation of sound in mammalian kidney tissue was measured at 8 100 MHz and 220 MHz.
The tissue samples were either fresh, fresh-frozen-
thawed, or fixed in formalin.
The measured attenuation values were found
to be independent of preparation.
The result, presented graphically in
Figure 4, indicates that the acoustic attenuation in tissue continues to behave linearly up to a frequency of about 100 MHz.
Beyond this frequency
a square-law dependence (or greater) takes over, thereby indicating that the hypothetical crossover point may never actually be reached.
This is
more in line with intuition. The use of acoustic microscopy to delineate mechanical variations in hard tissues has been examined, and preliminary qualitative results were 17 published for sections of dental tissue.
Dentin and enamel components
of this tissue differ greatly in their visual and mechanical properties and this is clearly brought out in the acoustic micrographs. Furthermore,
Fig. 3
Composite images of a 10*5 day mouse embryo^4 histologically fixed in ethanol but unstained and uncleared. Similar images are produced of live specimens. a)
Optical through transmission image
b)
Acoustic micrograph at 100 MHz
c)
Acoustic interferogram demonstrating variations in acoustic index of refraction of the various tissues. These variations appear as lateral position shifts of the vertical fringe lines. The fringe-to-fringe spacing, in terms of acoustic transmission time, is 10 nanoseconds. By means of the formulas in the text the elastic properties of the tissues can be calculated.
686 1
1—i
i i i i 11
1
1—i
i
3.0
2.0 1.5
T
T
i
i i i i i j
i
A) Striated Muscle B) Kidney and Brain C,D) Kidney
3.5
2.5
i i i T-j —
-i A mum
1.0
^
III,
,,
i
III
B
//////////////////////////////////
C
0.5
0
1
0.1
1
1—1 M i l l
1
1
1
1 i i i i i1
|
|
|
10
I 1 i i i1
,
i
.
. .
100
. 1
1000
FREQUENCY IN MHz. Fig. 4
Ultrasonic attenuation in mammalian kidney tissue at extended frequencies.7 Data A and B are taken from reference 10 and data C and D taken from an acoustic microscopy study described in reference 8.
in the region of dentin-enamel junction the acoustic interference mode demonstrates a smooth gradation of density and elasticity.
This study
serves as the forerunner to a series of experiments on bone and plaque formations particularly. Investigation of tissue elastic component involvements in the diseased state is being conducted.
With regards to sound velocity variations within
and among soft tissues at low frequencies (1-10 MHz) only a 100 m/sec range has been observed.
On the microscopic scale, preliminary evidence has
shown that there are localized regions which are characterized by velocities of sound 200-800 m/sec above that of neighboring tissue structures.
This
means that microelasticity variations in tissue are great at the microscopic level and implies that these may be quite characteristic of the tissue type and pathology.
687 REFERENCES 1.
A. Korpel, L. W. Kessler and P. R. Palermo: at 100 MHz, Nature 232 (1971) pp. 110-111.
2.
L. W. Kessler, A. Korpel and P. R. Palermo: Simultaneous Acoustic and Optical Microscopy at Biological Specimens, Nature 239 (1972) pp. 111-112.
3.
L. W. Kessler, P. R. Palermo and A. Korpel: Practical High Resolution Acoustic Microscope, Acoustical Holography, Vol. 4, Plenum Press, New York (1972) ed. by G. Wade, pp. 51-71.
4.
L. W. Kessler, P. R. Palermo and A. Korpel: Recent Developments with the Scanning Laser Acoustic Microscope, in Acoustic Holography, Vol. 5, Plenum Press, New York (1974) ed. by P. Green, pp. 15-23.
5.
L. W. Kessler: The Sonomicroscope, Proceedings 1974 IEEE Ultrasonics Symposium, ed. by J. de Klerk,IEEE, New York, N.Y. Cat. #74-CH0896-l SU, pp. 735-737.
6.
7.
An Acoustic Microscope Operating
TM SONOMICROSCOPE 100 , Manufactured by Sonoscan, Inc. 7 52 Foster Avenue, Bensenville, Illinois 60106, U.S.A. under exclusive license from Zenith Radio Corporation. . L. W. Kessler:
High Resolution Visualization of Tissue With Acoustic Microscopy, in Proceedings of the Second World Congress on Ultrasonics in Medicine, Rotterdam, June, 1973, ed. by M. de Vlieger, Excerpta Medica Foundation (1974) pp. 248-256.
8.
L. W. Kessler: VHF Ultrasonic Attenuation in Mammalian Tissue, J. Acoust. Soc. Amer. 53 (1973) pp. 1759-1760.
9.
L. E. Kinsler and A. R. Frey: Inc. (1962), Chapt. 6.
10.
See for example: D. E. Goldman and T. F. Hueter: Tabular Data of the Velocity and Absorption of High-Frequency Sound in Mammalian Tissue, J. Acoust. Soc. Amer. 28 (1956) pp. 35-37.
11.
P. R. Palermo and L. W. Kessler:
12.
R. C. Eggleton and L. W. Kessler: Ultrasound in Medicine, Vol. 1, ed. by D. N. White, Plenum Press, New York, N.Y. (1975) pp. 537-542.
13.
R. C. Eggleton, L. W. Kessler, F. S. Vinson and G. B. Boder : Proceedings 1975, IEEE Ultrasonics Symposium, Los Angeles, California, Published by IEEE New York, Catalog #75-CHO-994-4SU, pp. 57-58.
14.
W. D. O'Brien, Jr., and L. W. Kessler: Examination of Mouse Embryological Development with an Acoustic Microscope, American Zoologist 15 (1975) p.807A.
15.
M. Ahmed, and L. W. Kessler: Microanatomy of a Histologically Unstained Embryo as Revealed by Acoustic Microscopy, in Acoustical Holography, Vol. 6, ed. by N. Booth, Plenum Press, New York, N.Y. (1975).
16.
L. W. Kessler, S. I. Fields and F. Dunn: Acoustic Microscopy of Mammalian Kidney, J. Clinical Ultrasound, 2 (1974) pp. 317-320.
17.
L. W. Kessler: A Review of Progress and Applications in Acoustic Microscopy. J. Acoust. Soc. Amer. 55 (1974) pp. 909-918.
Fundamentals of Acoustics, John Wiley & Sons,
Unpublished.
Introduction The application of acoustic microscopy to biological tissues is a
new field which deals with the problem of delineating the physical characteristics of tissue at a microscopic level.
Knowledge of the physi-
cal characteristic of tissues in their viable state instead of histologically fixed and stained, can be rendered directly with acoustic microscopy. This is especially important when trying to identify subtle changes in tissue architecture which may exist with pathological states. The technique of acoustic microscopy is analogous to optical microscopy in that high resolution, magnified images are produced, however, ultrasonic energy, rather than an "optical frequency" of electromagnetic energy, is transmitted through the sample.
The very high ultrasonic frequencies em-
ployed to produce high resolution are new to the medical ultrasonics arena. The high frequencies are associated with large acoustic attenuation values in tissue, thus making small samples necessary.
On the other hand, the
obtainable resolution is several orders of magnitude better than conventional diagnostic ultrasound.
The applications of acoustic microscopy are
somewhat different than conventional medical ultrasound.
In particular,
at frequencies of a few megahertz, i.e., the "macro-range", ultrasonic diagnosis profits from being able to penetrate optically opaque bodies and differentiate soft tissue interfaces without ionizing radiation.
On the
other hand, at frequencies two orders of magnitude greater, the "microrange" is defined.
Here, in addition to providing the same capabilities
on a much finer scale, acoustic microscopy provides direct access to elastic properties of tissue at the microscopic level. At this level, localized
674 variations in mass density, modulus of elasticity and acoustic attenuation, are much more prevalent than for bulk tissue. Although there may be many possible techniques for producing acoustic micrographs of tissue, for example, see the review article reference 17 t this paper concentrates on the various aspects of tissue characterization, per se, rather than on instrumentation.
A brief discussion of the instru-
ment employed to produce the results contained herein is presented for general review. 2.
TECHNIQUE The following brief description of the technique employed for acoustic
microscopy is presented for reference. listed references
Interested readers may review the
for more detailed accounts.
In general terms, pro-
ducing an acoustic micrograph involves placing a sample between sending and receiving ultrasonic transducers, and displaying the point by point pattern of sonic transmission on a CRT.
Figure 1 schematically illustrates
the operation of a commercially available acoustic microscope at 100 MHz and produces resolution of 20 microns.
which operates
Biological specimens are
placed on a sonically activated glass stage and are covered with a mirrored coverslip which also allows a small amount of light transmission.
The
acoustic energy transmitted through the sample imparts oscillatory mechanical pertubations to the coverslip surface.
These disturbances occur at the
acoustic frequency and are proportional to the acoustic amplitude in each region.
These are detected by a focused, scanning laser beam probe which
drives an opto-acoustic receiver.
The laser beam acts as a non-contacting
high resolution microphone. An optical image of the specimen is produced simultaneously with the acoustic by means of the through-transmitted laser light, i.e., through the mirror and sample.
By virtue of this point by point scanning method, the
optical and acoustic signals are in perfect register electronically and corresponding images may be either presented on separate side-by-side TV monitors or superimposed on separate color channels of a single color TV
675
Laser
Beam Scanners
V Mirrors/
Imaging Optics ι
î a
Ultrasonic Transducer
Φ
Φ
Acoustic Signal Processor
**
Mirrored Coverstipj, ^ Acoustic Frequency Generator
Demodulator and Photodetector
Specimen Stage
*
Photodetector
▼
Optical Signal Processor
Acoustic Image Display
j Fig. la
Schematic diagram of a 100 MHz acoustic microscope which produces optical and acoustic micrographs of a specimen simultaneously. A focused scanning laser beam is employed as a high resolution acoustic receiver.
monitor.
The latter display method has been especially useful for sorting
through and precisely correlating the abundant acoustic and optical image details. In addition to displaying the sound field intensity or amplitude throughout the field of view, i.e., the acoustic micrograph, the S0N0MICROSCOPE provides an acoustic interference mode of operation. Here, the phase of the acoustic beam is measured as it propagates through various structures within the field of view.
Thus, velocity of sound measure-
ments can be made in each region of the sample and, under the appropriate conditions, fluctuations of mass density and elasticity can be observed on a microscopic basis. The procedures for mounting specimens in the acoustic microscope are very similar to those normally encountered in optical microscopy.
The
acoustic energy is coupled directly to the specimen without need for an
676
677 acoustically lossy water bath, although the specimen is usually moistened with a thin layer of liquid (saline, culture medium, etc.) to insure good acoustic transmission. 3.
SONOMICROSCOPIC CHARACTERIZATION OF TISSUES The characterization of tissue microstructure is traditionally ac-
complished by optical, X-ray or electron microscopy methods. However, these methods are intrinsically capable of revealing only the physical "electronic properties" of structure as defined by the dielectric constant. (The magnetic properties are usually unimportant).
A great number of use-
ful histological methods have been developed which clarify, fix, and selectively stain tissues in order to render visible, otherwise unobservable, aspects of structure. tissues.
However, these methods alter the natural state of
Classically speaking, the size, shape, position, and staining
characteristics of tissue components provide the body of well documented information which is diagnostically available at present. The important aspect of acoustic microscopy is that it intrinsically differentiates the elastic and density features of structure. parameters are not directly accessible by other techniques.
These
Elasticity
and density can be determined through the acoustic impedance value Z and the velocity of sound, C.
The familiar relationships which define these
quantities can be rearranged slightly to show that k = ZC
(1)
P = Z/C
(2)
where k is the elastic modulus (the reciprocal of the compressibility), and p is the mass density. The essence of the characterization procedure is to experimentally de-
Fig. lb
Photograph of a commercially available acoustic microscope apparatus, the SONOMICROSCOPE™ 100, employing the principles outlined in Figure la. 6
678 termine Z and C, with acoustic microscopy. city can be determined.
From this, the density and elasti-
A more detailed description of the methods for de-
termining acoustic attenuation, acoustic impedance and velocity of sound is presented below. 4.
ACOUSTIC ATTENUATION AND IMPEDANCE DISTRIBUTION In general, the attenuation suffered by an acoustic beam propagating
through tissue results from several possible mechanisms.
As an acoustic
beam propagates through an inhomogeneous medium, reflection and refraction at boundaries divert energy away from the primary beam.
However, as the
spatial extent of the interfaces becomes comparable to or smaller than the dimension of the acoustic wavelength, the energy diversion is referred to as scattering.
It should be recognized that structures, which at clinical
diagnostic frequencies may be considered as scatterers, at the higher frequencies employed in microscopy, may be specular reflectors. Frictional losses in tissue cause a fraction of the acoustic energy to change into heat.
Furthermore, since the physical state of the environ-
ment is altered with sound beam propagation, shifts may occur in chemical or structural equilibria at a molecular level.
The frictional and relax-
ational losses are termed absorption. Acoustic attenuation measurements are made by means of a comparison 8 technique.
With the specimen in place and the acoustic image being
presented on the CRT, the appropriate region of interest is selected. Positive identification of the region is made with the assistance of the corresponding optical image as well.
The relative image brightness in the
region of interest, compared to that when no specimen is in place, is a measure of the attenuation.
Experimentally, an image brightness level is
established with an optically apertured light meter to confine the field of view of the sample.
With the specimen removed, the microscope is re-
focused on the illuminating sound field alone.
Known values of electrical
attenuation are then inserted into the signal path of the insonification transducer in order to restore the brightness to the previous level.
The
679 inserted electrical attenuation is equal to the acoustic attenuation provided by the specimen.
Confidence in the method, established by measuring
the absorption coefficient in water, indicates that an overall accuracy of + 10% can be achieved. In order to separate transmission losses due to impedance mismatch from absorption and scattering losses, a measurement procedure may be performed on several samples of the same tissue.
The sample can be made suf-
ficiently thin so as to insure that absorption and scattering losses are an insignificant fraction of the total observed attenuation.
By way of
example, if the absorption of sound within tissue at 100 MHz is of the order of 1 db/cm/MHz, then for sample thickness of 10, 100 and 1000 microns, the anticipated absorption losses would be 0.1, 1.0 and 10 db, respectively. Since typical sections prepared for optical microscopy are sliced 5-10 microns thick to produce some measure of optical transparency, obtaining such thin samples is no problem. In order to evaluate the loss in tissue due to the localized level of acoustic impedance, consider the simple case of an acoustic beam emerging from the acoustic microscope stage, impedance Z , propagating through the sample, impedance Z , and then entering the coverslip, impedance Z . The general equation for the intensity transmission coefficient a is given by
9
4 Z Z (Z3 + Ζ χ ) 2 cos 2 k2l +
Z
+ Z3 Zl ] Z
2
2
sin2 k2l
J
In this expression 1 is the sample thickness and k the sample, i.e., 2 π/wavelength.
(3)
is the wave number of
Although expression (3) appears to be
rather cumbersome, reasonable simplifications can be made based on certain assumptions on the tissue, stage and coverslip without loss of accuracy. 5.
VELOCITY OF SOUND DISTRIBUTION The velocity of sound has been measured in many soft tissues over the
frequency range of 1-10 MHz and the values observed don't seem to vary by more than 100 m/sec or less, even though there may be substantial differences
680 10 in the tissue types.
It may be reasoned that at the level of "bulk tissue"
the velocity of sound characteristics present in small components are masked by the predominant aqueous constituents.
Microscopically, however, the
velocity of sound may be measured in the small components of the tissue by 3 4 means of the acoustic interferometry mode. ' The principle of measuring changes in velocity of sound can be explained as follows.
Consider the intersection of two plane, coherent sound beams
at a detector plane, viz., the coverslip.
Depending upon the angle between
the two beams a series of regularly spaced fringe lines, caused by mutual interference, will appear.
If an object, whose velocity of sound charac-
teristics differ from that of the surrounding medium, interrupts one of the beams, localized shifting of the fringe lines will result.
The displace-
ment of a fringe, measured graphically, is related to the localized disturbance in transmission time of one sound beam compared with the unperturbed beam. A simple formula has been derived in an "unknown" sample.
to calculate the velocity of sound
In the acoustic microscopy apparatus described,
the geometry required by the high acoustic attenuation precludes two acoustic beams. Instead, the reference beam is simulated electronically at normal incidence, and the acoustic beam is angled slightly with respect to the normal.
According to Snell's Law, an acoustic beam, incident upon the
specimen at angle Θ
from the normal will be refracted to angle Θ
within
the sample according to this relationship sin Θ
sin Θ (4)
C x
where C and C are the velocities of sound in the initial medium and the o x sample, respectively.
If the thickness of the sample, or of the region
of interest is denoted by Δτ, then the lateral shift of the fringes, normalized by the unperturbed spacing of the fringes, N, is given by Δ T sin Θ N<
L o
tan Θ o
VI
tan
(5)
681 where L is the wavelength of sound in the initial material, viz., the o microscope stage.
Note that N is a dimensionless number, corresponding to
the number of fringes of shift in the interferogram. Θ
and then using the Snell's Law relationship, C
X
By solving Eq. 5 for
is readily calculated.
2C
Typically Θ
is 10°, however, it depends upon the particular configuration
of the stage employed. The capability of this technique for differentiating velocity of sound regions within the matrix of a tissue, is dependent upon the region thickness.
This is consistent with other methods of velocity measurements such
as "time of flight" techniques.
For example, if fringe line displacements
can be measured to the nearest 1/10 fringe, then in a section of tissue, say, 300 microns thick, i.e., 20 wavelengths at 100 MHz, 7.5 m/sec variations in C
may be discriminated.
X
6.
RESULTS AND DISCUSSION During the development of the very young field of acoustic microscopy
a variety of biomédical investigations have been conducted.
A few of the
highlights are discussed below. Isolated mouse embryo hearts have been maintained alive for several days on the stage of the SONOMICROSCOPE 100 using an organ culture tech12 13 nique. '
Simultaneous optical and acoustical viewing in real time
permitted more complete observation of the contractile event than heretofore possible.
An example of micrographs produced is shown in
Figure 2. Internal and surface structures of the heart such as valves, chambers, and coronary arteries are imaged on a TV monitor and precise measurements of atrial, ventricular and sinus originated beats are measured and recorded on video tape. tinuous perfusion.
Drugs are introduced either by pulsing or by con-
Various states of contractility, e.g., synchrony,
arrhythmia or fibrillation, is observed either spontaneously or through the introduction of drugs.
Effectiveness of cardioactive drugs such as
lidocaine, epinephrine, aconitine, etc. in modifying such induced or in-
682
Fig. 2
Still frame photographs taken from video tapes of a live mouse embryo heart maintained in organ culture on the stage of the acoustic microscope.-'-3 The cross marks represent spacings of 1 millimeter. a)
Optical through transmission image demonstrating the opacity of the organ.
b)
Corresponding acoustic micrograph at 100 MHz. The functional anatomy of the cardiac cycle can be viewed clearly in the acoustic micrograph. The instantaneous positions of the muscle fibers and valves in diastole are shown here.
trinsic contractile states is measured by this non-invasive, non-destructive technique. The embryological development of CFI mice has been studied with an acoustic microscope.
14
The embryos were removed from time-mated mice and
placed on the microscope stage where they remained alive for substantial periods of time (hours) without the assistance of culture techniques. fetal mice were at gestational ages ranging from 10 to 12 days.
The
Major
morphological differentiation of tissues has occurred immediately prior to this period.
By the tenth day, the heart and its functional aspects
are completely discernible. embryos were produced.
Video tape recordings and movies of live
These illustrate the high degree of tissue differen-
683 tiation at this stage of development, as well as the great amount of structural detail that can be observed acoustically compared with that observed optically.
Figure 3 shows acoustic and optical micrographs of
a 10^ day specimen. Optical visualization of whole-mount embryos is usually very difficult since the specimens are thick and optically opaque.
Therefore, it is
necessary to employ fixing, clearing and staining techniques which alter the natural state of the tissue.
However, the microanatomy of a fixed but
unstained, optically opaque 72-hr. chick embryo is nearly completely revealed with an acoustic microscope.
Soft tissue structures have been
differentiated and information on elasticity and density at the microscopic level have been obtained.
Comparison of the acoustic micrograph with the
known, optically revealed, microanatomy of the chick embryo indicates that a rather complete visualization of organ primordia has been achieved without clearing and staining the specimen.
It was further suggested that
the effects of the fixative may be selective in the manner in which the elastic properties of the tissues are altered.
This can, perhaps, be used
to advantage. Acoustic micrographs of unstained specimens of mouse kidney have been produced.
The acoustically revealed structure has been examined in re-
lation to established microanatomy.
High contrast details corresponding
to connective tissue boundaries of supporting elements of the nephron are exhibited.
In addition, various regions of the kidney such as the cortex
and the three medullary regions can be differentiated.
The acoustically
exhibited structures result from wave scattering at connective tissue interfaces.
The scattering is caused by localized elasticity and density dis-
continuities . Acoustic attenuation measurements have been made in kidney tissue. It has been well established that for mammalian tissues such as kidney and brain, the attenuation coefficient exhibits a linear dependence over the frequency range of investigation, i.e., 0.3-10 MHz, and the magnitude is
684
685 of order of 1 db/cm/MHz.
The absorption coefficient for water is signifi-
cantly less than for tissue, and exhibits a square-law frequency dependence. Projecting the linear dependence for tissue to higher frequencies, and comparing this to the known absorption in water, leads to speculation that beyond about 500 MHz, the attenuation for water might exceed that for tissue.
Since tissue is composed mainly of water, this would not ordinarily
be expected. The attenuation of sound in mammalian kidney tissue was measured at 8 100 MHz and 220 MHz.
The tissue samples were either fresh, fresh-frozen-
thawed, or fixed in formalin.
The measured attenuation values were found
to be independent of preparation.
The result, presented graphically in
Figure 4, indicates that the acoustic attenuation in tissue continues to behave linearly up to a frequency of about 100 MHz.
Beyond this frequency
a square-law dependence (or greater) takes over, thereby indicating that the hypothetical crossover point may never actually be reached.
This is
more in line with intuition. The use of acoustic microscopy to delineate mechanical variations in hard tissues has been examined, and preliminary qualitative results were 17 published for sections of dental tissue.
Dentin and enamel components
of this tissue differ greatly in their visual and mechanical properties and this is clearly brought out in the acoustic micrographs. Furthermore,
Fig. 3
Composite images of a 10*5 day mouse embryo^4 histologically fixed in ethanol but unstained and uncleared. Similar images are produced of live specimens. a)
Optical through transmission image
b)
Acoustic micrograph at 100 MHz
c)
Acoustic interferogram demonstrating variations in acoustic index of refraction of the various tissues. These variations appear as lateral position shifts of the vertical fringe lines. The fringe-to-fringe spacing, in terms of acoustic transmission time, is 10 nanoseconds. By means of the formulas in the text the elastic properties of the tissues can be calculated.
686 1
1—i
i i i i 11
1
1—i
i
3.0
2.0 1.5
T
T
i
i i i i i j
i
A) Striated Muscle B) Kidney and Brain C,D) Kidney
3.5
2.5
i i i T-j —
-i A mum
1.0
^
III,
,,
i
III
B
//////////////////////////////////
C
0.5
0
1
0.1
1
1—1 M i l l
1
1
1
1 i i i i i1
|
|
|
10
I 1 i i i1
,
i
.
. .
100
. 1
1000
FREQUENCY IN MHz. Fig. 4
Ultrasonic attenuation in mammalian kidney tissue at extended frequencies.7 Data A and B are taken from reference 10 and data C and D taken from an acoustic microscopy study described in reference 8.
in the region of dentin-enamel junction the acoustic interference mode demonstrates a smooth gradation of density and elasticity.
This study
serves as the forerunner to a series of experiments on bone and plaque formations particularly. Investigation of tissue elastic component involvements in the diseased state is being conducted.
With regards to sound velocity variations within
and among soft tissues at low frequencies (1-10 MHz) only a 100 m/sec range has been observed.
On the microscopic scale, preliminary evidence has
shown that there are localized regions which are characterized by velocities of sound 200-800 m/sec above that of neighboring tissue structures.
This
means that microelasticity variations in tissue are great at the microscopic level and implies that these may be quite characteristic of the tissue type and pathology.
687 REFERENCES 1.
A. Korpel, L. W. Kessler and P. R. Palermo: at 100 MHz, Nature 232 (1971) pp. 110-111.
2.
L. W. Kessler, A. Korpel and P. R. Palermo: Simultaneous Acoustic and Optical Microscopy at Biological Specimens, Nature 239 (1972) pp. 111-112.
3.
L. W. Kessler, P. R. Palermo and A. Korpel: Practical High Resolution Acoustic Microscope, Acoustical Holography, Vol. 4, Plenum Press, New York (1972) ed. by G. Wade, pp. 51-71.
4.
L. W. Kessler, P. R. Palermo and A. Korpel: Recent Developments with the Scanning Laser Acoustic Microscope, in Acoustic Holography, Vol. 5, Plenum Press, New York (1974) ed. by P. Green, pp. 15-23.
5.
L. W. Kessler: The Sonomicroscope, Proceedings 1974 IEEE Ultrasonics Symposium, ed. by J. de Klerk,IEEE, New York, N.Y. Cat. #74-CH0896-l SU, pp. 735-737.
6.
7.
An Acoustic Microscope Operating
TM SONOMICROSCOPE 100 , Manufactured by Sonoscan, Inc. 7 52 Foster Avenue, Bensenville, Illinois 60106, U.S.A. under exclusive license from Zenith Radio Corporation. . L. W. Kessler:
High Resolution Visualization of Tissue With Acoustic Microscopy, in Proceedings of the Second World Congress on Ultrasonics in Medicine, Rotterdam, June, 1973, ed. by M. de Vlieger, Excerpta Medica Foundation (1974) pp. 248-256.
8.
L. W. Kessler: VHF Ultrasonic Attenuation in Mammalian Tissue, J. Acoust. Soc. Amer. 53 (1973) pp. 1759-1760.
9.
L. E. Kinsler and A. R. Frey: Inc. (1962), Chapt. 6.
10.
See for example: D. E. Goldman and T. F. Hueter: Tabular Data of the Velocity and Absorption of High-Frequency Sound in Mammalian Tissue, J. Acoust. Soc. Amer. 28 (1956) pp. 35-37.
11.
P. R. Palermo and L. W. Kessler:
12.
R. C. Eggleton and L. W. Kessler: Ultrasound in Medicine, Vol. 1, ed. by D. N. White, Plenum Press, New York, N.Y. (1975) pp. 537-542.
13.
R. C. Eggleton, L. W. Kessler, F. S. Vinson and G. B. Boder : Proceedings 1975, IEEE Ultrasonics Symposium, Los Angeles, California, Published by IEEE New York, Catalog #75-CHO-994-4SU, pp. 57-58.
14.
W. D. O'Brien, Jr., and L. W. Kessler: Examination of Mouse Embryological Development with an Acoustic Microscope, American Zoologist 15 (1975) p.807A.
15.
M. Ahmed, and L. W. Kessler: Microanatomy of a Histologically Unstained Embryo as Revealed by Acoustic Microscopy, in Acoustical Holography, Vol. 6, ed. by N. Booth, Plenum Press, New York, N.Y. (1975).
16.
L. W. Kessler, S. I. Fields and F. Dunn: Acoustic Microscopy of Mammalian Kidney, J. Clinical Ultrasound, 2 (1974) pp. 317-320.
17.
L. W. Kessler: A Review of Progress and Applications in Acoustic Microscopy. J. Acoust. Soc. Amer. 55 (1974) pp. 909-918.
Fundamentals of Acoustics, John Wiley & Sons,
Unpublished.