A high resolution ultrasonic microscope

A high resolution ultrasonic microscope A. PENTTINEN and M. LUUKKALA

The construction and imaging capabilities of a scanning high resolution ultrasonic microscope are considered in this paper. Theoretical expressions for the resolution limits of the microscope and some of the most important properties of the ultrasonic lenses are given. Ultrasonic through-transmission and back-scattering images of different objects are presented and a quasi-three-dimensional display mode is introduced. A resolution of about 4 um is demonstrated at 300 MHz by the present ultrasonic microscope.

I ntroduction The development of ultrasonic transducer techniques has made it possible to generate ultrasonic waves in vhf and uhf ranges. At these frequencies the wavelength of ultrasound may be only a few microns. If such a wave is used in a diffraction limited imaging system, a very good resolution, comparable to that of an ordinary optical microscope, can be achieved. There are two ways to pick up the image information in an acoustical microscope. One uses optical laser pick-up, 1,2 and has the important property of being able to produce both optical and acoustical image at the same time. Another method involves a two-lens system, a-6 where the image information is transduced into an electrical signal by a piezoelectric element. This method has turned out to produce the best resolution and has no resolution limit determined by the optical wavelength. It is also capable of image formation based on the information carried out by the wave reflected from the object. Other important properties of an ultrasonic microscope are the possibilities to use both amplitude and phase sensitive detection, 7 dark-field viewings and non-linear microscopy.9 The contrast of an acoustic image has turned out to be good also for objects that are hardly visible without staining in an optical microscope. Many biological tissues and cells are such subjects. The staining procedure of these samples may be time consuming and can also change the properties of the material. Using an ultrasonic microscope an image of the untouched object can be obtained and in much less time that would be required for optical imaging. In many cases it is reasonable, however, to take both the optical and acoustic image of the sample, because the two methods give different results. The acoustic image is sensitive to the elastic properties of the object while the content of the optical The authors are at the University of Helsinki, Department of Physics, Siltavuorenpenger 20 D, 00170 Helsinki 17, Finland. Paper received 16 February 1977.

ULTRASONICS. SEPTEMBER 1977

image is determined by the dielectric properties. So some new details may be revealed when the two images are compared.

Equipment The design of our ultrasonic microscope is based on a scanning principle wherein the image is formed point by point. The object is moved in the focal plane of a high quality ultrasonic lens and the sweep of a display tube is synchronized with this movement. The image information that is picked up by another ultrasonic lens is converted into an electrical signal and used to intensity modulate the image point on the display tube screen. The block diagram of the system is seen in Fig. 1. The transmitted signal is a gated sine wave of frequency up to 300 MHz. This eliminates the standing waves that otherwise would appear between the lenses at lower frequencies. It also helps to separate the reflected signal from the transmitted one, when the system is used to take reflection images of the objects. The signal is then transformed into a longitudinal ultrasonic wave by the transmitting piezoelectric transducer. The acoustic wave is focused by a single surface ultrasonic lens and allowed to penetrate through the object. On the other side of the sample another identical lens-transducer pair is set confocaUy with the first lens. This collects the through-transmitted ultrasound and converts it again into electrical form. This signal is observed by a sensitive am-receiver. A sample and hold circuit is used to store the amplitude information of the received rf-pulse. The image signal is then led to the z-modulator which generates a suitable voltage for the brightness modulation. Because we used a bistable storage tube, a special circuit was required. 1° This generates a greyscale image by changing the effective spot size of the display tube according to the image signal. A logarithmic amplifier is also provided to increase the dynamic range of the display. For phase contrast micrographs the receiver must maintain the phase information of the image signal. For this reason the signal is only amplified by a high-gain preamplifier and then

205

P O

I

I

Lenses

Preamplifier

Io

~l. O

Directional coupler

j-

J Superheterodyne receiver

y scan Error ampli

Fig. 1 The ultrasonic microscope

fed into a doubly balanced mixer which takes the reference signal from the transmitter oscillator. The output of the mixer is again stored by the sample and hold circuit. When reflection mode images are taken, the receiver must be connected to the transmitting transducer lens system. This is accomplished via a directional coupler or a hybrid junction that isolates the transmitted pulse from the receiver. In this case only one lens is needed. The two-dimensional scan of the object is performed electromechanically. In the y-direction the object holder is driven back and forth by a loud-speaker type electromagnetic system, involving a capacitive motional feedback unit. The whole system input is a triangle voltage and the deviation of the true position of the scanner from this is detected by an error amplifier. The feedback ensures a linear scan in the y-direction regardless of the scanner load and friction conditions. The triangle voltage is also used to generate clock pulses to the x-scanner which works digitally. When the start signal is applied these pulses begin to accumulate in the up/down counter. The content of this counter is converted into an analogue voltage by a digital to analogue converter and this voltage is led to the display circuits. Depending on the required magnification the step pulses of the x-scan stepper

Oblect-~

Water

LiNDO3

LiNb03 I YAG Fig. 2 The lens system of the microscope

206

YAG

motor are taken from different points in the counter chain. The steps of the motor are transformed into linear motion by a screw-drive system that carries the whole y-scan assembly and moves along steel rails. When a certain number of clock pulses have passed, the counter circuit provides a direction signal that changes the direction of rotation of the motor and the scanner returns to the original position. After that re-imaging of the same object area is possible. It takes about ten seconds for our present equipment to scan the whole image area (2 mm x 2 mm at a magnification of 50). No significant reasons have appeared that would require to make it faster. Because the grey-scale capabilities of the display tube are quite limited, a display control circuit is included in the system. This circuit generates a 'three-dimensional' display by adding a third axis to the display. ~ If the y-scan of the object is directed along this diagonal axis in the display, the image information can be led to the y-axis of the display. This three-dimensional image gives a much better idea of the dynamic behaviour of the image contents.

Lenses and transducers The lens system of the microscope is seen in Fig. 2. The two single-surface lenses are identical and made of YAG (yttrium. aluminium-garnet) rod that has a very low value of ultrasonic attenuation (~ 3 dB m -1 at 300 MHz) and a high value of ultrasonic wave velocity (8600 m s -1 ). At the other end y-cut LiNbO3 is attached. An epoxy bond was used between the transducer and the lens. The platelet has its fundamental resonance near 30 MHz but is polished to generate harmonic waves at much higher frequencies. The highest observed harmonic frequency was at 362 MHz (eleventh harmonic) but the highest usable frequency was about 300 MHz (ninth harmonic). Inside the water drop, where the object is immersed for imaging, the wavelength of the ultrasonic wave is thus about 5 pm.

ULTRASONICS

. SEPTEMBER 1977

The lenses were made by grinding a spherical surface at the free ends of the YAG rods. The radius of curvature of the surface is 1 mm. The focal length and possible spherical aberrations of the lens are determined by the ratio of the wave velocities in water and in the lens material. In our case this ratio is # = 0.174 and the focal length of the lens is f = 1.2 mm. The size o f the lens surface was so chosen that its f-number is unity.

200

The spherical aberration of a lens is proportional to the square of the ratio #. For optical waves /a = 0.67 typically and the aberrations are so large that the focal spot size is determined by them. In our ultrasonic lens the velocity ratio is much smaller and the lens is practically diffraction limited. This is also clearly revealed when the sound pressure distribution near the focal area is computed. This can be done on the basis of the calculations made for curved ultrasonic transducers. Some results of these computations 12 are shown in Fig. 3. These show a strong concentration of ultrasonic energy into a very small area. Def'mition of a focal point of ultrasonic lenses is consequently at least as justified as it is in optics. Because of powerful focusing, high pressure (intensity) gains can be obtained. Expressions for the most important lens properties can be written in a relatively simple form, if the material and shape of the lens are known. If/a is the ratio of wave velocities in the surrounding medium and in the lens, Ro the radius of curvature of the lens, and a m half the apex angle of the cone that determines the boundary o f the lens, then the expressions for the focal length and maximum pressure gain are:

I so

Ioo

5o

O .,

f

l°

= Ro/(1 --U)

(1)

-60

-40

-20

a

0

20

40

go

z-I C/~m]

and Gm =

(2rrRo/X) (1 -- cos am) (1 -- 0.976 U)

(2)

where X is the wavelength o f the ultrasound. The size of the focal area is described by the distances between the ttrst pressure minima on both sides of the greatest pressure maximum. In the focal plane distribution of pressure this distance gets the form: Ap

A p o e COS (ba~n)/cos (barn)

=

(3)

where Apo is the corresponding property for a curved radiator o f the same size: Apo

=

1.22 X cos (bam)/Sin am,

b

=

0.39

and

a~n = e =

sin -1 (sin am/e)

[1 -- # ( 1 - - /a) ( 1 - - cOS a m ) l / ( 1 - - /a)

The corresponding measure for the axial distribution is: =

X(2fd -

-- X2)

(4)

,%( V,

where d

= f--

(f2_a2),/=

and a = f s i n a m / e

Usually the -- 3 dB points of pressure are considered. Approximate values for these are obtained when the values

U L T R A S O N I C S . S E P T E M B E R 1977

-,; b

-,o

-5

6

,V ,V" ,;

s

,o

~; t ~

Fig. 3

a -- the axial and b -- focal plane distributions of sound

pressure for a Y A G ultrasonic lens w i t h R o = 1 mm and ~.=

5#m

207

mm -1 leading to about 43 dB loss in our microscope. The loss can be reduced, if the radius of curvature of the lenses is made smaller. This, in turn, makes the handling of the object more inconvenient, which is not desirable. In our present system imaging is easy and rapid, because there is room enough for the object without moving the lenses. The lens system is firmly installed and only one adjustment is needed to set the object to the common focal plane of the lenses. Experimental

results

When the object is fastened to the object holder between the lenses, a small drop of water is brought on both sides of the object. These provide the necessary path of ultrasound from the lenses to the object. Then the location of the object is adjusted until the best resolution and contrast is achieved. This can be done while imaging. If thin biological objects or other non-selfsupporting samples are imaged, they must be fixed on a thin plastic membrane attached to the object holder. If reflection (back-scattering) imaging is used, the thickness of the object may be up to 1 mm.

7! 4 1

t

Fig. 4 Acoustic through transmission images of a calibration mesh. a - 50/~m div-' ; b -- 12.5 #m div -t

of the above expressions are divided by two. For the lenses of our microscope these quantities get the values: f=

1.2mm

G m = 211 Ap(--3dB)

=

5.hpm

Az(-3dB)

=

32/zm

when an ultrasonic wavelength of 5 tam is assumed. The lateral resolution of the microscope thus is equal to the ultrasonic wavelength and is only limited by diffraction. As a matter of fact, a slightly better resolution is obtained if two such lenses are set in succession. This is the case in our microscope and that is why a resolution of about 4/am is to be expected. The selection of the lens type to the microscope was a compromise between a good resolution and easy operation. It is possible to improve the resolution by increasing the frequency of the ultrasonic transducers. By doing this the attenuation of ultrasound in water becomes, however, very significant. The attenuation of ultrasound at 300 MHz is about 18 dB

208

Fig. 5 a -- Q u a s i - t h r e e - d i m e n s i o n a l d i s p l a y o f the calibration mesh ( 1 2 . 5 # m d i v -1 ); b -- through-transmission image of human red blood ceils ( 1 2 . 5 / ~ m d i v -1 )

ULTRASONICS

. SEPTEMBER

1977

an image of the surface of an analogue integrated circuit. The surface structure is formed by thin aluminium conductors that are deposited on the surface. On the fight edge there are a couple of connecting pads for external wires and a '+' sign that is clearly recognizable on the front corner of the image. The three dimensional display is, perhaps, the best one for this kind of image. The lower picture shows tile surface of a piece of polished stainless steel. The small details on the surface are clearly visible. If phase sensitive detection is used to construct these images, the real topography of the corresponding surface can be obtained. In Fig. 7 an image of a thin paper sheet is presented. Magnification was about 100 (100/an div -I ) while imaging. The usual intensity modulated image gives a better view of the structure of the object but the corresponding three-dimensional image tells much more about the dynamic content involved in the image.

Conclusions

The construction and most important properties of a high resolution ultrasonic microscope have been treated. Special attention has been given to the ultrasonic lens system and its limitations. Theoretical expressions were given for the

D Fig. 6 images taken by reflection method, a -- quasi-threedimensional image of the surface of an integrated circuit. b - surface of polished stainless steel (both 50 ~um div- 1 )

The first thing considering a new microscope is to demonstrate its resolution. This is easily done by taking an image of a calibration mesh intended for electron microscopes. In Fig. 4 there are two images of such a mesh taken at different magnifications. The upper image was taken with a nominal magnification of 200 (50/am div -1) and in the lower image it is about 800 (12.5/an div -I ). A resolution of a few microns is clearly demonstrated in these pictures. The rounding of the mesh holes is probably due to the limited resolution and small air bubbles that may be located in the corners of the holes. In Fig. 5a the three-dimensional display is used for the object of Fig. 4b. This indicates much better the intensity fluctuations of the through-transmitted ultrasound. The high peaks in the image correspond to bright spots in the normal display. Fig. 5b is a through-transmission image of a few human red blood cells. It confirms the resolution of about 4/am, because the diameter of these cells is typically 6-7/am. It also gives an idea of the good contrast of ultrasonic images that is obtained without staining the object. The pictures in Fig. 6 have been taken in the reflection mode and displayed in three dimensions. The upper one is

ULTRASONICS. SEPTEMBER 1977

Fig. 7 a - Through-transmission image of thin paper; b -- quasithree-dimensional display of the same image (both 100 # m div -1 )

209

lateral and axial resolutions of the microscope and also for the most useful properties o f the lenses. The lenses were shown to be diffraction limited and able to produce a lateral resolution that is slightly less than the ultrasonic wavelength used. This makes it possible to achieve a resolution of 4 / l m at an ultrasonic frequency of 300 MHz. Many images of different objects were presented to demonstrate the resolution and magnification capabilities of the microscope. Resolution limited, high contrast images o f human red blood cells could be achieved and the alternative three-dimensional display mode was noticed to be suitable for reflection or back-scattering images. In its entirety an ultrasonic microscope seems to produce a supplementary and even easier way to image microscopic samples o f various materials. The high contrast images of biological objects without staining give a good example o f its possibilities. An ultrasonic microscope is easily connected to a m o d e m data acquisition system, because the image signal is in an electrical form. The frequency content of the image signal is in the audio range which enables one to use a low cost cassette tape recorder for storage of the microscopic images.

References Kessler,L.W., Palermo, P,R., Kotpel, A. Practical high resolution acoustic microscopy, Acoustical holography, Plenum Press, NY, 4 (1972) 51 2 Kessler, L.W. Review of progress and applications in acoustic microscopy, JASA 55 (1974) 909 3 Lemons, R.A., Quate, C.F. Acoustic microscope-scanning version, Appl Phys Lett 24 (1974) 163 4 Lemons, R.A., Quate, C.F. A scanning acoustic microscope, 1973 Ultrasonics Symposium Proc, IEEE, 18 5 Lemons, R.A., Quate, C.F. Integr-ted circuits as viewed with an acoustic microscope, Appl Phys Lett 25 (1974) 251 6 Lemons, 1LA., Quate, CAF.Acoustic microscopy: Biomedical applications. Science 188 (1975) 905 7 Wickxamasinghe, H., Hall, M. Phase imaging with the scanning acoustic microscope, Electronics Lett, 12 Nov (1976) 8 Bond, W.L., Cutlez, C.C., Lemons, R.A., Quate, C.G. Dark-field and stereo viewing with the acoustic microscope, Appl Phys Lett 27 (September 1975) 9 Kompfer, R. and Lemons R.A. Nonlinear acoustic microscopy, Appl Phys Lett 28 (March 1976) 10 Karman, J., Kzoese, N. Displaying grey-scale images on bistable storage tubes, Electronics (Nov 1973) 11 l.anz, IC Circuit adds diagonal axis to any scope, Electronics (Sept 19, 1974) 12 Penttinen, A., Luukkala, M. Sound pressure near the focal area of an ultrasonic lens, ] Phys D: Appl Phys 9 (1976) 1927 1

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