High-frequency harmonic imaging of the eye

Abstract

Purpose

Harmonic imaging has become a well-established technique for ultrasonic imaging at fundamental frequencies of 10 MHz or less. Ophthalmology has benefited from the use of fundamentals of 20 MHz to 50 MHz. Our aim was to explore the ability to generate harmonics for this frequency range, and to generate harmonic images of the eye.

Methods

The presence of harmonics was determined in both water and bovine vitreous propagation media by pulse/echo and hydrophone at a series of increasing excitation pulse intensities and frequencies. Hydrophone measurements were made at the focal point and in the near- and far-fields of 20 MHz and 40 MHz transducers. Harmonic images of the anterior segment of the rabbit eye were obtained by a combination of analog filtering and digital post-processing.

Results

Harmonics were generated nearly identically in both water and vitreous. Hydrophone measurements showed the maximum second harmonic to be −5 dB relative to the 35 MHz fundamental at the focus, while in pulse/echo the maximum harmonic amplitude was −15dB relative to the fundamental. Harmonics were absent in the near-field, but present in the far-field. Harmonic images of the eye showed improved resolution.

Conclusion

Harmonics can be readily generated at very high frequencies, and at power levels compliant with FDA guidelines for ophthalmology. This technique may yield further improvements to the already impressive resolutions obtainable in this frequency range. Improved imaging of the macular region, in particular, may provide significant improvements in diagnosis of retinal disease.

1. INTRODUCTION

The use of ultrasound for diagnostic imaging of the eye was first described by Mundt and Hughes¹ in the late 1950’s. Practical B-scan systems operating at 10 MHz became available in the 1970’s. In the early 1990’s, ultrasound biomicroscopy (UBM), at frequencies of 35–50 MHz, was introduced by Foster and Pavlin.² Because of the effect of attenuation, UBM systems are limited to evaluation of the anterior segment, consisting of the cornea, anterior chamber, iris, ciliary body, and anterior lens. UBM systems have made a significant impact in evaluation of pathologies including glaucoma, corneal scars, hypotony and tumors. It is also proving useful in surgical procedures for vision correction such as laser in situ keratomileusis (LASIK) and phakic lens implantation. Most recently, transducers operating at about 20 MHz have been introduced for evaluation of the posterior segment, providing a sensible improvement in resolution compared to 10 MHz systems (75 micron versus 150 micron wavelength).^3–5

In 1991, Huang and his coworkers first described the use of optical coherence tomography (OCT) for high resolution imaging.⁶ By the mid-1990’s, commercial OCT systems for evaluation of the retina became available, with continuing improvement in image detail until the present day. Recently, the extension of OCT to the anterior segment of the eye was described as well.⁷ With resolution on the order of 10 microns, such systems are significantly superior to ultrasound for evaluation of the retina. OCT, however, provides limited penetration in depth (about 1 mm), is degraded by any opacity (cataract, hemorrhage) in the eye, and can only be performed in the fundus, i.e., that part of the retina visible through the pupil.

The ability to visualize the retina at the highest possible resolution is crucial for diagnosis of conditions such as age related macular degeneration, diabetic retinopathy, retinal tears and posterior vitreous detachment. Ultrasound’s capability of visualizing deeper tissues such as the choroid and orbit is of great potential value, yet the gap between 20 MHz ultrasound and OCT resolution is substantial.

In the early 1990’s, it became widely appreciated that ultrasound resolution can be enhanced by use of tissue harmonic imaging (THI). The basis of this is the non-linear generation of harmonics of the fundamental transmitted frequency on a cumulative basis as the pulse propagates. Non-linear effects increase with the amplitude of the pressure in the acoustic pulse, and are also related to the properties of the propagation medium. THI is of advantage because the harmonic has reduced sidelobes compared to the fundamental, resulting in improved lateral resolution.^8,9 In ophthalmic applications, fundamental frequencies in the UBM range cannot be used for imaging the posterior segment due to attenuation. However, the second harmonic of a 20 MHz fundamental need only traverse the distance between the retina and the transducer in one direction, since the harmonic is generated primarily in the focal region. Because the posterior chamber of the eye is filled with vitreous humor, which is approximately 99% water, it can be expected both to support generation of harmonics and to have a low attenuation coefficient.

The aim of this study is to examine the use of harmonics for ophthalmic imaging.

2. METHODS

We measured the reflection of a flat glass plate placed in the focal plane and oriented normally to the beam axis of a spherically focused PZT 10 MHz transducer (aperture 12 mm, focal length 36 mm). Using a Panametrics 5900 pulser/receiver, we acquired the glass plate echo using a LeCroy digitizing oscilloscope operating with a sample rate of 250 MHz. We conducted the experiment using both degassed water and bovine vitreous as propagation media. Echo data were recorded at excitation energies of 1, 2, 4, 8, 16 and 32 μJ. We determined power spectra for each condition, and normalized (by subtraction) the spectra at each excitation energy against that obtained at 1 μJ. The experiment was then repeated, but instead of recording the glass plate echo, we digitized data from a calibrated 40 μm aperture needle hydrophone (Precision Acoustics, Ltd., Dorchester, UK) placed in the near field.

We next examined glass plate and hydrophone data from focused lithium niobate transducers with center frequencies near 20 MHz (10 mm aperture, 30 mm focal length) and 35 MHz (6 mm aperture, 12 mm focal length). In this case, we used a custom pulser/receiver which produced bipolar excitation pulses of adjustable frequency and voltage. Data were recorded as a function of excitation pulse voltage at an excitation pulse frequencies of 22 MHz and 35 MHz for the 20 MHz and 35 MHz transducers, respectively. We also recorded data with the hydrophone in the near field (about half the distance to the focal point) and the far field (about double the distance to the focal point) along the beam axis, averaging the echo data from 1000 successive pulses.

We used a 3D motion system (1 μm resolution) to measure the sound field in the region of the focal plane. We maximized the hydrophone response in three axes and then recorded data in a series of planes at 0.1 mm intervals from −2.5 mm to 2.5 mm relative to the focal plane. In this case, data were recorded using an Acqiris 12-bit digitizer operating at 400 MHz, with 100 successive signals averaged at each position.

Scans were performed on the anterior segment of a rabbit eye using a 35 MHz transducer and images generated of the fundamental and second harmonic. Scans were made of the posterior pole of a normal human subject using a 20 MHz transducer. A 40 MHz high pass filter was used to obtain images of the harmonic.

3. RESULTS

Figure 1 shows glass plate spectra for a nominal 10 MHz fundamental transducer at excitation energies from 2 to 32 1 μJ obtained in water and vitreous after subtraction of the 1 μJ spectrum. In both cases we observed a peak centered at approximately 22.5 MHz which increases in amplitude with increasing excitation energy. Hydrophone measurements performed in the near field 1 mm from the face of the transducer showed no such increase.

Figure 1.

Spectra of glass plate reflection of 10 MHz transducer at a series of increasing excitation energies (2 to 32 μJ) after subtraction of 1 μJ spectrum. A harmonic at 20 MHz occurs in both water and bovine vitreous propagation media as excitation energy increases.

The spectra of the glass plate reflections and hydrophone measurements at the focus for the 35 MHz transducer are shown as a function of excitation voltage in Figure 2. The plots demonstrate the generation of a peak at approximately double that of the fundamental that increases in amplitude with excitation voltage. In the hydrophone measurements, the harmonic is relatively larger than that observed in pulse/echo mode, probably as a consequence of one-way travel, which reduces attenuation of the high-frequency components.

Figure 2.

Plots of spectra for a 35 MHz transducer as a function of excitation voltage for signals recorded in pulse/echo mode (left) and using a needle hydrophone at the focal point (right).

The spectra in the near-field and far-field for the same transducer are shown in Figure 3. In this case, the second harmonic component is absent in the near-field, while remaining present in the far-field.

Figure 3.

Spectra of hydrophone signal obtained in near and far fields for 35 MHz transducer with 40V excitation..

Figure 4 is a plot of the peak negative pressure for this transducer about the focal point, and the sidelobe is readily seen.

Figure 4.

Plot of peak negative pressure in the region of the focal point for the 35 MHz transducer under 40V excitation.

A representation of the power spectrum as a function of range along the beam axis ±2.5 mm about the focal plane is presented in Figure 5. The fundamental and second harmonic are clearly evident, as well as an apparent subharmonic.

Figure 5.

Spectra along beam axis in region of focal plane for 35 MHz transducer under 40V excitation.

Similar plots of peak negative pressure and spectra about the focus of the 20 MHz transducer are provided in Figures 6 and 7. In this case, we can readily see the second, third and fourth harmonics, as well as the fundamental. Spectral scalloping along the beam axis is more notable in the 20 MHz transducer harmonic components than in spectrum of the 35 MHz transducer, but in all cases, the harmonics are present throughout the focal region.

Figure 6.

Peak negative pressure in focal plane of 20 MHz transducer excited at 40V.

Figure 7.

Spectra along beam axis in region of focal plane for 20 MHz transducer under 40V excitation.

Table 1 summarizes acoustic output parameters at the focal point of the 35 MHz transducer as a function of excitation voltage. For reference, the current FDA standards for ophthalmic exposure are 28 W/cm² for I_SPPA.3 (derated spatial-peak temporal-average intensity), 17 mW/cm² for I_SPTA.3 (derated spatial-peak pulse average intensity), and 0.23 for MI (mechanical index).

Table 1.

Acoustic parameters for 35 MHz transducer at various excitation voltages.

Volts	Pr (MPa)	ISPPA.3 (W/cm2)	ISPTA.3 (mW/cm2)	MI
5	0.853	1.147	0.007	0.027
10	1.899	6.785	0.032	0.061
20	3.860	19.189	0.084	0.104
40	5.409	42.012	0.181	0.162

In vivo fundamental and harmonic images of the anterior segment of a rabbit eye (35 MHz) and the posterior segment of a normal human eye (20 MHz) are presented in Figures 8 and 9, respectively. In general, the harmonic images appear to have somewhat reduced speckle, improved resolution, and better delineation of interfaces. The harmonic images tend to have reduced penetration in depth. This is especially true in the human eye, where the acoustic path penetrates the orbital tissue, and must traverse a path approximately 3 cm in length on its return trip to the transducer.

Figure 8.

35 MHz fundamental (left) and harmonic (right) mages of the ciliary body of a rabbit eye.

Figure 9.

Two sets of 20 MHz fundamental (left) and harmonic (right) images of the posterior segment of a normal human eye in the region of the optic nerve.

4. CONCLUSION

Optical techniques have long been used for diagnosis of diseases of the eye because the retina can be readily visualized due to the transparency of the intervening tissues (cornea, aqueous, lens, vitreous). Angiographic techniques allow real-time en face visualization of retinal perfusion. In the last decade, OCT has allowed visualization of the retina to the point of visualization of internal layering. Ultrasound, however, still has two advantages over optical methods. First, in situations where opacities are present (corneal scarring, cataract, hyphema or vitreous hemorrhage, pupillary constriction), optical methods are unusable. Secondly, optical methods are limited in terms of depth of penetration beneath the retina. OCT, for instance, barely penetrates into the choroid, the vascular tissue layer underlying and oxygenating the retina. It is probable that senile macular degeneration and other pathologies affecting retinal function originate with abnormal function of the choroid. However, the resolution provided by current standard ophthalmic ultrasound systems operating at 10 MHz (roughly 200 mm axially by 500 microns laterally) is an order of magnitude lower than that provided by OCT. Ultrasound resolution is improved by a factor of two in the recently introduced 20 MHz ophthalmic scanners. Further improvement in ultrasound resolution by increases in frequency are likely to be small, as acoustic pulses must traverse a distance of about 25 mm (the size of the globe) and tissues such as the sclera, all of which contribute to acoustic attenuation. Harmonic imaging represents a potential means for improving resolution. High frequency components generated during propagation are effectively attenuated only in one direction instead of two, as is the fundamental. The reduction in the size of the sidelobes leads to a significant improvement in lateral resolution.

Cherin and his coworkers demonstrated that harmonics can readily be generated at 20–40 MHz.^10,11 Our findings demonstrate that in this range harmonics are generated in both water and vitreous. For the 35 MHz transducer, acoustic parameters were within FDA safely limits except at the highest excitation voltage (40V). Harmonics were readily produced at lower excitation intensity. 20 MHz pulse intensity parameters were significantly lower than those produced with the more highly focused 35 MHz transducer. Thus, there is no regulatory limitation affecting use of harmonics.

The comparative fundamental and harmonic in vivo images of the rabbit anterior segment and human retina show an improvement in resolution, but at the cost of reduced sensitivity and signal to noise ratio. The latter is to a great extent a consequence of technical aspects of our imaging system that may be addressed in the future. Furthermore, the signal processing approach may be improved by use of pulse inversion instead of the more easily implemented filtering used here. While tissue harmonic imaging is unlikely to ever provide the resolution obtainable using OCT, it, together with other signal processing methodologies may impact upon what can and cannot be visualized in vivo. In particular, if resolution can be improved to allow imaging of choroidal structure, ultrasound might have a profound impact upon our understanding of the etiology of disease states including age related macular degeneration and neoplasms.