Optical and Acoustical Interrogation of Submicron

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ieee transactions on ultrasonics, ferroelectrics, and frequency control, vol. 49, no. 12, december 2002 1641

Optical and Acoustical Interrogation of Submicron Contrast Agents

Divia Patel, Paul Dayton, Jana Gut, Erik Wisner, and Katherine W. Ferrara, Senior Member, IEEE

Abstract—Unlike conventional ultrasound contrast agentswith a diameter of several microns, in this paper we explore

the use of submicron contrast agents for the detection andlocalization of lymph nodes. The submicron agents are gas-filled, double-walled microspheres that rupture when ex-

posed to ultrasound energy at megahertz frequencies. Inthis study, three experimental systems are combined with

model predictions to assist in understanding the responseof these unique agents to a range of signal transmissionparameters. Optical experimental results for each agent de-

lineate the relative expansion as a function of acousticalpeak negative pressure, pulse length, and center frequency.

The optical images demonstrate an order of magnitude ex-pansion in radius during the pulse rarefaction, in which the

expansion magnitude is dependent on the transmitted pres-sure and frequency. Simulations using a modified Rayleigh-Plesset model predict an increasing relative expansion for

the microbubbles (initial bubble radius ranging from 0.3–1.3

m) with increasing pressure and decreasing initial ra-dius. Acoustically recorded frequency spectra reveal the

presence of harmonics for a range of transmitted pulses.In addition, in-vivo results from a normal canine modeldemonstrate marked contrast enhancement of first order

lymph nodes. We hope to offer an alternative to presentintra-operative procedures for sentinel node detection.

I. Introduction

Until recently, accurate staging and appropriate treat-ment planning in breast cancer patients often has re-

quired excision of regional lymph nodes by radical axil-lary node dissection [1]. Sentinel nodes are the first lymphnodes to receive lymphatic drainage from a parenchymalneoplasm, and, therefore, are at greatest risk for regionaltumor metastasis [2]. These nodes are labeled for selec-tive excision using radiotracers or dyes that enter the lym-phatics. Current detection techniques for sampling of thesehigh-risk lymph nodes in breast cancer patients includepreoperative lymphoscintigraphy for regional node map-ping and intraoperative localization of specific nodes, us-ing perilesional injection of particulate radiopharmaceuti-

cals in conjunction with a hand-held intraoperative gammaprobe. Lymphoscintigraphy is used to identify the locationof the sentinel lymph node by making the node radioac-tive through injection of a radiocolloid into the breastparenchyma around the primary tumor [3]. A study by

Manuscript received April 5, 2002; accepted July 15, 2002.D. Patel, P. Dayton, J. Gut, and K. W. Ferrara are with the De-

partment of Biomedical Engineering, University of California, Davis,Davis, CA 95616 (e-mail: [email protected]).

E. Wisner is with the Department of Surgical and Radiological Sci-ences, School of Veterinary Medicine, University of California, Davis,Davis, CA 95616.

De Cicco et al. [4] involved 382 patients with operablebreast cancer and identified at least one sentinel node in98.7% of the cases using lymphoscintigraphy with opera-tive gamma probe detection. Lymphoscintigraphy is alsoa relatively inexpensive, straightforward nuclear medicinetechnique satisfactorily accepted by patients [5]. Perile-sional intradermal blue dye injection often is included inthe protocol to visualize sentinel nodes and draining lym-phatics arising near the primary tumor [6]. Intraoperativesentinel node excisional biopsy has been shown to be ac-curate for cancer staging [1] and is becoming the standardof care for staging breast cancer patients because radical

lymph node dissection can be avoided when sentinel nodesare determined to be free of metastatic disease [7].

Following injection into subcutaneous or interstitial tis-sues, small particles traverse the walls of the peripheralafferent lymphatic vessels and are transported to regionallymph nodes in which at least part of the population arephagocytized by intranodal macrophages that line the si-nus system [8]. The ideal size range for peripheral lym-phatic uptake of particles is 10–40 nm [9], although par-ticles hundreds of nanometers in diameter may effectivelycross through the lymphatic endothelium, as suggested byempirical studies [10]. Likely reasons for this occurrenceinclude the following possibilities: with a heterogeneoussize distribution, only the smallest particles are able to ef-fectively cross the endothelial lining; deformation of themicrobubble shell may provide a simple means for parti-cles to cross lymphatic wall fenestrations; and the transientincrease in the hydrostatic pressure gradient across the pe-ripheral lymphatic vessel walls directly subsequent to mi-crobubble injection may assist small particle transit [10].Small particles tend to diffuse freely through the extracel-lular fluid space and are dispersed by blood capillary flow.Larger particles tend to sequester at the injection site. Theobservation that submicron and near-micron bubbles gen-erate contrast enhancement more regularly than 4-micron

diameter bubbles supports the idea that smaller bubblesare more successfully transported to regional lymphatics;and, as a result, are uniquely suited to lymphatic imagingapplications.

The accuracy of optical studies of sentinel node pre-dictions of axillary node metastasis ranges from 96–100%[11]. A study by Rubio et al. [12] tested the hypothesisthat the histology of the sentinel lymph node is an accu-rate predictor of the axillary node status. The study usedradiolabeled 99Tc sulfur colloid (unfiltered) to label thesentinel lymph node for biopsy and obtained a 96.3% suc-cess rate for sentinel lymph node identification.

0885–3010/$10.00 c 2002 IEEE



1642 ieee transactions on ultrasonics, ferroelectrics, and frequency control, vol. 49, no. 12, december 2002

It is hypothesized that ultrasound contrast agents, whenadministered interstitially, could serve as an alternative toconventional sentinel node detection methods requiring ra-diotracers in breast cancer patients [6]. Previous researchhas reported on the use of ultrasound contrast agents toimage vascular volume [13]. We extend this work to con-sider submicron sized gas-filled microbubbles and focus ontheir unique responses as a function of the transmitted fre-

quency, pulse length, and acoustic peak negative pressure.The relative expansion ratio of a microbubble is de-

fined as the maximum radius Rmax divided by the initialradius R0. When the relative expansion ratio exceeds acritical value, inertial forces dominate, and the microbub-ble collapses with resultant fragmentation. A temperatureand pressure increase occurs when the work done on themicrobubble during collapse, from convergence of the sur-rounding liquid, exceeds the dissipation of energy from themicrobubble [14]. Cavitation of a microbubble has been ex-amined with regard to contributions from the inertial forceand the pressure driving force during collapse [15]. If therelative expansion ratio is less than the critical value, ei-

ther the pressure force alone controls bubble collapse orthe control occurs in conjunction with inertial forces [14].A large relative expansion ratio initiates transient collapse,and a restricted range of initial radius values produce sucha large expansion. The lower limit exists because the cav-ity must initially undergo rapid growth and is dependenton pressure amplitude, whereas the upper limit is set bythe minimum relative expansion ratio that will produce atransient collapse [14].

In this paper, we present the analysis of two experimen-tal submicron, gas-filled, double-walled agents. Our prin-cipal goal is to evaluate, characterize, and compare the re-sponse of each of the contrast agents to ultrasound, using

both optical and acoustical methods. We begin by pre-senting the three experimental systems, used along withsimulations from a theoretical model. Relative expansionfor each agent is determined from optical analysis and dis-cussed with respect to acoustical peak negative pressure,pulse length, and center frequency. Acoustic data are ana-lyzed to produce frequency spectra, a mean frequency, anda mean amplitude. Additionally, we present successful ap-plication of these agents for sentinel lymph node detectionin vivo in a normal canine model. Simulations from themodified Rayleigh-Plesset model [16] predict relative ex-pansion ratios as a function of initial radius. Discussion of the results includes a comparison between our experimen-tal findings and predictions from the theoretical model.

II. Methods

The ultrasound contrast agents examined are alyophilized suspension of microbubbles. These structuresare composed of a bilayered shell containing nitrogen gas.The biSphere is composed of a biodegradable polymer anda human serum albumin outer coating (M1134, M1091 &M1136 POINT Biomedical, San Carlos CA). Manipula-

tions in the manufacturing process generate a collectionof structures varying in wall thickness and size. M1134 in-cludes microbubbles with an initial diameter from 500 nmto 1.3 µm, with more than 50% having a diameter smallerthan a micron. M1091 includes microbubbles that areslightly smaller, with a mean initial diameter of 800 nm.The two agents are each made of the same polymer withthe same relative wall thickness, differing only in mi-

crobubble diameter.Three systems are presented in this paper to assist in

understanding the response of these unique agents to a va-riety of signal transmission parameters. We first describean imaging system that consists of a microscope coupled toa high-speed camera. This system permits the observationof radial oscillations of microbubbles during insonation.The second experimental system consists of an acousticsystem that allows for the recording of echoes from the mi-crobubbles. The third experimental system uses a clinicalimaging procedure for diagnostic analysis of microbubbleuptake in the canine lymphatic system. Then simulationsfrom the modified Rayleigh-Plesset model allow for the

evaluation of experimental findings.

A. Optical

Optical experiments are performed using an invertedmicroscope (IX 70, Olympus, Melville, NY) with a 100Xwater objective (Zeiss Achroplan 100X, Carl Zeiss Inc.,Thornwood, NY). Images are acquired before, during, andafter insonation with a high-speed digital camera (Imacon468, DRS Hadland Inc., Cupertino, CA) that generatesseven individual frame images and a streak image. Thepixel size in the resulting images is 200 nm, and the opti-cal resolution is on the order of 500 nm. We have verifiedour ability to detect calibrated 200 nm beads, and the crit-ical parameter of interest in this study is the detection of a small particle rather than the resolution of two smallparticles. Therefore, our measurements presented here arebased on the pixel size of the imaging system rather thanthe optical resolution of the system. The streak image is anM-mode image of a single line-of-sight through the bub-ble over time. This allows for initial, maximum, and fi-nal diameter measurements in conjunction with fragmen-tation observations. A pulse is directed from an arbitrarywaveform generator (AWG 2021, Tektronix, Wilsonville,OR), amplified with an RF power amplifier (3200LA, ENI,

Rochester, NY), and delivered to the 2.25 MHz transducer(Panametrics V305, 2 PTF), which has a focal lengthof 2 inches. The transducer is focused on an opticallyand acoustically transparent 200-micron ID cellulose tube(Spectrum Laboratories, Ft. Lauderdale, FL). The con-trast agent is prepared by reconstituting the vial, contain-ing on the order of 1011 bubbles, with 2 mL of distilled wa-ter. Two drops of the solution are then diluted with an ad-ditional 3 mL of distilled water for optical experiments. Amicro injector (IM-5B, Narshige Inc., East Meadow, NY)is used to control the flow of microbubbles, concentratedat 8× 108 bubbles/mL, through the tube. At this concen-



patel et al.: submicron ultrasound contrast agents 1643

tration, approximately 40 microbubbles are present withinthe optical field of view. Subsequent to each signal trans-mission, contrast agent solution is refreshed with another83 µL of solution with the same concentration. Microbub-ble response over a range of acoustic pressure and pulselength is assessed. Transmitted signals are calibrated bymeans of a needle hydrophone (Specialty Engineering As-sociates PZT-0200; Soquel, CA) and a preamplifier (Spe-

cialty Engineering Associates, A17dB) linked to a digitaloscilloscope (9310, Lecroy, Chestnut Ridge, NY)

B. Acoustical

Acoustical experiments use two mutually focused trans-ducers, at an angle of 90 degrees, with the focus in the cen-ter of the cellulose microtube. In each case, a broadbandtransducer (Panametrics V305) with a center frequencyof 2.25 MHz and focal length of 2 inches, was used totransmit the signal, and a broadband transducer (Pana-metrics V309) with a center frequency of 5 MHz and focallength of 2 inches was used to receive the signal. The agent

flows through the focal point in an acoustically transparentcellulose tube, controlled by a syringe pump, which flowsat a constant rate of 1 mL/hour. Contrast agent solutionis prepared by reconstitution of the vial, containing 1011

bubbles with 2 mL of distilled water. Two drops of thesolution then are diluted with an additional 10 mL of dis-tilled water for a concentration of 2.5 × 108 bubbles/mL,referred to as high concentration throughout or with anadditional 20 mL of distilled water to give a concentrationof 1.3 × 108 bubbles/mL, classified as low concentration.Final concentration of the sample volume is approximately1–2 orders of magnitude more dilute than the solution inthe original syringe due to the fact that many bubbles float

to the top of the tube. For phase inversion experiments,the contrast agent solution was diluted additionally untilacoustic interrogation indicated that an echo from a sin-gle microbubble was acquired per acoustic pulse, and anecho was obtained for one of every five pulses. Transmit-ted pulses were produced by an arbitrary waveform gen-erator (Tektronix AWG 2021) and amplified with an RFpower amplifier (ENI 3200LA). Transmitted signals werecalibrated with the same needle hydrophone and pream-plifier linked to the oscilloscope, as described previously. AGeneral Purpose Interface Bus (GPIB) interface betweenLabview (National Instruments Corporation, Austin, TX)

and an oscilloscope is used to digitize and store the re-ceived echoes for data analysis. Acquired echoes are cal-ibrated through normalization by the receive transducerspectrum prior to further analysis.

C. In Vivo

The in-vivo experiment involved injection of contrastmedia into the subcutaneous tissues of the hind limbof a normal anesthetized dog to enhance the ipsilateralpopliteal lymph node. The limb was injected with a totalof two reconstituted vials of agent M1136, each contain-

ing 1011 particles per vial. The images of the left popliteallymph node were acquired using continuous power Dopplermode on an ATL HDI 5000 US unit (ATL-Philips, Bothell,WA). A linear L7-4 transducer (ATL Ultrasound, Bothell,WA) probe was used, operating at a pulse repetition fre-quency (PRF) of 12 KHz. The mechanical index (MI) wasvaried up to a maximum of 1.3. The gain was set in orderto maximize contrast between the agent and the tissue.

D. Simulations

The theoretical model used for relative expansion pre-dictions of the microbubbles is based on the modifiedRayleigh-Plesset (R-P) equation [16]–[18]. This differentialequation predicts the time-dependent radial oscillations of an encapsulated microbubble subject to a driving pressure,Pdriv(t). The modified R-P equation, given by (1), differsfrom the Rayleigh-Plesset equation in that it incorporatesadditional terms to account for the bubble shell and acous-tic radiation damping. The gas pressure in the bubble andthe acoustic radiation damping are described by the first

and second terms on the right of the equation. The thirdterm accounts for the viscosity of the surrounding liquid.The surface tension of the bubble is approximated by thefourth term and the shell elasticity by the fifth term of the equation. The sixth term reports for the viscous lossesdue to the shell, and the final term describes the drivingpressure. In order to set (1) to a nondimensional form, thevariables R∗ = R/R0 and t∗ = ωt were used. The differen-tial equation then was solved with application of the ode45function in Matlab. This solver uses an explicit Runge-Kutta method. Initial conditions applied include R = R0

and R = 0 at time t = 0.Eq. (2) describes the pressure at the liquid interface,

used in (1). Notation for both equations is given in Table I.

ρ(RR+3

2R2) =

P o +

2σ

Ro

+2χ

Ro

R30 −R3

0

b

V m

R3(t) −R30

b

V m

γ

+R

c

d

dt p(R, t) −

4µR

R−

2σ

R

−

2χ

R

Ro

R

2

− 12µshεR

R(R− ε)− (P o + P driv(t)), (1)

p(R, t) = P o +2σ

Ro

+2χ

Ro

R30 −R3

0

b

V m

R3

(t) −R3

0 bV m

γ

−

2σ

R−

2χ

R

R0

R

2

. (2)

III. Results

A. Optical

Fig. 1 shows five two-dimensional images and a streakimage of a line through the center of the microbubble, in




TABLE I

Notations.

c Speed of sound in liquid

f Linear frequency of incident acoustic field

f mean Mean frequency of a magnitude spectrum

P Pressure in surrounding liquid

P driv(t) Time varying acoustic pressure

P o Hydrostatic pressure pi Internal pressure

R Instantaneous bubble pressure

R Wall velocity of bubble

R Acceleration of bubble wall

R0 Initial bubble radius

R1 Inner bubble radius

R2 Outer bubble radius

S (f ) Magnitude spectrum

r Distance from bubble

u Radial velocity at a distance r from bubble

β Damping coefficient for the bubble

χ Elasticity modulus of lipid shell

ε Thickness of lipid shell

φ Velocity potential

γ Polytropic gas exponentµ Viscosity

µsh Viscosity of lipid shell

ρ Liquid density

σ Interfacial tension coefficient

τ rr Radial component of stress tensor

ω Angular frequency of incident acoustic field

ωo Natural frequency of the bubble

ωr Resonance frequency of the bubble

(˙) Time derivative

Fig. 1. Optical frame images and corresponding streak image of con-

trast agent microbubble expansion for a 10-cycle, 2.25 MHz acousticpulse at a PNP of 1.7 MPa. The microbubble has a resting radius

of 0.5 µm. The streak image in (f) shows the transmitted pulse andthe lines that indicate the times at which the two-dimensional frameimages shown by (a)–(e) were acquired relative to the streak image.

which a streak image is an optical M-mode image. Thisparticular image is the result of insonation of a 0.5 µm ini-tial radius bubble by a 10-cycle, 2.25 MHz acoustic pulseat a peak negative pressure of 1.7 MPa. Although the cam-era captures seven individual images for each streak im-age, five are shown here as the first and last are priorto and post insonation, and the microbubbles are difficultto visualize for these particular frames. The five lines on

Fig. 1(f) designate the times at which the two-dimensionalframe images in Fig. 1(a)–(e) are acquired relative to thestreak image. These images were acquired during the rar-efactional portion of select acoustic cycles. Fig. 1(a) illus-trates the spherical initial expansion of the microbubbleto approximately 8 µm. During the second rarefactionalhalf-cycle, several microbubbles or fragments, with rest-ing radii below the optical resolution of the system, havebegun to expand, as shown by Fig. 1(b). In most of ourimages, a tight clump of fragments is observed after theapplication of one or more rarefactional cycles, with fewmicrobubbles observed to respond later in other regionsof the image. Thus, it is most likely that the appearance

of closely spaced fragments in this case is the result of fragmentation. One of these fragments is observed on thestreak image as this is the only fragment in the line-of-sightused to produce the streak image. In Fig. 1(c), the thirdrarefactional half-cycle of the pulse, the 3 microbubbles ap-pear to have fused with two indentations that portray thefusion. Fig. 1(d) may indicate fragmentation of the fusionof the expanded microbubbles or additional microbubblesmoving into the focus and expanding as a result of thepulse. In order to distinguish these events, very low con-centrations of the contrast agent are used. However, thesesmall microbubbles cannot always be visualized at restand, therefore, additional microbubbles that were slightly

out of the plane of focus can appear during insonation.Fig. 1(e) shows the continued fusion of microbubbles nearthe end of the pulse. Oscillation of the microbubble is ob-served over the entire 10-cycle pulse length. We are unableto detect the microbubbles during the compressional half-cycles, likely due to the resolution and pixel size of theoptical system.

Fig. 1(f) displays the transmitted pulse and the streakimage of a line through the center of a bubble over an8 µs window. We observe rapid oscillations with increasingdiameter for each rarefactional half-cycle. We also observea downward displacement of the microbubble position, on

the order of 10–15 µm, as a result of radiation force.Relative expansion is the maximum microbubble diam-eter normalized by the initial diameter. Fig. 2 presents therelative expansion of M1134 as a function of pressure for acenter frequency of 2.25 MHz, averaged over 15 microbub-bles per pressure setting. For the linear (least-squares fit)model shown in Fig. 2, the slopes are 4.9, 2.5, and 4.0for the 5-, 10-, and 20-cycle pulses, respectively. Given thelarge standard deviation observed for these measurements,a conclusion cannot be drawn concerning the effect of pulselength on the relative expansion. Assuming a linear fit,the maximum r-squared value calculated for relative ex-




Fig. 2. Effect of pulse length on mean relative expansion for agent

M1134. Each microbubble is insonated with a 2.25 MHz sinusoid of varying peak negative pressure and pulse length. Relative expansionis calculated from the maximum diameter achieved prior to fragmen-

tation.

pansion versus pressure for all pulse lengths in Fig. 2 is0.8035; therefore, a significant residual error is present.

The relative expansion for these submicron microbub-bles is much larger than that observed for ultrasound con-trast agents with a diameter of a few microns [16]. In allcases in which an expansion is observed, M1134 expandsby more than six times, resulting in an average expansionof 11 times the initial bubble diameter for experiments per-formed with a 2.25 MHz transducer. M1091 demonstratesan average expansion of approximately 17 times the ini-tial bubble diameter when insonated at a center frequency

of 2.25 MHz. With increasing pressure, there is a slightincrease in the maximum expansion for all pulse lengthsstudied, up to 18 times the original bubble diameter at thehighest pressure (2.5 MPa). With an increase in pressure of approximately 1 MPa, the maximum expansion increasesby 50%.

Fig. 3 presents the relative expansion of M1091 as afunction of pressure for the center frequency of 2.25 MHz,also averaged over 15 microbubbles per pressure setting.Fig. 3 shows the effect of acoustic pressure on relative ex-pansion for each pulse length. The linear (least squares fit)model, as shown on Fig. 3, has a slope of 3.2 and 8.2 for the5- and 10-cycle pulses, respectively. Based upon Figs. 2 and3, the relative expansion for agent M1091 is greater thanthat of agent M1134. We also observe that agent M1091requires a higher acoustic pressure (additional 0.4 MPa)to produce an expansion than agent M1134.

Table II summarizes the relative expansion for in-sonation at 2.25 MHz, in which the relative expansionis calculated for each pulse and averaged over pressuresbetween 1.4 and 2.5 MPa. We do not observe a signifi-cant dependence of expansion on pulse length with pulselengths greater than 5-cycles. However, Table II shows thatthere is a large difference in the relative expansion of the

Fig. 3. Effect of pulse length on mean relative expansion for agent

M1134. Each microbubble is insonated with a 2.25 MHz sinusoid of varying peak negative pressure and pulse length. Relative expansionis calculated from the maximum diameter achieved prior to fragmen-

tation.

TABLE II

Effect of Pulse Length on Relative Expansion for Both

Agents M1134 and M1091.∗

M1134 Relative M1091 Relative

Pulse length expansion expansion

5 cycles 10.85 16.6110 cycles 11.89 17.25

20 cycles 8.87 15.07

∗Each microbubble is insonated by a 2.25 MHz sinusoid of varyingpulse length.

two agents for the same pulse lengths, approximately 50%greater for M1091 as compared to M1134.

Pulse length does affect the probability of microbub-ble expansion within a given field. For a concentration inwhich 20 or more microbubbles are within the field of view,a 10-cycle pulse produces a response from more than 4 mi-crobubbles within the field of view for M1134 when aver-aged over 100 acquisitions, and approximately 3 microbub-bles within the field of view for M1091, also averaged over100 acquisitions. A 5-cycle pulse produces an expansion in2 or less microbubbles for both agents when averaged over100 acquisitions. There is a much greater probability of observing the microbubble expand with a 5-cycle pulse ascompared to a 1-cycle pulse, although the 1-cycle responsewas too rare to report quantitatively.

Transmission frequencies of 2.25 and 3 MHz (and higherfrequencies not reported here) were calibrated for the sametransmitted pressure with the same transducer. Experi-ments performed with a 2.25 MHz center frequency re-sulted in an average expansion of 11 times the initial bub-ble diameter for M1134. A center frequency of 3 MHzproduced a smaller average expansion of just under seventimes the initial bubble diameter for the same agent. A




Fig. 4. Received echoes as a function of time (a) phase inversion

pulse (180-degree phase followed by a brief delay and then a 0-degreephase); (b) 5-cycle pulse.

center frequency of 2.25 MHz produces a larger expansion

with both agents when compared with higher center fre-quencies.

In order to determine whether the shell remains in-tact and whether gas diffusion affects performance, exper-iments were performed with agent M1134 and multiplepulses. Experiments involved the application of two 10-cycle pulses, separated by time delays of 100 µs to 5 ms.Comparing an interpulse delay of 300 µs to a delay of 4 ms,the relative expansion decreases from 18 times the originalbubble diameter to 9 times, at a peak negative pressureof 1.8 MPa. There is a decrease in the probability of anoscillation in response to a second pulse with an increas-ing interpulse delay (100 µs to 5 ms). An average of 3.5microbubbles was observed to expand in the field of viewafter a delay of 100 µs for a set of 10 acquisitions. Theaverage count decreased to 1.5 microbubbles following adelay of 5 ms for a set of 10 acquisitions.

B. Acoustical

Fig. 4(a) presents the received echoes from a pair of phase inverted pulses (180-degree phase followed by a brief delay and then a 0-degree phase). The 180-degree phaseconsists of a 1-cycle waveform with the rarefactional (neg-ative) half-cycle first, and the 0-degree phase consists of

a 1-cycle waveform with the compressional (positive) half-cycle first. Although the transmitted pulse sequence in-cludes two single-cycle pulses with the phase inverted be-tween these pulses, the received echoes from the nonlinearbubble oscillations are not phase-inverted replicas.

The echoes from the two pulse phases are separatedprior to analysis. Fig. 4(b) displays the received echo whena 5-cycle pulse is transmitted. The y-axis of both graphsreveals the amplitude of the received echo in microvolts.We observe a greater peak echo amplitude (172.3 µV) forthe 5-cycle pulse as compared to the peak amplitude forthe pair of phase-inverted pulses (8.54 µV and 18.74 µV).

Fig. 5. Mean echo amplitude as a function of pressure for a pair

of phase-inverted pulses for agent M1134. Each microbubble is in-sonated by a single-cycle, 2.25 MHz sinusoid with varying peak neg-ative pressure.

Fig. 6. Mean echo amplitude as a function of pressure for a pair

of phase-inverted pulses for agent M1091. Each microbubble is in-sonated by a single-cycle, 2.25 MHz sinusoid with varying peak neg-ative pressure.

Figs. 5 and 6 present a summary of the received echoamplitude as a function of the transmitted pressure for theaverage of 50 echoes per pressure amplitude. The data were

acquired following the transmission of pulses with a centerfrequency of 2.25 MHz and peak negative pressures be-tween 0.7 and 1.4 MPa. The peak amplitude of the echoesdepends on the peak negative pressure applied for all wave-forms studied. Fig. 5 presents this dependence for agentM1134 showing the increase in amplitude with increas-ing acoustic pressure for a phase-inversion pulse sequence.A similar plot is observed for agent M1091 and shown inFig. 6. We observe that the echo amplitude from the secondpulse is greater than the first pulse for both agents. We alsoobserve greater echo amplitude at all acoustic pressures foragent M1091 when compared to agent M1134.




Fig. 7. Mean echo frequency as a function of pressure for a pair of

phase-inverted pulses for agent M1134. Each microbubble was in-sonated by a single-cycle, 2.25 MHz sinusoid with varying peak neg-

ative pressure.

Fig. 8. Mean echo frequency as a function of pressure for a pair of

phase-inverted pulses for agent M1091. Each microbubble was in-sonated by a single-cycle, 2.25 MHz sinusoid with varying peak neg-ative pressure.

In Figs. 7 and 8, we compare the mean frequency of thereceived echo for two different pulse sequences, averagedover approximately 50 echoes per pressure amplitude. The

first pulse sequence consists of a single-cycle, 180-degreepulse followed by a single-cycle, 0-degree pulse. The secondpulse sequence is identical to the first, except that theorder of the pulses is switched.

The mean frequency of the received echo for agentM1134 is plotted as a function of pressure in Fig. 7. Thefirst echo has a higher mean frequency, regardless of thephase of the transmitted pulse. Fig. 7 also shows that themean frequency is not dependent upon the acoustic pres-sure applied. Fig. 8 displays the mean frequency of thereceived echo for agent M1091 as a function of pressure,and in each case the first echo has a higher mean frequency

Fig. 9. Normalized amplitude spectrum (M1134) from experimental

echo for the 180-degree phase of a phase-inversion pulse sequence ata PNP of 1 MPa.

Fig. 10. Normalized amplitude spectrum (M1134) from experimental

echo of a 5-cycle pulse sequence at a PNP of 1.45 MPa.

than the second echo. One of the differences between theplots for the two agents is that M1091 shows an overallhigher mean frequency, by approximately 0.5 MHz, thanM1134 for each transmitted pulse, regardless of the phase.

Fig. 9 displays the mean frequency spectrum for the firstpulse (180-degree phase) of the phase inversion pulse se-quence, normalized by the transducer receive bandwidth.A broadband spectrum between 1 and 7 MHz for −20 dBis demonstrated, with the center frequency near 2 MHz.The echo spectrum obtained following transmission of a 5-cycle pulse is shown by Fig. 10, and demonstrates that har-monic components within the echo are down by 5–20 dBcompared with the echo center frequency of 2.25 MHz.The center frequency of the first harmonic component isapproximately 4.3 MHz.

Figs. 11 and 12 present spectra for data acquired andaveraged over 100 echoes per pressure amplitude. Themean echo frequency for agent M1134 following trans-mission of the 5-cycle pulse is presented in Fig. 11 as afunction of transmitted pressure. We observe two groups,




Fig. 11. Effect of solution concentration on mean echo frequencyfor agent M1134. Each microbubble was insonated by a 5-cycle,

2.25 MHz sinusoid with varying peak negative pressure.

Fig. 12. Effect of solution concentration on mean echo frequency

for agent M1091. Each microbubble was insonated by a 5-cycle,2.25 MHz sinusoid with varying peak negative pressure.

one in which the mean frequency ranges between 4 and5 MHz, and the other in which the mean frequency rangesbetween 2.5 and 3.5 MHz. The two groups are a resultof different concentrations of the agent. The sample withthe higher concentration results in a lower mean frequency,and a slightly more dilute concentration results in a highermean frequency. Fig. 12 shows the mean echo frequencyof M1091 following transmission of the 5-cycle pulse. Bothconcentrations, as used for M1134, were studied for M1091.There is no distinct difference in mean frequency for thetwo groups, with the entire range of mean frequencies be-tween 3.5 and 4.5 MHz.

Fig. 13 presents the mean echo amplitude as a functionof transmitted pressure with each value displaying the av-erage over 100 echoes per pressure amplitude. Echo ampli-tude for agent M1134 is first illustrated in Fig. 13. A cleartrend of increasing echo amplitude with increasing pres-sure is demonstrated for echo amplitudes up to 300 µV.

Fig. 13. Mean echo amplitude as a function of pressure for a pair of

phase-inverted pulses for both agents M1134 and M1091. Each mi-crobubble is insonated by a 5-cycle, 2.25 MHz sinusoid with varying

peak negative pressure.

Fig. 14. An in-vivo image obtained from the left popliteal lymphnode of a canine. Contrast agent M1136 was used for this specificimage.

The mean echo amplitude for agent M1091 is shown inFig. 13 to lie between 30 and 260 µV.

C. In Vivo

Fig. 14 is a still frame, in-vivo image showing markedcontrast enhancement of the popliteal lymph node. Con-trast media arrives from the afferent lymphatics, shown atthe top left, and surrounds the periphery of the node, fill-ing the subcapsular sinus. The agent progresses throughthe medullary sinus channels filling the tissue within thesentinel node within a few minutes. Uptake of contrastappeared to be limited to the first order lymph node in




Fig. 15. Predicted effect of pulse length on mean relative expansion,

evaluated using the modified Rayleigh-Plesset model. Open symbolsrepresent a PNP of 1 MPa, and closed symbols represent a PNP of 2 MPa.

this initial study. We observe a large population of bub-bles distributed in the lymph node sinus system. The colorresponse diminishes incrementally with each pulse. A re-sponse can be elicited for total interval of a few seconds.The time required to refill the node with the contrast agentinjected previously is variable and depends on factors suchas passive limb movement, massage of the injection site,and injection site to target node distance.

D. Simulations

Application of the modified Rayleigh-Plesset equationpredicts an increasing relative expansion with decreas-ing initial radius of the bubble, as a result of insonation.Fig. 15 shows the relative expansion predictions from themodified Rayleigh-Plesset model, using a χ2 value of 0.26and µsh calculated as in [16] (1.49 ∗ Ro/1e-6 − 0.86), asa function of initial radius. All other parameters are asdiscussed in [16].

The model predicts expansion up to 138 times the ini-tial radius when microbubbles with an initial radius lessthan 0.3 µm and more than 0.02 µm are insonated by a 5-cycle pulse at a peak negative acoustic pressure of 2 MPa.An increasing relative expansion is predicted with increas-ing pressure (initial bubble radius ranging from 0.01 to

1.5 µm), for all pulse lengths studied.Below 0.5 µm, a very steep slope for expansion rela-tive to initial radius is observed for some imaging param-eters, with the radius required to exceed a threshold forsignificant expansion to occur. For a 5-cycle pulse with apeak negative pressure of 1 MPa, the results illustrate athreshold radius for significant expansion of 0.08 µm. Fora 5-cycle pulse with a peak negative pressure of 2 MPa,the threshold radius decreases to 0.04 µm. The results fora 20-cycle transmitted pulse are very similar to those of the 5-cycle pulse for all acoustic pressures. Computationswere also performed for a 1-cycle pulse at peak negative

acoustic pressures of 1 and 2 MPa. The predicted relativeexpansion is approximately 10 for a 0.8 um bubble withthe transmitted pressure of 1 MPa.

IV. Discussion

In the optical experiments reported here, measurements

of relative expansion are compared with the predictions of the modified R-P equation. Due to the fact that we areunable to resolve the microbubble during each compres-sional half-cycle, we believe that the minimum diameteris less than the initial bubble diameter, which is near theoptical resolution limit of the system. Thus, diameter mea-surements for the minimum diameter are not reported andcannot be used as a basis of comparison.

Microbubbles analyzed in this study show a greater rel-ative expansion when compared to previously studied ul-trasound contrast agents that had an initial diameter of 4 µm. The diameter is observed to expand up to 18 timesfor M1134 and up to 30 times for M1091, for a peak neg-

ative pressure near 1.5 MPa, as compared to a relativeexpansion of 3.25 times the original bubble diameter at apeak negative pressure of 1.2 MPa for MP1950 [19]. Ex-pansion of these submicron (500 nm to 1 µm in diameter)agents increases with increasing pressure over the rangefrom 1.6 to 2.5 MPa.

The experimental observations are in agreement withsimulations based on the R-P model. We observe M1091(with a mean initial radius of 0.4 µm) to have an averagerelative expansion ratio of 17 with a standard deviation of 2, when insonated by a 5-cycle pulse with a peak negativeacoustic pressure of 2.2 MPa. M1134 (with a mean initialradius of 0.5 µm) demonstrates a relative expansion ratioof 11 with a standard deviation of 2, with the same driv-ing pulse. This compares well with the model that demon-strates a relative expansion ratio of approximately 19 and15 for an initial radius of 0.4 µm and 0.5 µm, respectively,with a 5-cycle transmitted pulse at a peak negative acous-tic pressure of 2 MPa.

The predictions from the R-P model and the experi-mental results agree in that the relative expansion ratio isnearly identical for 5- and 20-cycle pulses, for the rangeof initial radii between 0.1 and 1.5 µm at a fixed trans-mitted pressure. A repeated expansion and contraction isobserved throughout the pulse, for transmitted pulses from

5 up through 20-cycles.The relative expansion predicted by the R-P modelfor a 1-cycle pulse does not agree with the experimen-tal results, however. For the range of transmitted pres-sure used in these experiments, neither M1091 nor M1134responded consistently to a 1-cycle excitation. The rela-tive occurrence of an expansion in response to a 1-cyclepulse was small enough that adequate observations couldnot be made with the optical techniques. In the simulationsection, we applied shell parameters obtained for the lipid-shelled agent, MP 1950, to this agent with a polymer shell.Microbubble shells are believed to be disrupted following




insonation with an effective center frequency, pulse length,and acoustic pressure. Increasing the inter-pulse delay be-yond a few milliseconds decreases the number of microbub-bles that respond to the second pulse. Because a few mil-liseconds is the time required for the dissolution of freedand fragmented microbubbles of this diameter, it is be-lieved that the shell is ruptured by the first pulse, and thegas diffuses. Given that the agent does not respond to mul-

tiple pulses with a long inter-pulse delay, it is likely thatthe shell does not act as a barrier to static diffusion afterthe agent has been excited, and thus, does not constitutea rigid shell. Therefore, one interpretation of these resultsis that once the shell is disrupted, the microbubble oscilla-tion is similar in amplitude to a lipid-shelled microbubble.Before the shell is disrupted, oscillations do not occur. Forthe 1-cycle pulse considered in these experiments, it ap-pears that the shell is not typically disrupted; therefore,in this case, the model parameters and experimental con-ditions are not equivalent. If multiple pulses are used insignal processing, a high pulse repetition frequency (PRF)is needed in order to create an image with data acquired

in the time window during which the agent is active.Echoes received from acoustical experiments reveal har-

monic frequency components following the transmission of a short or long pulse. The echo from a second pulse trans-mitted a few microseconds after the first has a higher am-plitude and lower mean frequency. This is most likely dueto a change in the shell characteristics that occur with thefirst pulse and is explained by the optically observed oc-currence of fusion of several expanded microbubbles. Theechoes from the inverted pulses show a difference in bothmean frequency (lower for the second pulse) and maximumamplitude (higher for the second pulse) from one another.Harmonics are observed to be present for a range of trans-

mitted pulses.One of the significant differences observed between the

two agents studied is the pressure threshold required togenerate an echo from the microbubble. Agent M1091 re-quires a peak negative pressure of approximately 1 MPa tobegin oscillation; agent M1134 will respond to peak nega-tive pressures as low as approximately 0.8 MPa. This resultis observed with both acoustical and optical experiments.Increasing the transmitted acoustic pressure increases notonly the relative expansion, but also increases the pro-portion of microbubbles in the volume that respond. Thisobservation may be the result of the differences in mean

radius or a resultant difference in the shell properties.Lymph node macrophages phagocytize particles, andlarger particles are more likely to be retained within thenodes than smaller particles. Sentinel node detection usingradioactive tracers and/or intraoperative probes allow foridentification and selective excision of radiolabeled nodes,reducing the need for radical regional lymph node dissec-tion. A number of potential side effects are involved withthe use of tracers and dye. Hypersensitivity to Patent BlueV results in side effects ranging from hives to vasomotorcollapse. Also common is discoloration of the skin fromthe dye, affecting oxygen monitoring by the pulse oxime-

ter. Another potential advantage of ultrasound imaging isthat the node can be distinguished prior to incision. Ad-ditionally, a destructive pulse may allow assessment of thesentinel node multiple times with one injection in a situa-tion in which differentiation of the sentinel node from othernodes is ambiguous. Neither radiopharmaceuticals nor sol-uble blue dyes allow for such multiple injections. Further-more, if the agents are restricted to first order nodes, they

may be more accurate in localizing the sentinel node. Also,the currently used blue dye and radiopharmaceuticals dis-perse to more distant nodes, causing time to be a criticalfactor in assessing uptake.

Submicron contrast agents have several potential appli-cations in addition to sentinel node imaging, and diameterseven smaller than those examined here may be very use-ful. Microbubbles with initial radii on the order of tensof nanometers or greater are predicted to demonstrate agreater relative expansion than microbubbles with largerinitial radii. It is important to note that, if a stable agentwith a diameter on the order of 200 nanometers can bedeveloped, it is likely to provide a detectable ultrasound

echo. An agent with a diameter in this range is desirablefor imaging tumor vasculature, through which particles onthe order of several hundred nanometers can extravasate[20]. In addition, submicron agents are far more likely tobe successful for targeted imaging because they can becaptured by a binding ligand in the presence of a greatershear gradient.

V. Conclusions

Submicron contrast agents are evaluated for the firsttime using optical and acoustical experiments. The re-

sults indicate that these agents demonstrate a relative ex-pansion above 10 times and produce a strong acousticalecho. Maximum expansion, as predicted by the modifiedRayleigh-Plesset model, compares well with experimentalfindings, demonstrating large expansions for submicron di-ameter microbubbles. We believe that this agent may be asuccessful alternative to conventional sentinel node detec-tion methods if contrast uptake may be restricted to firstorder nodes.

Acknowledgments

The authors acknowledge the support of POINTBiomedical, Inc., the assistance of Jeff Gabe and GlennTickner in devising the experiments, and the assistanceof James Chomas and Dustin Kruse in the experimentalmeasurements.

References

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[6] E. R. Wisner, K. W. Ferrara, J. D. Gabe, D. Patel, T. G. Nyland,R. E. Short, and T. B. Ottoboni, “Contrast enhanced powerDoppler ultrasound with submicron bubbles for sentinel nodedetection,” Academic Radiology , vol. 9, suppl. 2, pp. 389–391,Aug. 2002.

[7] L. Barbera, L. Illanes, F. Terrier, and T. Dopta, “Sentinel nodein breast cancer. Multidisciplinary approach: Critical analysis of the learning curve: Results obtained with radiopharmaceuticalnot used previously,” Rev. Esp. Med. Nucl., vol. 20, no. 4, pp.269–275, Jun. 2001.

[8] G. W. Schmid-Schonbein, “Microlymphatics and lymphflow,” Phys. Rev., vol. 70, pp. 987–1028, 1990.

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[10] E. R. Wisner, K. W. Ferrara, J. D. Gabe, and D. Patel, “Sen-tinel node detection using contrast-enhanced power Doppler ul-

trasound lymphography,” Investigative Radiol., to be published.[11] D. Krag, “Minimal invasive staging for breast cancer: Clinical

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[13] M. Dunitz, Ultrasound Contrast Agents. Philadelphia, PA: Mar-tin Dunitz, 2001.

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[15] H. G. Flynn, “Cavitation dynamics. II. Free pulsations and mod-els for cavitation microbubbles,” J. Acoust. Soc. Amer., vol. 58,pp. 1160–1170, 1975.

[16] K. Morgan, “Experimental and theoretical evaluation of ul-trasonic contrast agent behavior,” Ph.D. dissertation, Char-lottesville, VA: University of Virginia, Jan. 2001.

[17] P. Dayton, “The effects of acoustic radiation force on contrastagents: Experimental and theoretical analysis,” Ph.D. disserta-tion, Charlottesville, VA: University of Virginia, Aug. 2001.

[18] J. Chomas, P. Dayton, D. May, J. Allen, A. Klibanov, and F. Fer-rara, “Optical observation of contrast agent destruction,” Appl.Phys. Lett., vol. 77, no. 7, pp. 1056–1058, Aug. 2000.

[19] J. Chomas, P. Dayton, D. May, and K. Ferrara, “Threshold of fragmentation for ultrasonic contrast agents,” J. Biomed. Opt.,vol. 6, no. 2, pp. 141–150, Apr. 2001.

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Divia Patel received a B.E. degree in 2000in biomedical and electrical engineering fromVanderbilt University, Nashville, TN. She iscurrently working toward an M.S. degree inbiomedical engineering at the University of California, Davis, CA. Her current researchinterests include contrast-assisted ultrasoundimaging, under the advisement of Dr. Kather-ine Ferrara.

Paul Alexander Dayton received a B.S.degree in physics and comprehensive sciencefrom Villanova University, Villanova, PA in1995. He received his M.E. in electrical en-gineering in 1998, and his Ph.D. in biomedi-cal engineering in 2001, both from the Uni-versity of Virginia, Charlottesville, VA. Asan undergraduate, he studied surface sciencein the physics department, and nuclear mag-netic resonance in the chemistry department.In graduate school, Dr. Dayton developed op-

tical and acoustical techniques to study theeffects of ultrasound on contrast agents and studied the physics of insonified microbubbles. In November, 2001, Dr. Dayton joined theDepartment of Biomedical Engineering at the University of Califor-nia, Davis, as an assistant research professor. His current interestsinvolve targeted imaging using contrast agents.

Jana Gut received her B.S. degree in 2002 in bioagricultural en-gineering from the University of California, Davis, CA. She will beattending Georgia Tech in the Fall of 2002 to pursue a graduatedegree in biomedical engineering.

Erik Wisner received his Doctor of Veteri-

nary Medicine Degree from the University of California, Davis, in 1983. After five yearsin general clinical practice, Dr. Wisner com-pleted a residency in veterinary diagnosticimaging at the University of California, Davis,in 1991. He served as an Assistant Profes-sor in the Department of Radiology, School of Medicine, UC Davis, from 1992 through 1997.From 1997 to 2000, he was an Associate Pro-fessor and Head of Radiology at the College of Veterinary Medicine, The Ohio State Univer-

sity. Dr. Wisner was appointed as Associate Professor in the Schoolof Veterinary Medicine, UC Davis, in August, 2000. He is currentlyProfessor of Radiology and the Director for the Center of ImagingScience at UC Davis and the President of the American College of Veterinary Radiology. His research interests are in contrast mediatargeting mechanisms with clinical interest in magnetic resonance

imaging and computed tomography.

Katherine Whittaker Ferrara (S’82–M’82–S’87–M’89–SM’99) received the BS andMS degrees in electrical engineering and sub-sequently worked for Sound Imaging, Inc.(Folsom, CA) and General Electric MedicalSystems (Rancho Cordova, CA) in the areas of magnetic resonance and ultrasound imagingfrom 1983 to 1988. She received her Ph.D. inelectrical engineering and computer science in1989 from the University of California, Davis.From 1989 to 1993, she was an associate pro-fessor in the Department of Electrical Engi-

neering at California State University, Sacramento. From 1993 to

1995, she was a principal member of the research staff at the River-side Research Institute (New York, NY) and an adjunct associateprofessor at Cornell University Medical School. From 1995 to 1998,she was an associate professor in the Department of Biomedical Engi-neering at the University of Virginia, Charlottesville. Since December1998, Dr. Ferrara has been Professor and Chair of Biomedical Engi-neering in the newly created Department of Biomedical Engineeringat the University of California, Davis.

Dr. Ferrara is an Associate Editor of the IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control. She chaired thetechnical program for the 1997 IEEE Ultrasonics Symposium. She isa member of Tau Beta Pi and Sigma Xi and a fellow of the AcousticalSociety of America. Her research interests are medical imaging andbiomedical signal processing, particularly in the areas of ultrasonicsand acoustics.

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Optical and Acoustical Interrogation of Submicron