CA2176206C - Harmonic ultrasound imaging with microbubbles - Google Patents

Abstract

A method for ultrasonic harmonic imaging is disclosed, which uses microbubbles particularly selected for their properties of reradiating ultrasound energy at frequencies other than the exciting frequency. A contrast agent for ultrasonic harmonic imaging having the property of reradiating ultrasound energy at frequencies other than the exciting frequency is also disclosed.

Description

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The present invention relates to an improved method for harmonic ulttaso~d knaging using a 5'' ' ctmtrast agent specialty designed to ntnrn harmonic frequencies.
Bac~ca~ound of the Art Uhrasound technology prov~dea an important and more econom~al ahernative to imaging techniques whicfi use ionizing radiation. Wade numerous convenfronal knagingtechnologies are available, e.g., magnetic resonance imaging (MRIi, computeraed tomography (CTh and positron emission tomography (PETh each of these techniques use extremely expensive equipment. Moreover, CT and PET
utilize loosing radiation. Unlike these other technaiogies, uhrasound imaging equipment is relatively inexpensive. Moreover, ultrasound imaging does not use ionizing radiation.
In using the uhrasound technique, sound waves are transmitted into an object or patient via a transducer. As the sound waves propagate through the object or body, they are either reflected or absorbed 15' by tissues and fluids. Reflected sound waves are detected by a receiver and processed to form an hnage.
The acoustic properties of the tissues and fluids determine the contrast which appears in the resulting image.
Ultrasound krtaging therefore makes use of differences in tissue density and composition that affect the reflecfron of sound waves by those tissues. Images are especially sharp where there are distinct variations in tissue density or compressibility, such as at tissue interfaces.
Interfaces between solid tissues, the skeletal system, and various organs andlor tumors are readily imaged with ultrasound.
Accordingly, in many imaging appbcations ultrasound performs suitably w'tthout use of contrast enhancement agents; however, for other applications, such as visualization of flowing blood in tissues, there have been ongoing efforts to develop agents to provide contrast enhancement.
One particularly significant application for such contrast agents is in the area of vascular imaging. Such uhrasound contrast agents could improve imaging of flowing blood in the heart, kidneys, lungs, and other tissues. This, in turn, would facilitate research, diagnosis, surgery, and therapy related to the imaged tissues. A blood pool contrast agent would also agow imaging on the basis of blood content (e.g., tumors and inflamed tissues) and would aid in the visualaation of the placenta and fetus by enhancing only the maternal circulation.
A variety of ultrasound contrast enhancement agents have been proposed. The most successful agents have generally consisted of microbubbles that can be injected intravenously. In their simplest embodiment, mic~obubb~s are miniature bubbles containing a gas, such as air, and are formed through the ~ use of foaming agents, surfactants, or encapsulating agents. The microbubbles then provide a physical object in the flowing blood that is of a different density and a much higher compressibility than the surrounding fluid tissue and blood. As a result, these microbubbles can easily be imaged with ultrasound.
i However, contrast agents developed thus far for use in ultrasound imaging have various problems.
Contre'st agents containing aqueous protein solutions require use of a foreign protein which may be antigenic ~;.~~ i~,~; ,~:.~ 2116206 ~1~1:~6~79~ ' t , , PCTIUBlI~J122A5 and wNulc~ - L~laoMlt !~øt ~ d~ having gas encapsulated therein, present problems due to unaveA ~s ~att.,a~ poor stability. Many of the existing contrast agents have fad to provide improved imaging, and furthermore, many of the methods ut~ad to~repare the contrast agents ere areff~ient, expentiva, and atherwi:e uasat~factory.
Conventional uhrasound syatems,work by transmitting pulsesdof ultrasoundof, a given frequency and measuring the time interval between this transmission and the detectipn of the rpf~cted echoes from w'tthin the object or body being imaged. Large n4ra4era of microbu6bles p~a~re collecfrvely as a large reflector. The system relies on measurement of reflected sound waves of the same ffequency as that tranam'rtted to produce the image.
It has been found to be advantageous, especially in biological ~ppli~ations, tD detect or image an uhraaound contrast agent while suppressing the uhrasound sj~al reflected by other objects such as tissue and bone (see Williams et al., WO 91115999). The ability to imaNe ultrasound contrast agent bubbles in the blood bY detecting harmonic frequencies in the echo when.,thay are excited bY
an ultrasound beam at a different frequency (the fundamental) greatly increases the sensitivity of canttast agent detection by ignoring the background fundamental frequency signal scattered by other non-bubble .objects in the organism, much like the detect'ron of fluorescent dyes by their frequency-shifted Ipht is jnhetently more sens'tt'rve than the detection of light-absorbing dyes by then modulation of the iUum~ating light intensity. Unlike microbubbles, animal tissues reWrn very little harmonic frequencies. Thus, background imaging is substantially eliminated through harmonic imaging.
As existing contrast enhancing agents and meti~Ods for ultrasound imaging have pot been found to be entirely.aatisfactory, there is a substantial need for providing an improved ultrasound contrast agent and method of uhrasound imaging which reauhs in the production of imprDVad ultrasound i~eges.
NUMMARIf OF T,~E ~11EAIIION
In accordance with one aspect of the present invention, there is ,provide, a method of ultrasonic harmonic imaging using ultrasonic energy transmitted by an uhrasonic source to an object or body to be imaged. The method comprises introducing into the object or body a contrast agent comprising microbubbles having generally spherical membranes and containing .gaseous material having,;vapor pressure at 37°C over 23 torn, less than about 1 % wt.lwt. solubilitylmiscibility In water, concentrati4n in gas phase when detected greater than 2% mole fraction, and a concentration greater than 50% of its saturation concentration. At leant a portion of said object or body is then uhrasonically imaged.
Preferably, the gaseous material contained in the bubbles is selected from the, group consisting of perfluorqbexane, perfluoropeatane, perfluorocyclopentane,,1,1,2-trichlorotrifluoroe,~hane, sulfur hexafluoride, , cyclopentane, methylene chbride, pentane and hexane. Preferably, the concentration in gas phase when detected is 25%, or 100%. In a .preferred embodiment, the membranes comprise a sprfactant.
In accordance with another aspect of the present invention, there is provided a method of ultrasonic harmonic imaging using ultrasonic energy transmitted by an ultrasonic source to an object or body to be ~, ~ , ~ , ~ P",~ _ Yr:
v - ~'~~~~'=~ 2 i ~6~U6 ind, compriaing~ introducing into sahl:o~ject o~ body a i~o~tca~ agent c~prg anicrobubblas. These micro have y =p~ical n~abflland. ~oM~in atttl that GontPrfses at least 2% mole frec~on. of a gas that has a solubibty of its liquid Phase in hexane at 37°C of more than about 10%
' malehnola aad a w~zohdylmaciy of it: kquid pbaee of lass than bout 1%
.wt.lwt. in water at 5- 37°G: At Isest a portion of the abject or body is than uhraaonically imaged.
' In a Pnfiarrad rodimeat, the ga: ~ a hlrdreaas~on or a fh~orocarbon. More preferably, the gas is selected from the group consisting of perfluorohexane, perfluoropentane, perfluorocyclopentane, 1,1,2-triabloratri~lnoraetlune. sulfur h~c$fluo~ide, clrclope~~na, me#hylenp chloride, pentane, hexane, dicbloros~b~~p~riatha~; triclaioraa~nof~~amqthet~aa; pe~flaarobut~e, parfluorecyclo~ut~ne, perfluoropropane, butane: c~yclabutnne, Pr~:-n~thane. and a~na.
P~efatably. the n~embraaaa con~riae a surfiactan~ The surfactant is Pr~ar~h fluorinated. In ~oth~ Ptnfe~rad ambit, the mateEiaal contained ~n the microi~ubblas c~ornpriee$ st least about 25% rtrole fraction of stud gas, ar about 100' mole fr~astien of said ,gas.
In accordance with yet another emlmdiment of tbo pteaent insel~~n, tbero~ is provided a method y5 of u~raton~ harmonic imaging #sing u~roaonic o~uargy transmitted by-ark ult~aaonic mource to an object or body to be iced, co~rising i~rQducing into the abject a< lredy a eortrast agent cempriaing microbubb~s atabiixed~ wfth at least.one surfactant: At h~i~at a p~artion of the object or ~dy is then ultrasonically imaged.
In a prefotred ombodimantthe aarfactant will chango the surface ta~sjan of water by more than 5 dynealcm when the area per melocule of surfactant h~ .c,hanged by 109 ~s maasursd, on a Langmuir fibn 20. b~ance. M anotfipr preferred eninwnt, fhR aurfa~tar~t has a cs~mponant ~aviag a hydrophigc-YpophiGc balance less than 11, or leas than or equal to 8. In yet another preferred embodiment, the surfactant has a compot with a c~ular wit oar 1:0~ artd capable of lowering the surface tension of water to q,0 dyne:fam or h~war. In still anethar ltreierred embodiment, the surfactant is 5c,apable of lowering the urface tension of water to 4i;? dynoslcm er lower end has a critical mice concentration of 0.3 or less 25 volume fraction in water. Preferably, the surfactant is non-Newtonian.
In accordance with stiN another aspect of the Present invention, in a method for ultrasonic harmonic irna~ing of ~. abject or body campris~g introducing ~to the ob~ct or body a, contrast, spent and transmitting ~tas~ic a~rgy from an uhrasanic a~urca to the abject ar body ~d detecting radiated energy from the pct or body; there. is groveled the impr~v~ment comprising tt~a contrast agar~t being comprised of 30 microb~b~s~ having the property of radiating imagaebk u~raaanic energy at a fre~quor~~y which is different frean that transmitted by tlrs ultrasonic source that is independent of the resonant frequency of the bubble.
~ t~ ~o~d, the ducting step utiY~s a efferent fnqueocy then the transmitting step.
M a preferred embodiment, the microbubbies certain a has or gas mixture, ~d the microbubbles area~tabihted by their pea er gas mi~etur~t c~tents. In another preferred ombodirnent, the microbubbles are 35- proceed by spray drying a liquid formuhrtion:.containing a biocort~patable membralno-forming material to form a- a~icrospherr powder tlrocefrem, combining the microspheres with a gas osmotic agent, and mixing an a t . yrt . '.<:~ °~. . ~ ~ ~. , i y;~~'~~~~~~~ ':2~?6206 .4.
,~ rider, ,,'~1~ , ~;~ablrtbe~ irt ~ the ~I~uuoua phase to form r,rs~ Pr~fdta6ly, the ~ inicr~ e~ cbat~ With a ~r~f of s~a~ta~t:
fn accotdenca with yet enothbr aspect of the present lnirbntion,'the~ i~,fed in method for . . ~ yarfhordbvuttreuo~rrd imagihg qty r~robubbiea° ll~ far~rtil~lit cdrai~''~pt'~kling at least one hydrocarbon gas or fleoroca~oe-ge~fn the niicrobifbbles in a conce~ati~ ~f at ~a:f 29G mole fraction.
,~ . , pETI~~9~RIP~R01~;OF 'IIIhI~R
In n . , ..
ps u~' v the ',pnirwllt d~crip~r claims.~'~the:=tetm~ ,~~t" ~uud "gas" are used t .My, eke,; when tetesfiing-te~ the tension of ~ssolv~li gad°i~
a'liquilh, tlle~nore familiar term "pressure" may be used interchangeabh~ with "tenaien' "bas tic -prdieute'" kt rr~lt~ i1111y defined below, but' r~ a a~hrrple approximation can be thought of as diff~en~se between the partial pressure of a gas in~idla 'a~microbobble alnd tfie paesaurlr~~tfienidon o~ that gad Ieither iA a gas: phase bE d~solved in a liquid phase) outside of the microbdbble; when the~~nicrobebble ~rem6~bae'Is a to~thb~ga'k. More precisely, ~;~ ~to e~~e~ ~ gee ditftrvtaiW a~rod~ ~ r~In. ~: tee r fiembrane" is used to refer te''the'ii~eterial surroun~g ~ defin~~a ~nict~ebbk,~w~ltdther~ ~ ~
~rf~ctaot'arretber film forming liquid, erg f~f ~ edpd~~rlid'qNl~obebble~" are~~onridited~'t~'bi~~iesha~arg a~diameter between ebo~ 0.9 arrd 300 ~nr~t; preferably h7wing~ e~~daNneter r~e''n~are~
thiur~abbut' 200 100;' sr 50 Nm, and for '-., ~Intrevastuser pteferi not imere' ~tha~i shoat 10;' 87~ ~r er 5 ~measured as average number wie~ht~ diameter of the ~icaob~ebb~ oempositio~ilv den rof~rin~ rc a "~s~*'h ~vu~ be understood that m~~~ of gasesvti~ather havP~ ~tl~e requi:'tta ~tc~tcy~'fag win the defiaithin, .excr~rt;~uhere th~context otherviriso tequKes.
~ p~resentarhreMibn~ pra~l e, e-. pe:eeo,~:,ue~g, specially designed dbbles as ulEta~und co~ltxoat~rnhanfager~ts. ~noptimi~rrg the abiof these gas bubbles to ~~r~~ the frequency of tf~e uhresot~iC~tadiaftion to rhieh thay~ure subjected=Ithe #undamentall, imaging is enhanced.
'~Ilhen ~a g'8s"bubble is ex~os~rl ~t~'ihigh tb~~twpktude!~Ittt~aund, which is not practical in :I systems bdcauae ~ef cevitation«aand oeH tisaue~'damago, or i~v ex~sed to ioyr~~ampfttude exciting ~; ~b$y' near the resea~nt irequorr~y of he dulbl~r, ft acts ins a ~eonx fashion. That is, the chati~e' in bubble v~lnme is no l!Onget prepertional to the chant in pr~esaure ~fvits aurroo~ngs. This nonlinear '~0 w belir' generates components of tt~e teradiatott~ohdeseo~nd, energy l~hat aro a~:frequerreies other than the .
dxeihng>ft~eqaency.'See Eatock, J. S~rc. Aa~wst ;gym: 77:1~2~1701w~19851;
de~Joap et al., Ultrasanics, ~g;~,~,~0 (1gg11~d : Ulttcs~ (19111: ~eee ~:at ftequmrci~bolh above and below .
tfie incident freqnetrcy are tfie result of the raedhanics e# -motion for the system. At medically useful ub~~dund exEiting empktudea: eignificeht 5~ilorifesEare anrty ger~sted by, bu~ae ~erithin;:a narrow sae range containing the resonant diameter. For example; fdr a 3 n~egaha~t~ excitation frequency4air bubble in water ''w'rtlt a diameter of 1.1 micron ~f reso~rete and generate harmonics, but ~e arrrplit~rde of these harmonics 'I""~"~ ~' '.'~ ~ .~ ~ rm': .
'W~ 9f~~ P"1~53'~45 fop b~ ~ facta~r of'2 fof rrltat ocdy 12%~dqffdrant'ftan the r~sorraatrtt diea~tar_ Bubbles hr this size range are' oily a amsA fraction of the relatively ttroad size distr~ut'ron of moat microb~bblea (de Jong at al., Ultresonics 3012):95t03 (1992r~. Bubbles produced w'tth aokd or semisolid slmBa e.g., denatured albumin (deac~ed in U:S. Patent No. 4,957".858; de Jong et al.; Ultrasonks 30(2k95-t03 (1992); and de Jong, 5 ' Acoustic Properties o~ Ultrasound Contrast Agents, Ph.D. thesis, Eraamus University, Rotterdam (1993)) exhib'tt increased damping due to the viscous shell and thus do not have the large radius excursions at resonance requhed to produce significant harmonic components in the scattered (reradiatedl signal. Thus, the ptasont inviention advaatageoualy provides for the,uae of microbubbNaa capable of generating harmonics at medicagy useful ultrasound exc'tting ampfttudes.

t0 To detect the reradiated uhrasound energy generated bY the microbubbles, the present invention mekes use of a modified conventional uhrasound scanner system:
The system is able to detect or select one or more of the new frequencies, or harmonics, radiated by the microbubbles for production of the uhrasound image. In other words, it detects a frequency dififerent from the emitted frequency. Equipment su'ttable for harmonic uhiasound unaging is disclosed in Wibiams et al., IIyO 911f5999. Many conventional ' uhrasound imaging devices, however, uti~ze transducers capable of broad bandwidth operation, and the outgoing wavefomr sent to the transducer is software controlled:
For this reason, reprogramming to emit a waveform different from the one detected is well w'tthin the level of skgl in the art.

In practicing the present arvention, the parameters of the uhrasound transmitted (e.g., the frequency, pulse duration, and intena'tty) can be varied according to the particular circumstances, and the optimal parameters for any particular case can be readily determined by one of 'orda~ary skill in the art.

While bubbles have been shown to be the most efficient ultrasound scatterers for use in intravenous uhresound contrast agents, the contrast enhancement agents of the present invention provide unexpectedly superior anaging; for example, clear, vivid, and distinct images of blood flowing through the heart and kidneys are achieved. The present invention is particularly su'tted for study of blood flow; but is equally applicable to the study of other kquids or tissues as well. Small, nontoxic doses can be administered in a peripheral vein or lymph vessel and used to enhance images of all or part of the body. Cavities or areas within a body into which microbubbha can be introduced can be imaged according to the method of the present invention.

Thus, the present invention provides means for 'anaging a variety of body cavit~s and vasculature which may be difficult to anage using conventional technpues.

It is not essential that the subject being imaged be an organic tissue. 'Rather, the method of the present invention can be used to image anything containing spaces ~to which the contrast agent can be introduced, so long as the material surrounding the contrast agent is permeabh to the ultrasonic radiation and does not 'itself resonate in a manner which obscures the selected harmonic of the microbubbles and does not hinder the resonance of the microbubbles.

The method of the present invention may use the measurement of a single recehred frequency, different from that of the frequency originally transmitted, to form a single: image. Alternatively, several i ~1 762A 8 different frequencies different from that of the exciting frequency can be detected and used to create multiple images, which can be viewed separately or electronically processed into a composite image.
The received frequency or frequencies can be processed by a variety of methods well known to one of ordinary skill in the art. These include, for example, making the receiving transducer selective toward the desired harmonic or harmonics so that it ignores the fundamental, or by using software or hardware filters to separate or isolate the various frequencies.
Microbubble Properties It was surprisingly discovered that certain properties of microbubble ultrasound contrast agents can enhance their ability to produce harmonics. While bubbles have been shown to be the most efficient ultrasound scatterers for use in intravenous ultrasound contrast agents, one practical drawback is the extremely short lifetime of the small (typically less than 5 microns diameter) bubbles required to pass through capillaries in suspension. This short lifetime is caused by the increased gas pressure inside the bubble, which results from the surface tension forces acting on the bubble. This elevated internal pressure increases as the diameter of the bubble is reduced. The increased internal gas pressure forces the gas inside the bubble to dissolve, resulting in bubble collapse as the gas is forced into solution. The LaPlace equation, p P-2y/r, (where p P is the increased gas pressure inside the bubble, y is the surface tension of the bubble film, and r is the radius of the bubble) describes the pressure exerted on a gas bubble by the surrounding bubble surface or film. The LaPlace pressure is inversely proportional to the bubble radius;
thus, as the bubble shrinks, the LaPlace "~' a1 720 8 - 6a -pressure increases, increasing the rate of diffusion of gas out of the bubble and the rate of bubble shrinkage.
It has been discovered that gases and gas vapor mixtures which can exert a gas osmotic pressure opposing the LaPlace pressure can greatly retard the collapse of these small diameter bubbles. Such technology includes the use of a primary modifier gas or mixture of gases that dilutes a gas osmotic agent to a partial pressure less than the gas osmotic agent's vapor pressure. The gas osmotic agent or agents are generally relatively hydrophobic and relatively bubble membrane impermeable and also further possess the ability to develop gas osmotic pressures greater than 75 or 100 Torr at a relatively low vapor pressure. The gas osmotic agent or agents act to regulate the osmotic pressure within the bubble. Through regulating the osmotic pressure of the bubble, the gas osmotic agent (defined herein as a single or mixture of chemical entities) exerts pressure within the bubble, aiding in preventing collapse of the bubble.
Bubbles of air saturated with selected perfluorocarbons can grow rather than shrink when exposed to air dissolved in a liquid due to the gas osmotic pressure exerted by the perfluorocarbon vapor. The perfluorocarbon vapor is relatively impermeable to the bubble film and thus remains inside the bubble. The air inside the bubble is diluted by the perfluorocarbon, which acts to slow the air diffusion flux out of the bubble. This gas osmotic pressure is proportional to the concentration gradient of the perfluorocarbon vapor .....,_ ..

.
x17820 g _ 7 _ across the bubble film, the concentration of air surrounding the bubble, and the ratio of the bubble film permeability to air and to perfluorocarbon.
As discussed above, the LaPlace pressure is inversely proportional to the bubble radius: thus, as the bubble shrinks, the LaPlace pressure increases, increasing the rate of diffusion of gas out of the bubble and the rate of bubble shrinkage, and in some cases leading to the condensation and virtual disappearance of a gas in the bubble as the combined LaPlace and external pressures concentrate the osmotic agent until its partial pressure reaches the vapor pressure of liquid osmotic agent.
Conventional microbubbles that contain any single gas will subsist in the blood for a length of time that depends primarily on the arterial pressure, the bubble diameter, the membrane permeability of the gas through the bubble's surface, the mechanical strength of the bubble's surface, the presence, absence, and concentration of the gases that are ordinarily present in the blood or serum, and the surface tension present at the surface of the bubble (which is primarily dependent on the identity and concentration of the surfactants which form the bubble's surface). Each of these parameters are interrelated, and they interact in the bubble to determine the length of time that the bubble will last in the blood.
It was surprisingly discovered that when the bubble contains a vapor which can condense at useful temperatures (e. g., 37°C for humans) and pressure, the change of phase, from gas or vapor to a liquid, causes the volume of the bubble to change much more rapidly than the change expected for linear systems. This nonlinearity results in the generation of harmonics. For this effect to be significant, the vapor must be present in the gas phase of the bubble at a mole fraction concentration of greater than approximately 2~, and preferably at about 5~, 10~, 255, 50~, or 100. The a~ ~s2o s _ 7a _ vapor inside the bubble is preferably near saturation under the conditions of examination, preferably at least about 50$, 75$ or 100$ of the saturation concentration. Thus, for microbubbles used for imaging in a human; the liquid phase of the vapor in the bubble must have a vapor pressure at 37°C greater than 2~ of the pressure inside the bubble (one atmosphere plus the blood pressure of the human being examined plus the pressure caused by the surface tension of the bubble, the LaPlace pressure). This total pressure for 3 micron bubbles could reach 1.5 bar and thus requires the liquid phase of the vapor in the bubble to have a vapor pressure at 37°C greater than approximately 23 torn, The liquid phase of the vapor should also have a low solubility in water, preferably less than 1~ wt./wt. Vapors of materials such as perfluorohexane, perfluoropentane, perfluorocyclopentane, Freon'~' 113, sulfur hexafluoride, cyclopentane, methylene chloride, pentane and hexane are particularly suitable.
While condensation of a vapor diluted with other gases involves diffusion of the gas to the forming film of liquid and thus requires a finite time to complete condensation, the fraction of vapor near the surface of the body temperature water surrounding the bubble can condense rapidly in less than the microsecond time frame of diagnostic ultrasound. A bubble containing pure vapor, e.g., perfluoropentane, can nearly instantly condense much like the water vapor bubbles that occur during high intensity ultrasound cavitation. These cavitation bubbles are known to produce high intensity harmonics, (see Welsby et al., Acustica 22: 177-182 (1969), although at biologically toxic intensities for water vapor bubbles.

2~~'~~206 '~ .. . ~ P~.'i'I~J>i,s ~~

T~ of phi rasult~ in ~acraaaed hiana~oni~ penmatian a~, daacribad, above can also occur when a gas is dissolved in or adsorbed to the hltdrophpp~ regions of tbe.aucfactants at the bubble surface.
This adaorpt~op pr,dissolutaon aqu~brium is effacttd,by the partial pressure of the gas inside the bubble and thus by the total pressure inside he bubble. An aicrease ~ prsstxre applied by the exciting ultrasound beam wig shift the ~uilibriwn toward dissolution or adsocpt~ in the wrfact~nt layer, causing a volume change different from that expected from a lipear:aystem. $u'ttabls gases are tines that bave a solubiNtylmiscibiGty of theb r~uid forma w'tth heacane, which ~ a modal for t~ hydrophobic re~ian of arfactants, of greater than 10% moh~lmaia at 37°C. ,for gases with a boibng point below 37°C~ a.g. butane and perfluorobutane, this messurement;must ba carried Qut at elevated pressure. The adsotdi~ pas ahmdd ba present in the bubble gas phase at a concentration of more than 2% by mole.,fraction of thp gas mixture and preferably at about 5%, 10%, 25%. 50%, or 100%. The use of fluoruiated surfactants wiEh fluorinated gases is preferred. The liquid phase of the, adsqrbing gas should be relatively insoluble in water, ass than 1 % wt.lwt.
solubilitylmiscibilixy.
Previous treatments of the mechanics of bubbles excited by ultrasound treat the surface tension 1~ of the bubb~ as a constant while the bubble sxpanda and ca~tracts. The surface tension at the surface of a small bubble~significantly effects the pressure u~aida the bnbb~ as defined by~,the law of LaPlace wherein the pressure inside a hobble (above the external ambien fi is ~vgrasty proportional to the diameter of the bubble and proportional to the surface tension of the,surfactant fibn. For example, a 3 micron bubble with a aurfaca tension of 40 dyneslsquare centinretBr itas an internal pressure more that one quarter bar above its surroundurpa, Some surfactant: are non-Newtonian, that is,, their surface tension Changes rapfdly when the surfactant layer is comptesaed, as mea~tura~ in a s~tandurd Lanpmuir trough. The change in surface tension ef a surfactant fikn, as the surface area is changed, is quantitatively characterized by the Surface Odation Modulus. This is defined as:
;..$~
dlnA
30. whale E is tha;aurface dilation modules, dy is the changs,in surface tension, and dlnA is the change in the natural op of the surface area. The additional change in surf ale tension due to the rate of area change is characterized by the Surface Dilational Yiscasity. This is, defined as:
35 py - K 1_ ,~
A dt , where ~y is the change in surface tension, K is the surface dilational viscosity, A is the area of surfactant 40 film surface , and dAldt is the rate of change of the surfactant film surface area (see Adamson, Physical ~1 7620 6 Chemistry of Surfaces, 5th ed., John Wiley & Sons, Inc. New York, 1990 ).
When a bubble is formed employing a non-Newtonian surfactant, its surface tension, and therefore its internal pressure will change as the surface area of the bubble changes in response to the exciting ultrasound pressures.
This additional expansion and contraction in response to changes in surface tension leads to a more nonlinear compressibility and therefore the generation of a higher intensity of harmonics. The expanding bubble increases the surface area of the surfactant film until the rising surface tension opposes further expansion. The compressing bubble, during the high pressure part of the exciting ultrasound cycle, will reduce the bubble surface area until the surface tension abruptly decreases, limiting further compression and distorting the sinusoidal volume change waveform, causing harmonics to be generated.
Suitable surfactants for use in the present invention are any surfactant or mixture, hydrocarbon or fluorinated, that has a component that will change the surface tension of water by more than 5 dynes/cm when the area per molecule of surfactant has changed by 10~ as measure on a Langmuir film balance.
Other suitable surfactants are those surfactants with a component having a hydrophilic-lipophilic balance less than 11, preferably less than or equal to 8.
High molecular weight surfactants (e. g. over 1,000) diffuse slowly compared to the microsecond timeframe of ultrasound, and thus surfactants with a component with a molecular weight over 1,000 and capable of lowering the surface tension of water to 40 dynes/cm or lower are suitable independent of the above.
Considering diffusion, solubility and microsecond time scales, other suitable surfactants include any surfactant capable of lowering the surface tension of water to 40 w a1 7s2o s - 9a -dynes/cm or lower and having CMC (critical micelle concentration) of 0.3 or lower volume fraction in water.
Examples of surfactants for use in the present invention include undenatured human albumin, phospholipids (e. g. phosphatidyl choline), sugar esters (e. g. sucrose stearate, sucrose distearate, sucrose tristearate), block copolymers (Pluronic F-68, Pluronic P-123), zonyl surfactants (e. g. FSK, FSC, FSO, FSN, FSE, FSP, FSA, FSJ, UR, TBS) fatty acids (e.g. stearic acid, oleic acid) and their salts (e. g. sodium stearate, potassium oleate).
With these particular operating constrains in mind, suitable microbubbles of the first type (containing a gas that partially condenses upon bubble excursion) may advantageously comprise perfluorohexane, perfluoropentane, perfluorocyclopentane, FreonT" 113, sulfur hexafluoride,.
cyclopetane, methylene chloride, hexane and pentane.
Similarly, suitable microbubbles of the second type (gas soluble in surfactant) may advantageously comprise perfluorohexane, perfluoropentane, perfluorocyclopentane, Freon'~'' 113, sulfur hexafluoride, cyclopentane, methylene chloride, hexane, pentane, perfluorobutane, perfluorocyclobutane, perfluoropropane, FreonTM 12 (dichlorodifluoromethane), Freon'''M 11 (trichloromonofluoromethane), butane, cyclobutane, propane, methane, and ethane.
Bubbles that are stabilized by viscous shells (eg.
denatured protein gel, U.S. patent No. 4,957,656;
~rr~

""~ X17820 8 saturated sugar solutions, U.S. Patent Nos. 5,141,738 and 4,657,756) damp the oscillation of the bubble at resonance and thus prevent the large volume excursions required to produce harmonics (de Jong et al., Ultrasonics, 29:324 330 (1991); de Jong et al., Ultrasonics 30(2):95-103 (1992); and de Jong, Acoustic~Properties of Ultrasound Contrast Agents, Ph.D. thesis, Erasmus University, Rotterdam (1993)). A
bubble stabilized by its gas phase contents as discussed above (e. g., a material such as a highly fluorinated compound such as perfluorohexane, perfluoropentane, perfluorobutane) requires only a monomolecular layer (monolayer) of surfactant to make it stable in the bloodstream long enough for practical utility see Quay, PCT/US92/07250 and PCT/US94/00422). Thus both at resonance and away from resonance, a bubble stabilized by its gas contents and with a monolayer of surfactant on its surface, has less damping to dissipate the exciting energy of the exciting ultrasound pressure waves and thus undergoes larger volume excursions to reradiate or scatter more of its energy as harmonic components at frequencies other than the excitation frequency. Sucrose stearate and Pluronic F-68 are two examples of surfactants which coat microbubbles with a monolayer and thus do not damp the gas/vapor stabilized bubble's oscillations.
With these particular parameters in mind, the particulars of construction of suitable microbubbles will be set forth below.
In addition to the particularly described microbubbles, other microbubbles, such as those described in Quay, PCT/US92/07250 and PCT/US94/00422, may be used, provided that the gas and/or surfactant is selected as described herein such that harmonic reradiation is sufficient for use in the present invention.
~'~ : f,~

,~ X17820 8 - l0a -Microbubble Construction A. The Membrane-Forming Liquid Phase The external, continuous liquid phase in which the bubble resides typically includes a surfactant or foaming agent. Surfactants suitable for use in the present invention include any compound or composition that aids in the formation and maintenance of the bubble membrane by forming a layer at the interface between the phases, and having the criteria discussed above. The foaming agent or surfactant may comprise a single compound or any combination of compounds, such as in the case of co-surfactants.
It will be appreciated that a wide range of surfactants can be used. Indeed, virtually any surfactant or foaming agent (including those still to be developed) capable of facilitating formation of the microbubbles and having the properties discussed able can be used in the present invention. The optimum surfactant or foaming agent or combination thereof for a given application can be determined through empirical studies that do not require undue experimentation. Consequently, one practicing the art of the present invention should choose the surfactant or foaming agents or combination thereof based upon such properties as biocompatibility, solubility of gas phase in surfactant, and their non-Newtonian behavior.
B. The Gas Phase A major aspect of the present invention is in the selection of the gas phase. As was discussed above, the present invention relies on the use of microbubbles which have the ability to generate harmonics ..,.,......
~,f~~

»>E~
.11.
of y aransmittard ulttd~ui~l fnqu~c~Such ~lib~e= can ~cor~iirr a gas or combaratian of gases to harness or cause differentials ar partial pressures and to gs~erata gas osmotic pressures which statue the bubbles. Further, the microbubbles contain matariab' which can change state from a gas to a gquid or solid .st bodlr tamperatura, (gully from about 35:5°G to about Y40°CI, mrd at useful pressures (generagy about 1-2 atm~ Ahernatively, the microbubbtes can contain material which adsorbs or solubiiizes in ~, hydroph~ic portico of the babble membrane ss di:a~ed above.
pccor~ngly; fhrorocarbons or other compounds that'ara rrot gases at room or body temperature can beused, luov~t tbas they have sufficient vapor pres~e, prefetab~r at kcal abmrt 1a-20 Torr, and more preferably 30, 40, 50 or 100 Torr °at body temperature, or more' preferably at least about 150 or 200 Torr.
However, with the first type of microbubble !condensing :gas?; the vaap~
p~tessure of at least one of the c~p~ts sirould b~ #wdow about 900 or 1000 Torr, such that when a sufficient concentration of the gas a prat kr a b~bla, the absokrte pressure applied to the gas in the bubble during imaging exceeds the vapor pressure of that component divi~d by the fractional partial pressure provided in the bubble by that gas: Thus,'P~ E P"tP~, where P" is the abso~ts pressure in the bubb~ when compre~ed by an ultrasound waves Pv is the vapor pressure at body tmnperatur8 of the relevant gaseous compound, and P,~ is the partial pressure of that gas expressed as a fraction of the. total gas pressure ~ the bubbhr. For example, if the pyre in the twbble is 1000 Torr 1760 Torr atmospheric pressure phrs 1'~ Torr contributed by systolic pry a wary 140 'Toy contributed by ~enic compr~saioAl then a condensing gas contributing 5016 of the partial gas preaure-in the bubble should have a vapor pressure at body temperature of under 500 Torr:
As wggested above, fluorocarbon gassy are particularly ~referted. The term fluorocarbon as used ~~ ~~~s -fugy fluorinated componnda (toearbonsl as wed as partially fluorinated hytkocarbontfluoroc~bon materials, all unsubstituted or substituted with anotfier halogen such as Br. CI, or F or another :ubatituent, such as 0, ~W; S, N0, and the pke. Substances possessing suitable solubility andlor vapor pressure criteria include perfworohexane; pef#luoropentane, perfluorocyc~pentane, p~u~ne, periluorocyc~butan~a: and perfluotoprtopene.
It wig be appreciated that one of ordinary skill in 'the art can readily determ~e other compounds tl~t would perform soit~ly in the preunt arvention that do not meet both the sok~ity and vapor pressure 'criteria; dears~ed a6eve. Rather, 'tt wib be understood thae certain compounds can be considered outside the preferred rank in either solub~ty or vapor pressure: if such compa~nda compensate for the .aberration irt the other category and previde a superior insolubigty in water or high vapor pressure or affinity to dissolve in the surfactant used.
It should also be noted that for medical uses, the gases should be biocompatible or not be da~terious. Uhimste~, the microbubbles caintaining the gas phase will decay and the gas base wig be released kilo the blood either as a dissolved gas or as submicron droplets of the condensed liq~d: -It wig be understood that gases wdl primarily bd remflv~ from the body through lung respiration or a> >s2o a through a combination of respiration and other metabolic pathways in the reticuloendothelial system.

Other Components It would be understood that other components can be included in the microbubble formulations of the present invention. For example, osmotic agents, stabilizers, chelators, buffers, viscosity modulators, air solubility modifiers, salts, and sugars can be added to fine tune the microbubble suspensions for maximum life and contrast enhancement effectiveness. Such considerations as sterility, isotonicity, and biocompatibility may govern the use of such conventional additives to injectable compositions. The use of such agents will be understood to those of ordinary skill in the art and the specific quantities, ratios, and types of agents can be determined empirically without undue experimentation.

Formation of the Microbubbles of the Present Invention.

There are a variety of methods which can be used to prepare microbubbles in accordance with the present invention. Rehydration of spray dried hollow microspheres is preferred. Sonication is also a preferred method for the formation of microbubbles, i.e., through an ultrasound transmitting septum or by penetrating a septum with an ultrasound probe including an ultrasonically vibrating hypodermic needle. However, it will be appreciated that a variety of other techniques exist for bubble formation. For example, gas injection techniques can be used, such as venturi gas injection.

Other methods for forming microbubbles include formation of particulate microspheres through the ultrasonication of albumin or other protein as described in European Patent Application 0,359,246 by Molecular Biosystems, Inc.; the use of tensides and viscosity increasing agents as described in U.S. Patent No. 4, 446,442; lipid coated, non-liposomal, microbubbles as ~1 7620 6 - 12a -is described in U.S. Patent No. 4,684,479; liposomes having entrapped gases as is described in U.S. Patent Nos.

5,088,499 and 5,123,414; and the use of denatured albumin particulate microspheres as is described in U.S. patent No.
4,718,433.
Sonication can be accomplished in a number of ways.
For example, a vial containing a surfactant solution and gas in the headspace of the vial can be sonicated through a thin membrane. Preferably, the membrane is less than about 0.5 or 0.4 mm thick, and more preferably less than about 0.3 or even 0.2 mm thick, i.e., thinner than the wavelength of ultrasound in the material, in order to provide acceptable transmission and minimize membrane heating. The membrane can be made of materials such as rubber, Teflon'', mylar, urethane, aluminized film, or any other sonically transparent synthetic or natural polymer film or film forming material. The sonication can be done by contacting or even depressing the membrane with an ultrasonic probe or with a focused ultrasound "beam". The ultrasonic probe can be disposable. In either event, the probe can be placed against or inserted through the membrane and into the liquid. Once the sonication is accomplished, the microbubble solution can be withdrawn from the vial and delivered to the patient.
Sonication can also be done within a syringe with a low power ultrasonically vibrated aspirating assembly on the syringe, similar to an inkjet printer. Also, a syringe or vial may be placed in and sonicated within a low power ultrasonic bath that focuses its energy at a point within the container.
., at ~s2o s Other types of mechanical formation of microbubbles are also contemplated. For example, bubbles can be formed with a mechanical high shear valve (or double syringe needle) and two syringes, or an aspirator assembly on a syringe. Even simple shaking may be used. The shrinking bubble techniques described herein are particularly suitable for mechanically formed bubbles, having lower energy input than sonicated bubbles. Such bubbles will typically have a diameter much larger than the ultimately desired biocompatible imaging agent, but can be made to shrink to an appropriate size by the loss of non-osmotic gases, thus concentrating the osmotic agent to near saturation.
In another method, microbubbles can be formed through the use of a liquid osmotic agent emulsion supersaturated with a modifier gas at elevated pressure introduced into a surfactant solution. This production method works similarly to the opening of soda pop, where the gas foams up upon release of pressure, forming the bubbles.
In another method, bubbles can be formed similar to the foaming of shaving cream, using perfluorobutane, freon, or another like material that boils when pressure is released.
However, in this method it is imperative that the emulsified liquid boils at sufficiently low temperatures or that it contain numerous bubble nucleation sites so as to prevent superheating and supersaturation of the aqueous phase. This supersaturation will lead to the generation of a small number of large bubbles on a limited number of nucleation sites rather than the desired large number of small bubbles (one for each droplet).
In still another method, dry void-containing particles or other structures (such as hollow spheres or honeycombs) that rapidly dissolve or hydrate, preferably in an aqueous solution, e.g., albumin, microfine sugar crystals, hollow spray dried sugar, salts, hollow surfactant spheres, dried porous polymer spheres, dried porous hyaluronic acid, or ~1 7820 8 - 13a -substituted hyaluronic acid spheres, or even commercially available dried lactose microspheres can be used to form the microbubbles of the present invention.
For example, a spray dried surfactant solution can be formulated by atomizing a surfactant solution into a heated gas such as air, carbon dioxide, nitrogen, or the like to obtain dried 1-10 micron or larger hollow or porous spheres, which are packaged in a vial filled with an osmotic gas or a desired gas mixture as described herein. The gas will diffuse into the voids of the spheres. Diffusion can be aided by pressure or vacuum cycling. When reconstituted with a sterile solution the spheres will rapidly dissolve and leave osmotic gas stabilized bubbles in the vial. In addition, the inclusion of starch or dextrins, a sugar polyester and/or inflating such as methylene chloride, 1,1,2-trichlorotrifluoroethane (Freon'p' 113, EM Science, Gibbstown, NJ) or perfluorohexane, will result in microbubbles with an increased in vito half-life.
Particularly preferred starches for use in formation of microbubbles include those with a molecular weight of greater than about 500,000 daltons or a dextrose equivalency (DE) value of less than about 12. The DE value is a quantitative measurement of the degree of starch polymer hydrolysis. It is a measure of reducing power compared to a dextrose standard of 100. The higher the DE value, the greater the extent of starch hydrolysis. Such preferred starches include food grade vegetable starches of the type commercially available in the food industry, including those sold under the trade-marks N-LOK and CAPSULE by National Starch and w A' ,~~w. .v 2 ~ ~ 6206 ~ . ~D 9Yf3 '. ''.,:~ ~ y ~ , , .- . P4~!">7)I~tS

'Chemical Co; IBrldreter; NJ); da~raxi~d at~~hes. suic1t as 'hlakaxy!eth~
starch (available under the trademarks fIETASTA~RCii and fiEN from du Poet Phartnaceuticelsl' ~M~Illydroxyethylstarch. Ajkrimoto, Td~,o, Japan). iNote that short ~a~ spray ~y, Wig; and caA be' used to produce microbubbles, but are not preferred because thos~r with a molacul~ wei~t ieaathan abwut ~fl0a do not stabilize the micrd~blea. Howerer: they can be used in the present invention in appilCations -in which additional stabiilZat'ron is not ratltdrad.) M the ah~mati~, a fyophiilaed clrice of aurllatrtant aad~ldedreagents produced w'tth a fine pore structure can be placed in a vfrl with a sterile sohttron and a lu~ad spaced w'tth an osmotic gas mixtere. The sobtioa can be frozen rapidly to produce a fine ~e crystal structure and, therefore, upon ly~gzation produces fine pores (voids where the ice cry'tals mere removed):
Alternatively, any diuohrabie or solubta void-farrr~g structwes may be used:
In this embodanent, where the void-forming material is not made from or does not contain surfactant: both surfactant and liquid .ere suppwed into the container witfi the at~ructures and the dashed gss or gases. Upon reconstitution these voids trap the osmotic gas and, with the dissolution of the sobd Bake, form microbubbfes with the gas or gases ~ them.
rne s for use ~ ~e p~~~tion can be< :farmed using a container enclosing the gas or gasaa described above for forming the microb~lbas, the fpuid; and the surfactant. The container can certain ail of he sterile dry components, and the gas; in ono chameer, with the sterile aqueous liquid in a second chamber of the same container. Suitable two-chamber vial containers are available, for example, under the trademarks WNFATON RS117FtW or S~1702F1 from Eton Glass Co., IMillville, NJ1.
Alternatively, an inverted two-chamber vital may be used for microbubble preparation. One advantage associate with thris method of microbubble. formation ~ that the aqueous ~raae can be instilled first and sterilized via autoclaving ar other meaos~' folowed by arstiilation of the spray dried microspheres. This will pravenrt~poteantist microbial growth in the aqueous pha:e prior to tea~ization;
Other suitable devices are known and are commercially avaiabie. For example; a two compartment glass sychrge such as 'the a-0 IfYPAK ~~~~pry 5+5 mf Dual Chamber prefiUed syrarge syst~ (Becton 0ickhrson, Frsnkiln lakes, NJ; deacr~ad in U. S. Patent 4,813,3281 can advantageously be used to reconstitute the sluay dried powder:
It can be appreciated by mte of or~sary skill in' the art that otwo~drember reconst'ttution vt~pap~,af combinhrg the spray dried pewdar: with the aqueous sokstion in a st~erife manner are also 3D within the scope of the present invention. in such systems, it a particularly advantageous if th9'squeous phase can be intwposed between the water-~sokrble~o~notic gas° and ha enviromnent, to increase shelf life of the produet.
Alternatively, the contacan contain the void fomung .material the gas or gases, and the surfactant and liquid can be add~ad to form th~ microbubbles. In one embodiment, the surfactant can be present with the other msterials in the container, so than only the Nquid creeds to be added in order to form the microbrrbbka. Where a material-nerxsaary for forming t#re microbubbles is not already present in the ~i r~~ p PT' ~ : 217 6 2 0 6 ~,0 3 r~'rr~f~sra~ases containers 'tt csrr be packaged t~e~~afhar coi~Vponents of a kh,-preferably ~n a form or container adapted to fac~tate ready ration with theother components of the kh.
The container used hr the kh may be of the type described'elser~here herein.
In one emboda~nt, rh~e coM~ine~'is a conventional aaptron~rsealed vial. In amather, h has a means for erecting or permitting apgbcation of sufficient bubble forming energy iMo the contents of the eorrteiner. The means can comprise, for example, the thin web or sheet de:cr~ed previously.
The atckraion of the surfectaMa and wetting agents iato the alien of the m~rosphere slaws the use of a lower surfactant concentrat'ron: As the ntrcrosphere shag is diaaiolv~g~ it temporarily surrounds the microbnbble formed in hs inter'ror whh a layer of aqueous phase that is saturated- with the surfactants, enhancing their depos'ttion on the microbubble's surface. Thus, spray-dr~d surfactant containing microspheres require only locally high concentrations of surfactant, and obviate the need for a high surfactant concentration in the eptire aqueou:.phase.
hnaninn Methodoloav Any of the microbubble preptarations of the preserrit awentien may be Irdm~a4terad to a vertebrate, such as a bttd or a mammal, as a contrast agent for uhrasonicaHy enaging portions of the vertebrate.
Preferably, the vertebrate is a human, and the port'ron that is imaged is the vasculature of the vertebrate.
In this embodiment, a amag qnaMhy of microbubbles (e.g.; 0.1 mlfKg based on the body weight of the vertebretef is urtroduced intravaacularhrinto the arixnal. Other quantities of microbubbles, such as from about 0.005 mllKg to about 1:0 mlfKg: can also be used. The fieart, arteries, veins, and organs, rich in blood, such as liver and kidneys can be uhrasanically imaged with this techn~ue: Assuming that the uhrasound knaging machine is set to anage at a particular frequency, the outgoing waveform supplied to the sonic transducer can be a numerical fraction of the anaging frequency (e:g., 1~2, 213, 1f3, and the bke) or a whole number or fractional muhiple of the imaging frequency (e.g., 2. 312, 3, 4, and the likel. With any particular combination of microbubble composition and excitation frequency, certain harmonics will be dominant. The second harrtronic is a common example. Those strongest harmonics are preferred for obvious reasons, although other harmonics may be selected for reasons such as preparation ef multiple images or elimination of background. Dominant harmonics can be determined by simple empirical testing of the microbubble solution.
The foregoing description wig be more fully understood with reference to the following Examples.
Such Examples, are, however, exemplary of preferred methods of practicing the preeeat invention and are not limiting of the scope of the invent'ron or the claims appended hereto.
Examole 1 Preoarat'ron of Ultrasound Contrast'Adent Throuoh Sonieation Microbubb~s whh an average number weighted sae of 5 microns were prepared by sonicat'ron of an isotonic aqueous phase containing 2% Pluronic F-68 and 1 % sucrose stearate as surfactants, air as a modifier gas and perfluorohexane at a concentration near saturation at 37°C.

'~a: ~ ~ ,. .-~., s.w~~$ 1, ~ _ ~ rls9sts -1s-.3 ml of a ateala watu salon co~ini~g Q~~%.J~I, ~,% .,~k~tpdi~ FaG~,~aa~ 1 %
sucrose stearate was added to a 2.0 ml rial The vial bad a r~a~ing bead s~aca pf, 0.2,.~ ~plly containing air. Air saturated witp perfluorobwcana rappr (220 tprr of perfluorofwxane with 540 torr of air) at 25°C was used fb~ah the head:psce of tba vial The vial was tsakd w'tth a thin 0.22 mm Oqtytetrafkrbroethylene (PTFE) septum. The vial was turpad baraontagy, sad .a 118" 13 mm1 sonication probe attached to a 50 watt sonicator model IIC50, availab~ from Sonics & MatoNale, was pressed gently against the septum. In this pin, he septum ;iWatates the prpbe from the solution. Power was than applied to the probe and the :awtioa was :anicated for 15 :second:, forn~ng a white :okition, of finely divided microbubbles, having an average number wsigbtad sae of 5 microns as measured by Horiba LA-700 laser tight scattering particle .~p analyZBr.:
Use of ' ~l~~a.co~6~rr~-:Anent Two rabbits were in~cted with doses ranging from 0.1 to 0.3 ml of coatrast agent prepared aacwdinp to Example 1 for a total of 5 injections per rabbit. The rabbits were then imaged with an i5 sxperanental tdtraaound instrument at the University of Toronto, Sunnybrook Heahh Science Center, 2075 :.Oayview Avenue, North York, Ontario, Canada. This instrument was capable of imaging in normal gray-scale and Doppler modes as well as harmonic enhanced gray-scale and Doppler modes..
,Images of the heart, int~ior vwta cave, aorta, kidneys and Gver ware examined. Images o~ the rabbit were greatly enhanced when this contrast agent was injected while imaging in the harmonic enhanced modes.
Small vessels were clearly 20. visible after coatrast injection, while the rwnvascular clutter signals were;greatly reduced. This enhancement lasted approximately 2 to 3 minutes. The enhanced image was the result of the contrast agent generating wperier harmonic uhrasound signals.
~"~p~r~l tion of Forna~l 2h , ~prav Dried Uhr~sound Contrast Aoe One plot of each of the following solutions was prepared with water for,,injection: Solution A
captaining 4.0% wlv N-lok vegetable starch (National Starch and Chemical Ca., Bridgewater, NJ) and 1.9%
wlv sodium chloride (Mallinckrodt, St. Louis, M0) and Solution B containing 2.0% Superonic F-68 (Serve, Heidelberg, Germany) and 2.0% wlv Ryoto $uaose Steuate S-1610 (Mitsubishi-Kasei Food Corp., Tokyo, 3D : Ja~anl. Solution B was added to a high shear muter and cooled in an ice bath. A coarse suspension of 40 ml 1,1,2-trichlorotriflupr99thane (Freon 113; EM Science, 6ibbstpwn, NJ1 was made in the 1 liter of solution B. This suspension was emulsified using a Microfluidizer (Microfluidics Corporation, Newton, MA; model M-11pE1 at 10.000 psi, 5°C for 5 passes. Tba resulting emu~ion was added to solution A to produce the following formula for :pray drying:

2 i ~6z06 W~f~~6~97"~3 P~°r9"~i1~I45 .17.
2.0% wIv r~-!fE$:tdroxyethy~ttarch (Ajinanoto, Tokyo, Japan) 2.0% rlrlv sodium chloride (MaUinckrodt) 0.87% sodium ~bhatb, dibasic (Mall~ckrodt!
0:26% sodNm ph~aphata, monobaaic (MaUinckrodt) 1.7% wlv Superosiic F-B8 (Serve) 0.3% wlv Sucrose''Stearate S-1670 (Mftsubishi-Kaaei Food'''Corp.) 0.1 % wlv S~croaa Stearata S-570 (Mi~tanbuhHKaa~ Fmod Gap:) 4.0% wlv 1,1.2-triChlorotrifluoroethane (Freon 113; EM Science) This emulsion was then spray dr~d in a Niro Atomizer Portable Spray Oryer'equ~ped with a two fluid atomizer (Niro Atomizer, Copenhagen, Denmark) employing,the following settings:
: hot ak flow rate 39.5 Cf~All inlet air temp. - 255C , outlet air temp. - 109C

atomizer air flow - 110 literslmin emulsion feed rate - 1 literlhr The drjf, hailo~r apheri~al product had a diameter between about 1,uM and about 15 NM and was collected ar the cyclone ~saparator as is standard for this dryer. Aliquots of powder (250 mg) were weighed into 10 ml tubing vials, spacged with perfluorohexane-saturated nitrogen (2 mg perfluorohexane per ml of gasl at 13°C and sealed. The nitrogen was saturated with perfluorohexane by passing, it through three perfluorohexane filled gas washing Bottles i~nmerssd in a 13°C water bath.
The vials were reconstituted for injection with 5 ml water to 400 mg of spray dried powder after inserting an 18-gauge needle as a vent to relive pressure as the water was injected.
Example 4 ~~a of Formula 1 Sorav Dried I~~,r,~ound Contrast Aoent A 1 ml injection of the contrast agent prepared as described in Example 3 was administered to two rabbits. The rabbits were then imaged as described in Example 2 above.
The formula enhanced the harmonic signal generated by the microbubbles. The Formula ~1 spray dried oantrast;agent,producad greater enhaatpanent than the soniceted contrast agent described in Example 1 above, and this enhancement lasted for approximately 4 minutes. This improved harmonic response and persistence are tba results of a more optimally chosen non-Newtonian surfactant system. The formula demonstrated that a copdanseble vapor, an absorbable vapor, a non-Newtonian surfactant and a fluorocarbon vapor stabil~ed monalayer surfactant bubble, generate enhenpsd harmonics for superior in vivo imaging.

n n. i s , ~ , ~. k '.'~ ~ , , ~ 2176206 ~voa rc~ivs~sas -1 s.
Predaration o~'fo~ule 2 sorav urrea ua:raaou ~~o~ra~ noent .,One ~ter of each of the foaowing solutions ~a~ prepared w'tth water for injection: Solution A
containiwg:4.0% whr N-lok rspetable starch iNational St~cb and~~Chemical Co., Bridgewater, NJ) and 1.9%
wlv sodaim chloride (Map'utckrodt, St. Louis, IIllO) and Solution B containarg 2.0% Superonic F-68 (Serve, Heidelberg, Germany) and Z.0% wfv Ryoto Sucrose Stearate S-1670 (Mitsubishi-Kasei Food Corp., Tokyo, K, , 3 Japan). Solution B was added to a high shear mixer and cooled in an ice bath.
A r~oarse suspension of 40 ml 1,1,2-trichlorotrifluoroethane (Freon 113; EM Sconce, Gibbstown, NJ) was made in the 1 liter of solution B. This suspension was emulsified using a Microfluidizer (Microfluidics Corporation, Newton, MA; model M
110F) at 10,000 psi, 5°C for 5 passes. The r~ultirr~ emrdsion was added to solufron A to produce the following formula for spray drying:
2.0% wlv m-HES hydroxyethylstarch (Ajinimoto, Tokyo, Japan) 3.0% wlv sodium chloride (MaUinckrodt) . 1.7% wlv Superonic F-68 (Serval 0.2% wlv Sucrose Stearate S-t670 (N'(itt~ib~lti-'Kgsei Fobd' Corp.1 a ~ ~ 20 ' _, : 0.1% wlv Sucrose Stearate S.570 (M~~~ei.: Foal~Corp.j 4.0% wlv 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science) ~' Nb, ' . 11 ~ ' i This emulsion was then spray dried in a'Niro At6Atize~~ti~gbt~'Sprgy'Dryer'~quipped with a two fluid atomizer (Niro Atomaer, Copenhagen, Denmark) emplo~fng~thA following settings:
~14ir flow rate ~ 39.5 CFM

inlet air temp. - 220C

outlet air temp. - 103C

r~ atomaer air flow - 1f0 I~ershnm . .

emulsion feed rate - 1 literlhr . , ., . ~.ii, The dry, hogow spherical product had a dierri~etiaw'betweett ~bont t~~jiill arid about 15 NM and was coljected at the cyclone separator as is standard fof'thi~ d~y~r:
''Aliquots of powder (250 mg) were weighed into 10 ml tubing vials, sparged with petfiuo~ohexane-saturated nitr0pen (2 tng perfiuorohexane per ml of gasl at 13°C and sealed. The nitrogen was saturatedvnith perfhi6rohexane'by passing it through three perfluorohexane figed gas washing bottles hnme~sed in a 13°C
water bath.
The vials were reconstituted for injection with 5 ml water to 350 mg of spray dried powder after inserting an 18-gauge needle as a vent to relieve pressure as the water was injected.

_ ,.
r~w~~ '_ y .; , * , : 21 ~ C~ 2 0 r~rr~.s Use of ,~orn~ula 2 ~orav tfried Uhrasound CQgtrf styAaent Two 1 ml arjections of the contrast agent descried ~ Example 5 was adm~istered to two rabbits. The rabb-tta were then imaged as described in Example 2 above.
The formula enhanced the harmonic s~nal generated by the microbubbles. The Formula #2 spray dried contrast agent produced greater enhancement than the sonicated contrast agent described in Example 1 above and the Formula #1 spray dried contrast agent described in Examph3 3, and this enhancement lasted for approxknately 5 minutes. This improved harmonic response and persistence are the results of an improved surfactant formulation. The formula again demonstrated that a condensable vapor, an absorbable vapor, a non-Newtonian surfactant and a fluorocarbon vapor stabilized monolayer surfactant bubble, generate enhanced harmonics for superior in vivo imaging.
Examole 7 Pre~~,fion of Formula 3 Sorav Dried Ultrasound Contrast Aoent One fitter of each of the following solutions was prepared with water for injection: Solution A
contaa~ing 4.0% wlv N-Lok vegetable starch (National Starch and Chemical Co., Bridgewater, NJ) and 1.9%
wlv sodium chloride (Maginckrodt, St. Louis, M0) and Solution B containing 2.0% Superonic F-68 (Servo, Heidelberg, Germany) and 2.0% wlv Ryoto Sucrose Stearate S-1670 (Mitsubishi-Kasei Food Corp., Tokyo, Japanl. Solution B was added to a high shear mixer and cooled in an ice bath.
A coarse suspension of 40 ml 1,1,2-trichlorotrifluoroethane IFreon 113; EM Science, Gibbstown, NJ) was made in the 1 liter of solution B. This suspension was emulsified using a Microfiuidizer (Microfluidics Corporation, Newton, MA;
model M-110F) at 10,000 psi, 5°C for 5 passes. The resulting emulsion was added to solution A to produce the following formula for spray drying:
3:8% vn~ir m-HES hydroxret~lstarch ~A~nirneto. Tokyo. Japan) 3.Z5% wlv sodium chlpride IMalknckro~t) 2.83% sodium phosphate, dibasic (Mellinckrodt) 0:42'% sodium phosphate, monobesic ~Maltimckrodt) ' 2.11 % wlv Superonic F-68 (Servo) 0.32% wlv Sucrose Stearate S-1670 (Mitsubishi-Kasei Food Corp:f 0.16% wlv Sucrose Stearate S-570 (Mitsubishi-Kasei Food Cerp:1 3.0% wfv 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science) This emulsion was then spray dried in a Niro Atomizes Portable Spray D~yei equipped with a two fluid atomizer (Niro Atomaer, Copenhagen, Denmark) employing the following settings:

x i a r , . ~ t n~..
~~~'~ ~;~ '.:: 2 ~ 7 6 2 0-6 .,~ ~~, :. rc-~n~s~~s~s .~

hot ae flow rate - 31 CFM
inlet:a~ temp. - 3~'~°C
outlet air temp. - 120°C
atomizer ab flow - 290 fiters/min emu~ian feed rate - 1.5 ~teralh~ ;~E
. The dry, hollow spherical product had a diameter between about 1 pM and about 15 NM and .. ;~, " . ..
was ~cohected at the cyclone separator as is standard for this dryer. Aliquots of powder 1250 mg) were weighed arto 10 ml tubing vials, aparged w-'tth perfluorohexane-saturated nitrogen (2 mg perfluorohexane per ml of gas) at 13°C and sealed. The nitrogen was saturated with perfluorohexane by passing it through three perfluorohexane filled gas washing bottles immersed in a 13°C
water bath.
The vials were reconstituted for inaction w'tth 5 ml water to 100 mg of spray dried powder after inserting an 18-gauge needle as a vent to relieve pressure as the water was injected.
xam h: 8 Use of Formula 3 Sorav Dried Ultrasound Contrast Anent ~, ,.
An anesthetized dog wephing approximately 20 Kg was prepared for examinat'ron at the Mayo Clinic, 200 First Street Southwest, Rochester, Minnesota, w'tth an experimental ultrasound imaging instrument capable of imaging in normal gray-scale and Doppler modes, as well as harmonic enhanced gray scale and Doppler modes. This instrument was designed independently from the one used. in the examples above. The heart of the dog was imaged in all modes before and after injection of 0.5 ml, 1.5 ml and 2 ml doses of the microbubble ultrasound contrast agent described in Example 7.
The harmonic enhanced mode images were far superior to the normal mode images in defining the motions of the heart wall, the volume of the chambers, and visualizing the contrast agent perfusing the heart muscle. Individual perforator vessels in the septum of the heart were observed. The conuast had a useful lifetime in the blood of approxanately 5 minutes.
The experanente~deml0tibed app d9~on~trate the ~an~ed harmonic generation of a microbubble contrast agent that (1) contains pa~fhtotohexgne with'a vapor piresserre at 37°C over 23 torn, less than 1 %
wt.lwt. solubility in water, and present at a concentration in the;;gas greater than 2% mole fraction and above 50% of its saturation concentration that enhances harmonic generation by condensation; that (2) contaars gas wit# more than 2% mole fraction of perfluorohexana that (a) has a solubility of its liquid phase in hexane at 37°C of more than 10% molelmole and (b) has a solubility of its liquid phase< in water of less than 1 % wt.lwt. at 37°C, and therefore enhances harmonic generation by adsorbingldissolving in the s4cfactant layer; that (3) contains a non-Newtonian surfactant that enhances harmonic generation; or t that (4) contains microbubbles stabilaed by perfluorohexane, a gas osmotic agent, and a monolayer of surfactant that enhances harmonic generation.

.
~~..,,~
~Q 3 P~T~~45 .21., Example 9 U,ea ~~11'~4 J~~~~'pg~;Gnntrast Aaent A microbubble coatreat agent is prepared by sonication as in Example 1 above except that the ~tmo~are is the vial prior to aonicatioa (and tlmrefore t~ pas n the microbubbles) is 100%
perfluorobutaas and the aob~tiao in he aonicatad vial is 0.9% sagne plus 3%
Pluronic F-68 (a Newtonian ' water aol~le turfactaat that demonstrates only :mall chanpas in at~faca te~ian when the monolayer is ~o~.
A rabbi ~ amaged a: ~ Example 2 above after injecting 0.3 rtd of this contrast agent. While the vascular persistence and harmonic generation of thin: ~eparafron are not optimal; hirer levels of harmonic tp ~rhar~ment are aeh~vtd, relative to ab filled micra~bu~las, because of the ability of perfluorobutane to dissolve in or adsorb to the hydrophobic rpion of the Pluronic F-88 monelayer.
This is because perfluorvbutane has a solubgity in hexane of greeter than 10% molelmole and a water solubgity of less than 1 % wt.Iwt. In eddhion, the bubbles are stabilized by thea~ gas contents and have a monolayer of ,surfactant.
The contrast agent described ~ Examp~ 9 is made with bubbles containing nitrogen saturated with p~fiuorohexans at 13°C. Rabbits are bnaged g-the,contraa agent as deacr~ed above. The harmonicenhancement-of this fomwlatien is better than in Example 9 because, in addition to the adsorption and gas stabgized monaiayer effects de$cribed in Example 9; the perfluorohexane can condeuae when excited, e.g., it is present in more than 2% mole fraction of the gas phase and at a concentration above 50% of its saturation concentration, is less~than 1% wt./wt. soluble in water, and has a vapor pressure at 37°C over 23 torr.
E,~mnle 11 Use of Ultrasound Contrast Aaent Containino Albumin-Coated Microbubbles The cortunercial avagable microbubble preparation Albunex (Molecular Biosystems Inc., San Diego, CAI is prepared mnploying nitrogen saturated with perfluerohexane at 13°C as the gas mixture present during the formation of the bubbles by sonication according to the method described in U.S. Patent No.
4,957,658. This contrast agent is injected into a rabbit and imaged as in Example 2 above. Even though this agent is damped by many layers of albumin surfactant and does not contain a non-Newtonian surfactant, its harmonic enhancement is increased by the presence of perfluorohexane which will condense and adsorb during excitation, producing enhanced ultrasound images.

.t t,' ,, ,.5~ , 2176206 m 1 Ira rot t~ta~ed Cadst dent Containa~a Fluorinated Surfactant w'tth lipoid Per~er~lme~ itmuhgons ' A parfkior~antaneis preparodaccording to the rrrtthod~is~lobi«facample 42 of Quay, ~ Pit appbcati~ non~er PCTf~84100422. The emuls~n is sdnNniatered to a rebbitvwhich is imaged as d~~ ixan~le 2 a~wen~ioyiagvaNtianic enhanced niodea: ltpon ir~ection;=tfiis perfluoropentane emulsion boils at the body temperature of the rabbit to form microbubbles dot~ir~g approximately 100%
~~or~Mane gas. -'fhie gaa w~~ tx~ ardor exatati~n as descried in the foregoing disclosure, g~arat)ng enhance barm~ic signals.
~.: lp The sohibdity of epeotaae in water a~td bex~°me~t tike ~cr'tt~rie a for adsi~tption in the surfactant layer of the miaobnbblt; generating. enhanced harmonic signets:
Tfiis preparation also contains Zonyl FSA surfact~t, a floerinated nea.Nesvtordan sur#a~tdr~; wAose autfece tension changes rspidly when the rronofayer is compressed: gating enhanced harmonic signals.
The surfactant mixture employed in the contrast agent (Pluronic P-123 and Zonyl FSO) is one that forms monolayers on bubbles 15 and the bubble is stabilaed by 'tts gas contents, therefore avoiding damping of the volume oscillations of the bubble when e~ndted; and rlsultinp ar ealhexed I~monic signal generation-The foregoing description dtatails certain preferrad'embodiments of the present invention and describes zhe best mode ~ateatplated: It wiR be a~reciated,~howe~er, thot-I!~
matter how detailed the foregoing appear in text, the invention can be practiced in many ways and the invention should be ~0 w~-construed in accordance with the appended tea and any e~ivalents thereof.

Claims (56)

WHAT IS CLAIMED IS:

1. A method of ultrasonic harmonic imaging using ultrasonic energy transmitted by an ultrasonic source to an object or body to be imaged, comprising:
introducing into said object or body a contrast agent comprising microbubbles having generally spherical membranes and containing gaseous material having vapor pressure at 37°C over 23 torr, less than about 1% wt./wt.
solubility/miscibility in water, concentration in gas phase when detected greater than 2% mole fraction, and a concentration greater than 50% of its saturation concentration; and ultrasonically imaging at least a portion of said object or body.

2. The method of Claim 1, wherein said material is selected from the group consisting of perfluorohexane, perfluoropentane, perfluorocyclopentane, 1,1,2-trichlorotrifluoroethane, sulfur hexafluoride, cyclopentane, methylene chloride, pentane and hexane.

3. The method of Claim 1, wherein said concentration is at least about 25% mole fraction.

4. The method of Claim 1, wherein said concentration is 100% mole fraction.

5. The method of Claim 1, wherein said membranes comprise a surfactant.

6. A method of ultrasonic harmonic imaging using ultrasonic energy transmitted by an ultrasonic source to an object or body to be imaged, comprising:
introducing into said object or body a contrast agent comprising microbubbles having generally spherical membranes and containing material that comprises at least 2% mode fraction of a gas that has a solubility of its liquid phase in hexane at 37°C of more than and 10% mole/mole and a water solubility/miscibility of its liquid phase of less than about 1% wt./wt. in water at 37°C; and ultrasonically imaging at least a portion of said object or body.

7. The method of Claim 6, wherein said gas is a hydrocarbon or a fluorocarbon.

8. The method of Claim 6, wherein said gas is selected from the group consisting of perfluorohexane, perfluoropentane, perfluorocyclopentane, 1,1,2-trichlorotrifluoroethane, sulfur hexafluoride, cyclopentane, methylene chloride, pentane, hexane, dichlorodifluoromethane, trichloromonofluoromethane, perfluorobutane, perfluorocyclobutane, perfluoropropane, butane, cyclobutane, propane, methane, and ethane.

9. The method of Claim 6, wherein said membranes comprise a surfactant.

10. The method of Claim 9, wherein said surfactant is fluorinated.

11. The method of Claim 6, wherein said material comprises at least about 25% mole fraction of said gas.

12. The method of Claim 6, wherein said material comprises about 100% mole fraction of said gas.

13. A method of ultrasonic harmonic imaging using ultrasonic energy transmitted by an ultrasonic source to an object or body to be imaged, comprising:
introducing into said object or body a contrast agent comprising microbubbles stabilized with at least one surfactant and comprising a gas or vapor selected from the group consisting of fluorocarbons and hydrocarbons wherein said gas or vapor has a gas phase concentration of greater than about 2% mole fraction, said stabilized microbubbles generating harmonics with an efficiency greater than a free air bubble; and ultrasonically imaging at least a portion of said object or body.

14. The method of Claim 13, wherein said surfactant will change the surface tension of water by more than 5 dynes/cm when the area per molecule of surfactant has changed by 10s as measured on a Langmuir film balance.

15. The method of Claim 13, wherein said surfactant has a component having a hydrophilic-lipophilic balance less than 11.

16. The method of Claim 15, wherein said hydrophilic-lipophilic balance is less than or equal to 8.

17. The method of Claim 13, wherein said surfactant has a component with a molecular weight over 1,000 and capable of lowering the surface tension of water to 40 dynes/cm or lower.

18. The method of Claim 13, wherein said surfactant is capable of lowering the surface tension of water to 40 dynes/cm or lower and has a critical micelle concentration of 0.3 or less volume fraction in water.

19. The method of Claim 13, wherein said surfactant is non-Newtonian.

20. In a method for ultrasonic harmonic imaging of an object or body comprising introducing into said object or body a contrast agent, transmitting ultrasonic energy at an insonating frequency from an ultrasonic source to said object or body and detecting radiated energy from said object or body at a frequency other than said insonating frequency, the improvement comprising:
said contrast agent comprising microbubbles of a gaseous or vaporous compound having a vapor pressure at 37°C
over 23 torr and less than about 1% wt./wt.
solubility/miscibility in water and having the property of radiating imageable ultrasonic energy when said ultrasonic source is transmitting at a frequency other than the resonant frequency of the microbubbles.

21. The method of Claim 20, wherein said microbubbles contain a gas or gas mixture.

22. The method of Claim 21, wherein said microbubbles are stabilized by their gas or gas mixture contents.

23. The method of Claim 20, wherein said microbubbles are produced by spray drying a liquid formulation containing a biocompatible membrane-forming material to form a microsphere powder therefrom; combining the microspheres with a gas osmotic agents and mixing an aqueous phase with the powder, wherein said powder substantially dissolves in the aqueous phase to form microbubbles.

24. The method of Claim 22, wherein said microbubbles are coated with a monolayer of surfactant.

25. In a method for harmonic ultrasound imaging ultilizing microbubbles, the improvement comprising:
providing at least one hydrocarbon gas or fluorocarbon gas in said microbubbles in a concentration of at least 2%
mole fraction.

26. A method for enhancing images obtained by ultrasound, said method comprising the steps of:
providing a contrast agent having a plurality of microbubbles comprising a gaseous or vaporous compound selected from the group consisting of perfluorohexane, perfluoropentane, perfluorocyclopentane, 1,1,2-trichlorotrifluoroethane, sulfur hexafluoride, cyclopentane, methylene chloride, pentane, hexane, dichlorodifluoromethane, trichloromonofluoromethane, perfluorobutane, perfluorocyclobutane, perfluoropropane, butane, cyclobutane, propane, methane and ethane wherein said microbubbles generate harmonics with an efficiency greater than a free air bubble;
exposing said contrast agent to ultrasonic energy comprising an incident frequency and detecting ultrasonic energy radiated from said contrast agent at a frequency other than said incident frequency.

27. Use of a surfactant in the preparation of a contrast agent for ultrasonic harmonic imaging by introducing said contrast agent having a plurality of microbubbles comprising a gaseous or vaporous compound selected from the group consisting of perfluorohexane, perfluoropentane, perfluorocyclopentane, 1,1,2-trichlorotrifluoroethane, sulfur hexafluoride, cyclopentane, methylene chloride, pentane, hexane, dichlorodifluoromethane, trichloromonofluoromethane, perfluorobutane, perfluorocyclobutane, perfluoropropane, butane, cyclobutane, propane, methane and ethane and stabilized with at least one surfactant into an object or body where said microbubbles generate harmonics with an efficiency greater than a free air bubble, exposing at least a portion of said object or body to ultrasonic energy comprising an incident frequency and detecting ultrasonic energy radiated from said contrast agent at a frequency other than said incident frequency.

28. A contrast agent for ultrasonic harmonic imaging;
comprising microbubbles having normally spherical membranes and containing gaseous material that have vapor pressure at 37°C over 23 torr, less than about 1% wt./wt.

Solubility/miscibility in water, a concentration in gas phase when detected greater than 2% mole fraction, and a concentration greater than 50% of its saturation concentration, or that comprises at least 2% mole fraction of a gas that has a solubility of its liquid phase in hexane at 37°C of more than about 10% mole/mole and a water solubility/miscibility of its liquid phase of less than about 1% wt./wt. in water at 37°C.

29. The contrast agent for ultrasonic harmonic imaging according to Claim 28, wherein said material is selected from the group consisting of perfluorohexane, perfluoropentane, perfluorocyclopentane, 1,1,2-trichlorotribluoroethane, sulfur hexafluoride, cyclopentane, methylene chloride, pentane, hexane, dichlorodifluoromethane, trichloromonofluoromethane, perfluorobutane, perfluorocyclobutane, perfluoropropane, butane, cyclobutane, propane, methane, and ethane.

30. The contrast agent for ultrasonic harmonic imaging according to Claim 28, wherein said membranes comprise a surfactant.

31. The contrast agent for ultrasonic harmonic imaging according to Claim 28, wherein said gas is a hydrocarbon or a fluorocarbon.

32. The contrast agent for ultrasonic harmonic imaging according to Claim 30, wherein said surfactant is capable of changing the surface tension of water by more than 5 dynes/cm when the area per molecule of surfactant has changed by 10% as measured on a Langmuir film balance.

33. The contrast agent for ultrasonic harmonic imaging according to Claim 30, wherein said surfactant has a component having a hydrophilic-lipophilic balance less than 11.

34. The contrast agent for ultrasonic harmonic imaging according to Claim 30, wherein said surfactant, capable of lowering the surface tension of water to 40 dynes/cm or lower, has a component with a molecular weight over 1,000, or has a critical micelle concentration of 0.3 or less volume fraction in water.

35. The contrast agent for ultrasonic harmonic imaging according to Claim 30, wherein said surfactant is fluorinated or non-Newtonian.

36. The contrast agent for ultrasonic harmonic imaging, according to Claim 28, wherein said microbubbles have the property of radiating imageable ultrasonic energy when exposed to transmitted ultrasonic energy at a frequency other than the resonant frequency of the microbubbles, said imageable ultrasonic energy comprising a different frequency than said transmitted ultrasonic energy.

37. The contrast agent for ultrasonic harmonic imaging according to Claim 28, wherein said microbubbles are obtained by a method comprising the steps of: spray-drying a liquid formulation containing a biocompatible membrane-forming material to form a microsphere powder therefrom;
combining said microsphere with a gas osmotic agent; and mixing an aqueous phase with said powder, wherein said powder substantially dissolves in said aqueous phase to form microbubbles.

38. A method of enhancing the contrast in ultrasonic harmonic imaging, comprising:
preparing microbubbles having normally spherical membranes and containing gaseous material that have vapor pressure at 37°C over 23 torr, less than about 1% wt./wt.
solubility/miscibility in water, a concentration in gas phase when detected greater than 2$ mole fraction, and a concentration greater than 50% of its saturation concentration, or that comprises at least 2% male fraction of a gas that has a solubility of its liquid phase in hexane at 37°C of more than about 10% mole/mole and a water solubility/miscibility of its liquid phase of less than about 1% wt./wt. in water at 37°C; and administering to an object to be imaged said microbubbles as a contrast agent, prior to ultrsonic harmonic imaging.

39. The method of Claim20, wherein said gaseous or vaporous compound comprises perfluoropropane.

40. The method of Claim 39, wherein said microbubbles further comprise protein microspheres.

41. The method of Claim 40, wherein said protein microspheres comprises albumin.

42. The method of Claim 20, wherein said gaseous or vaporous compound comprises perfluorobutane.

43. The method of Claim 20, wherein said gaseous or vaporous compound comprises perfluoropentane.

44. The method of Claim 20, wherein said gaseous or vaporous compound comprises perflorohexane.

45. The method of any of claims 39 to 44 wherein said microbubbles~further comprise a surfactant.

46. The method of Claim 26 wherein said gaseous or vaporous compound comprises perfluoropropane.

47. The method of Claim 46, wherein said plurality of microbubbles further comprise a protein microsphere.

48. The method of Claim 47, wherein said protein microsphere comprises albumin.

49. The method of claim 26, wherein said gaseous or vaporous compound comprises perfluorobutane.

50. The method of Claim 26, wherein said gaseous or vaporous compound comprises perfluoropentane.

51. The method of Claim 26, wherein said gaseous or vaporous compound comprises perfluorohexane.

52. The method any of claims 46 to 51 wherein said microbubbles further comprise a surfactant.

53. The use of claim 27 wherein said gaseous or vaporous compound comprises perfluoropropane.

54. The method of claim 27 wherein said gaseous or vaporous compound comprises perfluorobutane.

55. The method of Claim 27 wherein said gaseous or vaporous compound comprises perfluoropentane.

56. The method of Claim 27 wherein said gaseous or vaporous compound comprises perfluorohexane.