Michiel Postema
email: m.postema@erasmusmc.nl

Annemieke Van Wamel

Charles T Lancée

Nico De Jong

Ultrasound-induced encapsulated microbubble phenomena

Keywords: Encapsulated microbubble phenomena Encapsulated microbubbles, ultrasound contrast agent, radiation forces, coalescence, fragmentation, jets

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Blood does not reflect ultrasound well, but its response may be enhanced by gas microbubbles of a few micrometers in diameter that are introduced into the blood pool (Goldberg et al., 2001). These microbubbles oscillate upon insonification, and generate as such a characteristic acoustic response.

An ultrasound contrast agent (UCA) is a liquid containing gas microbubbles which are encapsulated by a shell. UCAs are widely used in clinical diagnostic imaging [START_REF] Becher | Handbook of Contrast Echocardiography: LV Function and Myocardial Perfusion[END_REF]Goldberg et al., 2001). The intravascular use of UCA enhances the quantification of perfusion and blood flow [START_REF] Klibanov | Targeted delivery of gas-filled microspheres, contrast agents for ultrasound imaging[END_REF][START_REF] Wei | Blood flow and perfusion measurements: background and clinical utility[END_REF], and lowers the detectability threshold of tumor and tissue vascularization, using conventional Doppler instruments [START_REF] Burns | Intravenous contrast agent for ultrasound Doppler: In vivo measurement of small tumor vessel dose-response[END_REF]). An overview of UCA detection procedures in ultrasound imaging has been presented by de Jong et al. (2000b), and an overview of UCA imaging innovations has been put forward by [START_REF] Chiou | Clinical applications of contrast echocardiography[END_REF].

To understand encapsulated microbubble behavior better in order to develop or enhance applications in diagnostics and therapeutics, UCA microbubbles have been subjected to high-speed photography. Postema et al. (2003a) gave an overview of publications on high-speed optical observations of insonified UCA.

Observations of dynamic UCA microbubble behavior have resulted in new insights and novel approaches in diagnostics and therapeutics: The nonlinear behavior of oscillating bubbles has led to the development of contrast harmonic imaging [START_REF] Schrope | Second harmonic ultrasonic blood perfusion measurement[END_REF][START_REF] De Jong | Higher harmonics of vibrating gas-filled microspheres. Part one: simulations[END_REF][START_REF] De Jong | Contrast harmonic imaging[END_REF]Bouakaz et al., 2002a,b), whereas observations of bubble destruction have led to the development of high-MI imaging techniques [START_REF] Uhlendorf | Nonlinear acoustical response of coated microbubbles in diagnostic ultrasound[END_REF][START_REF] Frinking | A new ultrasound contrast imaging approach based on the combination of multiple imaging pulses and a separate release burst[END_REF].

Targeted UCA delivery is based on microbubbles with ligands attached to them [START_REF] Klibanov | Targeted delivery of gas-filled microspheres, contrast agents for ultrasound imaging[END_REF][START_REF] Unger | Thrombus-specific contrast agents for imaging and thrombolysis[END_REF]. These bubbles circulate through vessels and accumulate at a target tissue, thereby marking the target in ultrasound images. [START_REF] Klibanov | Targeted delivery of gas-filled microspheres, contrast agents for ultrasound imaging[END_REF] put forward that targeted microbubbles may be applied for selective delivery to the areas where selective enhancement of the action of ultrasound would be required, and if such a bubble would contain plasmid DNA, enzyme, or another therapeutic agent, it might be released at the site of interest during insonification. Another means of targeting was suggested by Dayton et al. (2001b). They provided optical verification that microbubbles oscillate differently when inside neutrophils in response to insonification, and emit acoustic signals that are distinct from free microbubbles. Thus, microbubbles within neutrophils might be detected, providing a potential method to image activated neutrophils as they accumulate at sites of inflammation.

Recently, [START_REF] Unger | Therapeutic applications of microbubbles[END_REF] gave an overview of the therapeutic applications of UCA microbubbles. They presented experimental results using UCAs for thrombolysis, drug delivery, and gene delivery. Other recent advances in local drug and gene delivery include the in vivo delivery of a virus vector, attached to albumin microbubbles [START_REF] Shohet | Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium[END_REF], the in vitro and in vivo transfection of endothelial cells with plasmid DNA mixed with UCA [START_REF] Taniyama | Local delivery of plasmid DNA into rat carotid artery using ultrasound[END_REF], and the accelerated cellular drug uptake in vitro when UCA microbubbles are present [START_REF] Van Wamel | Effects of diagnostic ultrasound parameters on molecular uptake and cell viability[END_REF]. [START_REF] Tachibana | Induction of cell-membrane porosity by ultrasound[END_REF] subjected cells in the presence of a photosensitive drug to continuous ultrasound, and noticed surface pores on the cells. Inducing porosities in cells with ultrasound, generally referred to as sonoporation, has potential applications in (tumor) cell lysis and selective delivery of drugs and genes into cells. Independently, it was demonstrated that moderate microbubble oscillations are sufficient to achieve rupture of lipid membranes, in a regime in which the bubble dynamics can be accurately controlled [START_REF] Marmottant | Controlled vesicle deformation and lysis by single oscillating bubbles[END_REF]. This might enable the transport of drugs through the cell membrane.

At high acoustic amplitudes, destructive effects of UCA microbubbles have been observed, such as hemorrhaging [START_REF] Skyba | Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue[END_REF][START_REF] Maruvada | Optical monitoring of ultrasound interaction with blood vessels in transparent fish after injection with contrast agents[END_REF][START_REF] Maruvada | Optical monitoring of ultrasound interaction with blood vessels in transparent fish after injection with contrast agents[END_REF]lysis (van Wamel et al., 2002). [START_REF] Kudo | Study on mechanism of cell damage caused by microbubbles exposed to ultrasound[END_REF] performed an in vitro study on bovine arterial cell damage by ultrasound in combination with microbubbles. They found that the locations of damaged cells were similar to the locations where violent bubble collapse was observed, suggesting that bubble collapse is responsible for cell membrane damage.

In this paper, we present an overview of dynamic encapsulated microbubble behavior observed at high acoustic amplitudes. The mechanisms for each type of behavior, found in literature on free (not encapsulated) gas bubbles, acoustics, and cavitation, are correlated to our high-speed optical observations. Furthermore, we address the potential clinical applications of the phenomena.

Previously, some UCA microbubble destruction phenomena were categorized by Chomas et al. (2001a) and Postema et al. (2002a). From our optical observations, we discriminate the following categories: oscillation, translation, coalescence, fragmentation, sonic cracking, and jetting.

In the following subsections, we give a brief overview of these phenomena, and an explanation of their occurrence. Most theories on bubble behavior were validated for big, free (not encapsulated) microbubbles.

Oscillation

When a microbubble is exposed to an oscillating acoustic signal, it undergoes alternate expansions and contractions with the same amplitude and duration at low driving pressures [START_REF] Strasberg | Gas bubbles as sources of sound in liquids[END_REF]. This regime of pulsation amplitudes has been referred to as moderate [START_REF] Vokurka | Comparison of Rayleigh's, Herring's, and Gilmore's models of gas bubbles[END_REF]. Bubble activity which may occur at relatively low-amplitude pressures has been denoted as stable cavitation [START_REF] Miller | Theoretical investigation of the response of gas-filled micropores and cavitation nuclei to ultrasound[END_REF]. As the driving pressure increases, more complex nonlinear interactions occur: greater bubble expansion amplitude than contraction amplitude, and relatively slow expansion followed by rapid contraction (collapse).

This behavior has been referred to as violent [START_REF] Vokurka | Comparison of Rayleigh's, Herring's, and Gilmore's models of gas bubbles[END_REF] or inertial (or transient) cavitation [START_REF] Miller | Theoretical investigation of the response of gas-filled micropores and cavitation nuclei to ultrasound[END_REF][START_REF] Flynn | Transient pulsations of small gas bubbles in water[END_REF][START_REF] Barnett | Nonthermal issues: cavitation -its nature, detection and measurement[END_REF]. It has been associated with the production of harmonic signals (Postema et al., 2003a).

The transition from the moderate to the violent regime is rather abrupt: For any driving pressure, there exists a transitional equilibrium microbubble radius R tr 0 , above which microbubbles pulsate like inertial cavitaties [START_REF] Flynn | Cavitation dynamics. II. Free pulsations and models for cavitation bubbles[END_REF][START_REF] Flynn | Transient pulsations of small gas bubbles in water[END_REF][START_REF] Hilgenfeldt | Analysis of Rayleigh-Plesset dynamics for sonoluminescing bubbles[END_REF]. This transition is referred to as the cavitation threshold. A bubble is judged to have grown into an inertial cavity when its maximum radius is greater than approximately twice its equilibrium radius [START_REF] Flynn | Cavitation dynamics. II. Free pulsations and models for cavitation bubbles[END_REF][START_REF] Flynn | Transient pulsations of small gas bubbles in water[END_REF].

A number of models for computing radius-time curves of insonified gas bubbles has been developed. These models differ both in complexity and in the range of acoustic amplitudes for which they can be used [START_REF] Vokurka | Comparison of Rayleigh's, Herring's, and Gilmore's models of gas bubbles[END_REF].

To model the behavior of UCA microbubbles, the presence of an encapsulation around the gas core has to be accounted for. Hence, we have to introduce parameters that describe the dynamic behavior of the shell. et al., 2000a;[START_REF] Morgan | Experimental and theoretical evaluation of microbubble behavior: Effect of transmitted phase and bubble size[END_REF]Postema et al., 2003a). Currently, models on UCA microbubble oscillating behavior at high driving pressures have been under

investigation [START_REF] Stride | On the destruction of microbubble ultrasound contrast agents[END_REF].

Translation

Translations of UCA microbubbles in the direction of the sound field and towards each other have been frequently observed with high-speed cameras [START_REF] Dayton | Action of microbubbles when insonified: experimental evidence[END_REF](Dayton et al., , 1997b[START_REF] Dayton | Acoustic radiation force in vivo: a mechanism to assist targeting of microbubbles[END_REF](Dayton et al., , 2001a;;Postema et al., 2003c). Microbubble translation in the direction of the sound field has been attributed to a primary radiation force resulting from a pressure gradient across the bubble surface. The translation is maximal in contraction phase [START_REF] Postema | Coupled dynamics of translation and collapse of acoustically driven microbubbles[END_REF]. It was demonstrated that the velocity v of a bubble in a steady fluid subjected to an ultrasound field can be expressed by the following ordinary differential equation [START_REF] Tortoli | On the interaction between ultrasound and contrast agents during Doppler investigations[END_REF]:

m dv dt = F rad + F d , (1)
where F rad is the primary radiation force, F d is the drag force, m ≈ 2π 3 ρR 3 0 is the mass of the translating bubble, equivalent to half the mass of the displaced surrounding fluid [START_REF] Leighton | The Acoustic Bubble[END_REF], in which R 0 is the ambient bubble radius, and ρ is the density of the surrounding fluid. The primary radiation force is given by [START_REF] Tortoli | On the interaction between ultrasound and contrast agents during Doppler investigations[END_REF]:

F rad = p 2 ac R 0 ρcf δ t f r f f r f 2 -1 2 + δ t f r f 2 , ( 2
)
where c is the speed of sound, p ac is the peak rarefactional acoustic pressure, δ t is the dimensionless total damping coefficient [START_REF] Medwin | Counting bubbles acoustically: a review[END_REF], and f r is the bubble resonant frequency [START_REF] Medwin | Counting bubbles acoustically: a review[END_REF]. The drag force is given by [START_REF] Tortoli | On the interaction between ultrasound and contrast agents during Doppler investigations[END_REF]:

F d = - πη 4 C d ReR 0 v(t), (3)
where η is the shear viscosity of the fluid, Re = [START_REF] Pain | The physics of vibrations and waves[END_REF][START_REF] Leighton | The Acoustic Bubble[END_REF][START_REF] Postema | Coupled dynamics of translation and collapse of acoustically driven microbubbles[END_REF], causing the bubbles to recede from each other. The mean approach velocity v a of two identical bubbles is given by Dayton et al. (1997b):

v a = - (2πf p ac ) 2 27η ρκ 2 R 5 0 d 2 0 , ( 4
)
where κ is the compressibility of the bubble, and d 0 is the initial distance between the centers of the two bubbles.

Coalescence

Ultrasound-induced microbubble coalescence is the fusion of two or more microbubbles. As adjacent bubbles expand, the pressure in the film between them increases, resulting in a deformation (flattening) of the bubble surfaces.

The continuing bubble expansion causes drainage of the interposed film. This thinning continues until a critical thickness below 0.1 µm is reached, at which the Van der Waals attractive forces results in film rupture and the coalescence of the bubbles [START_REF] Duineveld | Bouncing and coalescence of two bubbles in water[END_REF]. Thinning and rupture of thin liquid films have been described by [START_REF] Sheludko | Thin liquid films[END_REF], [START_REF] Kralchevsky | Thin liquid film physics[END_REF], and [START_REF] Narsimhan | Structure, drainage, and coalescence of foams and concentrated emulsions[END_REF].

Flattening of the adjacent bubble surfaces occurs if and only if the bubble system has a Weber number We > 0.5 [START_REF] Chesters | Bubble coalescence in pure liquids[END_REF].

The Weber number for a fluid containing two bubbles with radii R 1 and R 2 , respectively, is given by the inertial force relative to the surface tension force:

We = ρu 2 σ Rm , ( 5
)
where u is the relative approach velocity of the bubble walls: u ≈ Ṙ1 + Ṙ2 , ρ is the fluid density, σ is the surface tension, and R m is the mean bubble radius for which holds:

2 Rm = 1 R 1 + 1 R 2 .
In our results, Weber numbers are relatively high because of the rapid bubble expansions, with maximal radius increases of several m s -1 . If the Weber number is lower than 0.5, bubble coalescence will always occur, without flattening of the adjacent surfaces prior to contact [START_REF] Chesters | Bubble coalescence in pure liquids[END_REF].

In a separate paper, we investigated the coalescence mechanism of microbubbles, based on high-speed optical observations of insonified UCA (Postema et al., 2003c). We investigated the mechanism of film drainage by comparing the observed coalescence times with calculated film drainage times from the Reynolds equation ( 6). The Reynolds drainage time τ d of the interposed film until a critical thickness h c is given by:

τ d = 3ηR 2 f R m 8σh 2 c , (6)
where R f is the film radius, and R m is the mean bubble radius.

The calculated drainage times prove too long to be compatible with the observations. Stated differently, if the film between bubbles thinned by Reynolds drainage alone during the observed time span, it would only reach a minimum thickness h m that is much larger than that needed to induce film rupture. Hence, a mechanism for film rupture must be at work that overrules the drainage. We suggest that shape instabilities of the bubble can cause local corrugations that bridge the film and rupture it, before thinning of the whole film. We show that small perturbations on the bubble surface may grow tremendously, a phenomenon known as parametric instability. The initial perturbations may come from thermal undulations and external radiation forces. We show that the latter are dominant, and lead to sufficiently large undulations to bridge the minimum thickness h m within half an ultrasonic cycle.

Bounce is the process where bubbles approach and flatten, but do not coalesce (Postema et al., 2003c). This behavior has been attributed to the drainage given in eq. ( 6) taking more time than the expansion phase.

Fragmentation

Fragmentation is the fission of a bubble into smaller bubbles (Postema et al., 2002a). Although acoustical observations have been associated with microbubble destruction [START_REF] Shi | Destruction of contrast microbubbles and the association with inertial cavitation[END_REF], fragmentation of UCA microbubbles was first visualized with high-speed cameras in 2001 (Chomas et al., 2001a;[START_REF] Postema | Real-time optical imaging of individual microbubbles in an ultrasonic field[END_REF]. In the same year, [START_REF] Brennen | Fission of collapsing cavitation bubbles[END_REF] computed the number of fragments for a cavitation bubble distorted by spherical harmonics. He demonstrated that for any positive Γ = ρR 2 R σ , where R denotes the second time derivative of R, there must be a mode n for which the spherical harmonic distortion has a maximum.

This mode depends on Chomas et al. (2001b) showed that fragmentation occurs around peak contraction, when the bubble collapse is driven by inertial forces, since the inward acceleration continues to increase as the bubble approaches its minimum radius and suddenly changes sign as the bubble begins a rebound. They suggest that if the energy that is transferred during collapse is not sufficiently dissipated during one ultrasonic cycle, the bubble will become unstable and fragment.

Γ as n = 1 3 √ 7 + 3Γ -2 3 . The number of fragments N is estimated N ≈ n 3 .
Longuet-Higgins (1992) related the intrinsic energy of a bubble fragment population with a size distribution P (R) to the initial intrinsic energy of the mother-bubble with equilibrium radius R 0 :

E * = ∞ 0 R R 0 2 P (R)dR, (7)
where E * is the intrinsic energy ratio, and R is the radius of a fragment. For bubble fragments of equal sizes,

E * = R 0 R = 3 √ N .
Longuet-Higgins (1992) also presented an elegant method to compute the size distribution of bubbles generated by "shattering a single air cavity".

Sonic cracking

Sonic cracking is the ultrasound-induced formation of a shell defect causing gas to escape from UCA microbubbles. It has been observed with rigid-shelled UCA microbubbles (Dayton et al., 1997a;Takeuchi, 1999;Postema et al., 2002a). The physical mechanism behind sonic cracking is yet not known. Tiny flaws in the shells may account for the fact that certain bubbles crack while others stay intact (Postema et al., 2002a).

Jetting

When a bubble rapidly contracts (collapses) near a boundary, this collapse will be asymmetrical. A high-speed liquid jet may form which projects through the bubble towards the boundary [START_REF] Barnett | Nonthermal issues: cavitation -its nature, detection and measurement[END_REF], pulling a slight volume of the bubble content along. This remarkable phenomenon is called jetting.

The jetting phenomenon for cavitation bubbles was described by [START_REF] Philipp | Cavitation erosion by single laser-produced bubbles[END_REF] and depicted by Ory (cf. Figure 1): The asymmetric collapse causes the velocity of the upper bubble wall to exceed the velocity of the lower wall in order to conserve the impulse of the bubble/fluid system. Consequently, the fluid volume above the bubble is accelerated and focused during collapse, leading to the formation of a liquid jet directed towards the boundary. This jet hits the lower bubble wall, causing a funnel-shaped protrusion and finally impacts on the boundary.

Jets have regularly been observed with cavitation bubbles (Lauterborn, 1976;[START_REF] Philipp | Cavitation erosion by single laser-produced bubbles[END_REF]). An illustrative enlargement of a high-speed photograph of a cavitation jet in the millimeter range was published by [START_REF] Lauterborn | Cavitation bubble dynamics[END_REF]. [START_REF] Ohl | Aspherical bubble collapse -comparison with simulations[END_REF] calculated the shapes of asymmetrically collapsing bubbles. Their results are consistent with photographs of a collapsing cavitation bubble developing a jet. [START_REF] Kodama | Dynamic behavior of bubbles during extracorporeal shock-wave lithotripsy[END_REF] studied the impact of jets on gelatin surfaces. They found that the radius of the jet R j is approximately related to the radius of the bubble on the verge of collapsing R c as R j Rc ≈ 0.1 [START_REF] Kodama | Dynamic behavior of bubbles during extracorporeal shock-wave lithotripsy[END_REF][START_REF] Ohl | Shock-wave-induced jetting of micron-size bubbles[END_REF]. The length of the jet l j , defined as the full travel path of the propelled liquid, is approximately related to R c as

l j
Rc ≈ 3 [START_REF] Ohl | Shock-wave-induced jetting of micron-size bubbles[END_REF]. From these two ratios the amount of liquid within the jet, V j , is estimated V j ≈ 0.1R 3 c [START_REF] Ohl | Shock-wave-induced jetting of micron-size bubbles[END_REF].

The impact of a jet on a surface generates a high-pressure region. The pressure in this region was dubbed water-hammer pressure by [START_REF] Cook | Erosion by water-hammer[END_REF]. For a perfectly plastic impact, the water-hammer pressure of a cavitation jet was estimated by de [START_REF] De Haller | Untersuchungen über die durch Kavitation hergerufenen Korrosionen[END_REF] and [START_REF] Ohl | Aspherical bubble collapse -comparison with simulations[END_REF]:

p wh ≈ 1 2 ρcv j , (8)
where p wh is the water-hammer pressure, v j is the jet velocity, ρ is the fluid density, and c is the speed of sound.

The damage caused by a jet can be estimated by comparing the water-hammer pressure to the maximum stress the impact surface can withstand before rupture.

The maximum stress τ max is given by:

τ max = Eε r , ( 9
)
where E is the elastic (Young's) modulus of the material, and ε r is the relative deformation of the surface before rupture.

Clinical applications

UCA microbubble oscillating behavior is the basis of most diagnostic imaging techniques [START_REF] Becher | Handbook of Contrast Echocardiography: LV Function and Myocardial Perfusion[END_REF]Goldberg et al., 2001). Since oscillating microbubbles provide a means to discriminate blood from tissue, they are applicable in perfusion and flow imaging, and tumor detection [START_REF] Burns | Intravenous contrast agent for ultrasound Doppler: In vivo measurement of small tumor vessel dose-response[END_REF][START_REF] Wei | Quantification of myocardial blood flow with ultrasound-induced destruction of microbubbles administered as a constant venous infusion[END_REF][START_REF] Wei | Blood flow and perfusion measurements: background and clinical utility[END_REF].

Medical applications of the manipulation of the movement of UCA microbubbles by means of acoustic energy will be primarily in directing UCA toward cells (Dayton et al., 2001a).

If small UCA microbubbles, having passed through the narrowest vessels, coalesce, they may be controlled to obtain resonant sizes. Especially for subharmonic imaging [START_REF] Shankar | Subharmonic backscattering from ultrasound contrast agents[END_REF], where twice the resonant bubble size is needed, and for tracking the diffusion of free gas bubbles with subharmonics, a promising technique in noninvasive blood pressure measurements (Postema et al., 2003b), controlled microbubble coalescence may be applicable. If coalescence of a lipid-shelled microbubble and a cell membrane can be induced, this will imply a promising technique in targeted drug delivery.

Bouncing behavior of UCA microbubbles has got no potential medical application yet.

UCA microbubble fragmentation has been associated with violent effects, such as cell membrane permeabilization and lysis. The reason for this may lie in the fact that fragmenting bubbles produce shock-waves. Because of the strong acoustic signal generated, fragmentation finds applications in high-MI imaging.

The applicability of microbubble fragmentation for local drug delivery is fully dependent of the presence of a shell after fragmentation. If the shell is absent after fragmentation, the fragments will behave as released gas bubbles. Thus, the potential therapeutic applications will then be identical to those for sonic cracking.

Sonic cracking may find applications in drug delivery, if the released bubble content has therapeutic properties (Frinking et al., 1998). [START_REF] Frinking | A new ultrasound contrast imaging approach based on the combination of multiple imaging pulses and a separate release burst[END_REF] suggested a technique they called release burst imaging: Upon transmission of a high-MI ultrasound burst, there will be a strong scattering response from the released gas bubbles. The free gas will rapidly dissolve, and with it the strong scattering response will fade away. [START_REF] Wei | Quantification of myocardial blood flow with ultrasound-induced destruction of microbubbles administered as a constant venous infusion[END_REF] proposed that the acoustical measurement of the reappearance of UCA microbubbles may provide a means for determining tissue perfusion in any organ accessible to ultrasound.

The diffusion rate of a gas bubble with a certain size is known for every hydrostatic pressure. By making use of the changing acoustic properties of the dissolving gas bubbles, hydrostatic pressures may be determined in a minimally invasive clinical setting [START_REF] Bouakaz | Noninvasive pressure measurement using microbubble contrast agent and wavelet transforms[END_REF]Postema et al., 2003b).

It has been speculated that jets may be formed when UCA microbubbles in the bloodstream are insonified with high enough acoustic pressures. These jets would be directed towards a boundary like a cell or a capillary wall with adjacent cells.

As such, jets might function as microsyringes [START_REF] Miller | Gene transfection and drug delivery[END_REF][START_REF] Ohl | Shock-wave-induced jetting of micron-size bubbles[END_REF], if they could penetrate cells. The maximum extension measured in human cells is less than 50% (ε r < 0.5) [START_REF] Lendon | Atherosclerotic plaque caps are locally weakened when macrophages density is increased[END_REF]. Therefore, we may assume that for any human cell or cell part, ε r 1. Elastic moduli of human cells were measured E 7.3 kPa for endothelial cells [START_REF] Mathur | Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells[END_REF], and E 12 kPa for fibroblasts [START_REF] Rotsch | Dimensional and mechanical dynamics of active and stable edges in motile fibroblasts investigated by using atomic force microscopy[END_REF]: less than half the elastic modulus of murine skeletal muscle cells (E ≈ 24 kPa) [START_REF] Mathur | Endothelial, cardiac muscle and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy[END_REF]. Rabbit cardiac cells are among the stiffest, with E ≈ 100 kPa [START_REF] Mathur | Endothelial, cardiac muscle and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy[END_REF]. It is safe to assume that even for the stiffest human cell E 200 kPa. Combining both assumptions in eq. ( 9) gives τ max 200 kPa. If the water-hammer pressure of a jet exceeds this stress, the jet will be able to penetrate any human cell.

The porosities observed on cell-membranes by [START_REF] Tachibana | Induction of cell-membrane porosity by ultrasound[END_REF] may well be attributed to jets. Probably, particles that were present in the photosensitive drug acted as cavitation nuclei, whereas the cell-membranes acted as the boundaries towards which the jets were directed.

Experimental setup Overview

For the high-speed observations, we made use of the Brandaris-128 system [START_REF] Chin | Brandaris 128: a 25 million frames per second digital camera with 128 highly sensitive frames[END_REF] and an Imacon 468 fast framing camera. An overview of this experimental setup is shown in Figure 2. A computer controlled the triggering of a waveform generator, a Xenon flash source, and the cameras. was not expected to interfere with the ultrasound transmitted. Without contrast agent inserted, we did not observe reflections from the tube.

Optics

Underneath the capillary tube an optic fiber was mounted. This fiber was connected to an MVS-7010 Fiber Optic Strobe (PerkinElmer Optoelectronics, Salem, MA), and to a KLS-201 continuous fiber light source (Olympus KMI (KeyMed Ltd), Southend-on-Sea, UK). The pulsed light source was triggered by a PM 5716 pulse/delay generator (Koninklijke Philips Electronics N.V., Eindhoven,

The Netherlands). The container was positioned beneath a customized BXFM microscope system (Olympus Optical Co., Ltd., Tokyo, Japan) with a U-CA magnification changer (Olympus Optical Co., Ltd.), switched to 2× magnification and a LUMPlanFl 60× water immersion objective lens (Olympus Optical Co., Ltd.). For the measurements with the Imacon 468 camera, we made use of a BH-2 model (Olympus Optical Co., Ltd.) with an SPlan 100 oil immersion objective lens (Olympus Optical Co., Ltd.). For image control purposes, an LCL-902K or a WAT-902HS CCD camera (Watec Co., Ltd., Yamagata, Japan) was fitted to the microscope. Focusing was done manually at the middle of the cellulose tube.

Because the tube was wide compared to the contrast microbubbles measured, its upper half was considered a flat surface between contrast microbubbles and object lens, not causing aberrations but lowering the dynamic range of the images.

Camera

The optical observations were recorded with a Brandaris-128 fast framing camera

Contrast agents

We investigated the ultrasound contrast agent Quantison TM (Upperton Limited). It consists of human serum albumin-encapsulated air bubbles with a mean diameter of 3.2 µm. Shell thicknesses are between 0.2 and 0.3 µm (Frinking and de Jong, 1998). The content of a Quantison TM vial was resuspended in 5 ml of Isoton R II (Beckman Coulter, Inc., Fullerton, CA), and shaken gently for 20 seconds before further dilution. The agent was inserted through the capillary tube using a syringe pressed by hand.

We also investigated an experimental UCA (supplied by Bracco Research SA, Geneva, Switzerland). It consists of phospholipid-encapsulated gas bubbles ranging in diameter from 1 to 6 µm with a median of 2 µm. The acoustic behavior of a very similar contrast agent was modeled and described in [START_REF] Gorce | Influence of bubble size distribution on the echogenicity of ultrasound contrast agents: A study of SonoVue TM[END_REF].

Undiluted UCA (5ml of a 0.9% sodium chloride dilution, added to a 25 mg UCA vial) was inserted through the capillary tube using either a syringe pressed by hand or a hose operated by a gravity fed or pumped infusion.

Ultrasound

For the experiments with the Brandaris-128 camera system, the UCA bubbles were insonified by 8 cycles of 1.7 MHz ultrasound at a peak rarefactional acoustic pressure of 2 MPa, corresponding to a mechanical index of MI = 1.5. For the experiments with the Imacon 468 camera, the UCA bubbles were insonified by 10 cycles of 500 kHz ultrasound at peak rarefactional acoustic pressures between 0.66 and 0.85 MPa, corresponding to mechanical indices in the range 0.9 < MI < 1.2. The mechanical index is defined as MI = p ac / √ f , where p ac is the peak rarefactional acoustic pressure in MPa and f is the center frequency of the ultrasound in MHz.

Acoustic pressures applied were measured with a calibrated MH28-10 hydrophone (FORCE Technology, Brøndby, Denmark) in a separate water tank.

An example of the acoustic signal measured at the transducer focus has been published in Postema et al. (2003a). The in-situ reverberant component due to the presence of the microscopic lens was observed to be less than -14 dB. Thus, it is assumed the in-situ acoustic signal is the same as the signal measured.

Procedure

We recorded 277 image sequences with the Brandaris-128 system. We performed 527 experiments at high acoustic amplitudes with the Imacond-468 camera.

Bubble sizes and distances were measured manually or by using a segmentation method described by Postema et al. (2003a).

Results and discussion

The following types of ultrasound-induced UCA microbubble behavior have been distinctly observed and categorized: oscillation, translation, coalescence, fragmentation, sonic cracking, and jetting. Inter-frame times for frames b to h are 0.33 µs. Each frame corresponds to a 88 × 58 µm 2 area. Frame a has been taken prior to ultrasound arrival. Maximal sizes are reached in frame d with bubble diameters of 7.6 µm, whereas minimal sizes are displayed in frame f with bubble diameters of 4.4 µm. Thus, the expansion and contraction of the bubbles are symmetric. We do not expect a violent collapse in this regime, since the maximum bubble radius is less than twice its equilibrium radius. The oscillating amplitude is represented in the left frame of Figure 6(iii). The solid line represents the radius-period curve of a free gas bubble [START_REF] De Jong | Higher harmonics of vibrating gas-filled microspheres. Part one: simulations[END_REF]. Apparently, the UCA bubbles have comparable excursions to a free gas bubble.

Oscillation

Figure 6(ii) and the right frame of Figure 6(iii) show a similar situation for a 1.5 µm strongly oscillating microbubble insonified at MI = 0.67. Again, the UCA bubble excursion is comparable to a free gas bubble. At the acoustic amplitude applied there may be some energy loss in the shell, causing increased thermal damping. Maximum size is reached in frame e with a bubble diameter of 10.0 µm, whereas minimum size is displayed in frame g with a bubble diameter of 0.57 µm. The bubble oscillates highly asymmetric. We expect the microbubble to grow into an inertial cavity in this regime, since the maximum bubble radius is much greater than its equilibrium radius.

Translation

Figure 7 shows a high-speed optical image sequence of Quantison TM insonified by 8 cycles of 1.7 MHz ultrasound with a mechanical index MI = 1.5. The ultrasound travels from the lower side of the frames to the upper side. Frame times are indicated in ns. Each frame corresponds to a 30 × 16 µm 2 area. In Figure 7(b,c,d) gas escapes from a 4 µm bubble. This free gas bubble expands and contracts, and translates towards the upper side of the frames. The displacement of the free gas bubble center has been measured 14 µm over 4 periods (frames e-h).

The resting diameter of the released bubble after insonification was 1.3 µm. The theoretical mean bubble displacement over time has been computed by integrating eq. ( 1) over 4 periods, taking c = 1480 m s -1 , ρ = 998 kg m -3 , R 0 = 0.65 µm, f = 1.7 MHz, f r = 5.3 MHz, δ t = 1.0, p ac (t) = [2 MPa] sin 2πf t, and η = 0.001 Pa s. The damping was computed using the parameters stated in [START_REF] Medwin | Counting bubbles acoustically: a review[END_REF] and de [START_REF] De Jong | Acoustic properties of ultrasound contrast agents[END_REF]. The theoretical displacement is 18 µm.

The measured displacement is lower than the theoretical value. At the acoustic pressure applied, the expansion phase is longer than the contraction phase, which may account for this difference.

Figure 8 shows 5 image frames of two 4 µm experimental UCA microbubbles, each captured after insonification by 10 cycles of 0.5 MHz ultrasound (duration T p = 20 µs). Each image frame corresponds to a 30×20 µm 2 area. During every ultrasound burst the bubbles draw nearer to each other. For each center-to-center distance d 0 measured the mean approach velocity v a has been computed from eq. ( 4), taking κ = 5×10 -6 m 2 N -1 . By combining d 0 with v a , the theoretical distances ∆d th = v a T p have been computed. These were compared to the distance ∆d m measured from Figure 8. The results are summarized in Table 1. The measured values ∆d are consistent with theory. The flattening of the adjacent bubble surfaces in Figure 9(i)b) is supported by a conservative estimate of the Weber number. Based on our analysis of the optical system (Postema et al., 2003a), we may assume that the optical resolution R 0.5 µm, and fragment diameters in Figure 9(i)b) < 0.5 µm. Even if the bubbles would expand linearly to the sizes observed in Figure 9(i)c, the approach velocity would still have to be u = 2 ∆Rm ∆t > 2.7 µm-0.5 µm 0.33 µs = 6.7 ms -1 . By taking ρ = 998 kg m 3 and σ = 0.072 N m -1 , we obtain a Weber number We > 0.84. As stated before, flattening of the adjacent bubble surfaces will occur if We > 0.5.

Coalescence

From eq. ( 6), it is expected that the drainage time of the film shown in Figure 9(i)c until a critical thickness h c = 0.1 µm is t d = 0.6 µm, taking η = 0.001 Pa s and R f ≈ 2 3 R m (Postema et al., 2003c). However, Figure 9(i)d

shows not only film drainage, but also continuing bubble expansion. For these bubble diameters of 4.6 µm, the interposed film would take 2.8 µs to drain. Still, 0.66 µs later the bubbles have coalesced, although the bubbles have continued to expand. This supports the theory that a mechanism for film rupture must be at work that overrules the drainage. fragments coalesce and form an ellipsoidal bubble in Figure 9(ii)e. Because the ellipsoidal shape can be discriminated, we may conclude that the second spherical harmonic mode is dominant. After collapse, the fragments in Figure 9(ii)f are too small to be counted, but in Figure 9(ii)g, captured while the fragments have begun to expand, 8 fragments can be discerned, which confirms the second mode instability.

Fragmentation

Sonic cracking

Figure 9(iii) shows the sonic cracking of a 3 µm Quantison TM microbubble.

Each frame corresponds to a 23 × 23 µm 2 area. In Figure 9(iii)d gas starts to escape from the bubble in the middle of the frame. It expands to approximately 8 µm in Figure 9(iii)g, and then begins to contract in Figure 9(iii)h.

Clearly, the gas bubble has been released from its rigid shell. However, in the image sequence, the pixel size (0.15 × 0.15 µm 2 ) corresponds in approximation to the shell thickness. Thus, even if the optical resolution were of the order of the shortest wavelength of the light, the presence of tiny flaws in the rigid shell would not have been detectable.

Jetting

We recorded two optical sequences showing jet development in UCA microbubbles (Postema et al., 2002b), one of which is shown in Figure 10. Rc is in agreement with the ratio put forward by [START_REF] Ohl | Shock-wave-induced jetting of micron-size bubbles[END_REF]. The volume of the jet is approximately V j ≈ 0.1R 3 c = 60 femtoliter.

Taking ρ = 998 kg m -3 and c = 1480 m s -2 , the water-hammer pressure is around 60 MPa. Since p wh 200 kPa τ max , such a jet may penetrate any cell.

Influence of the capillary tube [START_REF] Ishida | Cavitation behavior near solid boundaries[END_REF] demonstrated with simulations and high-speed optical observations, that if the distance between the walls of their solid test vessel was greater than 5 times the maximum free-field expansion radius of a cavitation bubble, the bubble would remain spherical while expanding. In our setup, the diameter of the water-soaked capillary tube is approximately 20 times the largest microbubble radius measured. Thus, the phenomena described other than jetting cannot be attributed to the boundaries imposed by the capillary tube, since our imaging plane is at the center of the tube.

Conclusions

An overview of dynamic behavior of ultrasound insonified encapsulated microbubbles has been presented. The following types of behavior have been observed and categorized: oscillation, translation, coalescence & bounce, fragmentation, sonic cracking, and jetting.

Oscillation is the expanding and contracting bubble response to an oscillating acoustic signal. At low acoustic amplitudes bubbles pulsate moderately, at high amplitudes their longer expansion phase is followed by a violent collapse.

Microbubble translation has been associated with primary and secondary radiation forces. Our optical observations of translating bubbles are consistent with theory.

Coalescence, the fusion of two or more bubbles, is mainly caused by the drainage of the liquid film separating expanding bubbles, whereas bounce -unsuccessful coalescence -is caused by the drainage taking longer than the expansion of the bubbles. Fragmentation is the fission of a bubble into smaller bubbles.

The number of fragments has been related to the dominant spherical harmonical oscillation mode of a bubble. In our observations, the second spherical harmonical mode appears to be the dominant mode. Remarkable are our observations of jetting through encapsulated microbubbles. For an observed jet, we computed a volume of approximately 60 femtoliter, generating a pressure at the tip of the jet around 60 MPa. This is high enough to penetrate any human cell. Hence, liquid jets may act as microsyringes, delivering a drug to a region of interest. (a-d) Sonic cracking of a 4 µm Quantison TM bubble (MI = 1.5).

(e-i) The released gas is subjected to a primary radiation force. (ii) A 1.5 µm bubble strongly oscillating (MI = 0.67).

(iii) Radius-period plots of both events. The solid line represents an oscillating free gas bubble. Each image frame corresponds to a 23 × 23 µm 2 area.

  The electrical signal was generated by an AWG 520 arbitrary waveform generator(Tektronix, Inc., Beaverton, OR), or by an LW 420A arbitrary waveform generator (LeCroy Corp., Chestnut Ridge, NY). The signal was adjusted by two variable 355C/D attenuators (Hewlett Packard Company, Palo Alto, CA) in series, and an A-500 60 dB linear power amplifier (ENI technology, Inc., Rochester, NY). It was converted to ultrasound by a V389-SU 500 kHz, or by a V397-SU 2.25 MHz single-element transducer (Panametrics Inc., Waltham, MA), both spherically focused at 7.5 cm. The transducers were mounted in a Perspex container at an angle of 45 • relative to the top of the container. This container was filled with saturated water. A 200 µm cellulose Cuprophan R capillary tube (Membrana GmbH, Wuppertal, Germany) was fixed in the focal area of the transducer, through which contrast agent was flowing. Because the capillary tube was watersoaked and much smaller in diameter than the acoustic wavelenght, it

system

  [START_REF] Chin | Brandaris 128: a 25 million frames per second digital camera with 128 highly sensitive frames[END_REF]. The Brandaris-128 captured sequences of 128 image frames at an average speed of 13 million frames per second. Typical frame sizes correspond to 89 × 68 µm 2 . In all observations, image frames were captured before, during, and after ultrasound insonification. A line drawing of the optical observation part of the setup is shown in Figure3and 4. Furthermore, we made use of an Imacon 468 fast framing camera (DRS Hadland, Ltd., Tring, UK), capturing eight two-dimensional frames at 3 MHz. In all observations, the first frame was taken a few microseconds before ultrasound waves reached the contrast agent. The other seven frames were taken during ultrasound insonification, with 330 ns interframe time for 500 kHz ultrasound, spanning a full ultrasound cycle. Frame exposure times ranged from 10 ns to 70 ns. A line drawing of the optical observation part of this setup is shown in Figure 5.

Figure 6

 6 Figure 6(i) shows an optical image sequence of two identical 6 µm moderately

Figure 9

 9 Figure 9(i) shows an example of microbubble coalescence. The first frame has

Figure 9 (

 9 Figure 9(ii) shows the repeated fragmentation, coalescence, and re-fragmentation

Figure 10 (

 10 Figure 10(a) shows a microbubble with a radius R c = 8.43 µm. Figure 10(b)

Figure 1 :

 1 Figure 1: Schematic evolution of jet development in a collapsing bubble near a boundary (below): bubble on the verge of collapse (a), asymmetric collapse (b), jet development (c), funnel-shaped protrusion (d).

Figure 2 :

 2 Figure 2: Basic setup of the high-speed photography system.

Figure 3 :

 3 Figure 3: Line drawing of the Brandaris-128 system.

Figure 4 :Figure 5 :Figure 6 :

 456 Figure 4: Line drawing of the optical observation part of the Brandaris-128 system.

Figure 7 :Figure 8 :

 78 Figure 7: (a-d) Sonic cracking of a 4 µm Quantison TM bubble (MI = 1.5).(e-i) The released gas is subjected to a primary radiation force. Each image frame corresponds to a 30 × 16 µm 2 area. Frames shown were selected from a sequence of 128 frames. The travel distance of the bubble center between (e) and (h) is 14 µm over 4 ultrasonic cycles.

Figure 9

 9 Figure 9: Experimental UCA microbubbles showing (i) coalescence, (ii) second mode spherical harmonical fragmentation, (iii) sonic cracking. Frames a have been captured prior to ultrasound arrival. Inter-frame times for frames b to h are 0.33 µs.Each image frame corresponds to a 23 × 23 µm 2

Table 2

 2

	gives an overview of potential clinical applications related to the
	encapsulated microbubble phenomena discussed. The phenomena have potential
	clinical applications in imaging, pressure measurements, tumor detection,
	permeabilization, lysis, targeting, and drug delivery.

Table 2 :

 2 Potential clinical applications for encapsulated microbubble phenomena observed.

	oscillation	translation	coalescence	fragmentation	sonic cracking	jetting
	imaging
	pressure measurements			?
	tumor detection
	permeabilization and lysis
	targeting
	drug delivery			?

Acknowledgments

This work has been supported by the Technology Foundation STW (RKG.5104).

We thank Kees Slager for the data on cell elasticity, Emmanuel Ory (Laboratory of Applied Thermodynamics, Helsinki University of Technology) for computing Figure 1, Leo Bekkering and Elmer Koene for technical assistance, and Cees Pakvis for drawing Figures 3, 4, and 5.

List of Figures

Each image frame corresponds to a 38 × 30 µm 2 area. Inter-frame time is 0.33 µs and exposure time is 10 ns.


Doron Swade
email: doron.swade@blueyonder.co.uk

Preserving the legacy of IT innovation

Keywords: Collecting IT, Museums and IT, Software preservation, IT Innovation legacy, Computer preservation, History of Computing

IT challenges museological conventions. There are issues of physical scale at both extremes -large mainframes on one hand and microscopic devices on the other. The social utility of collections is widely measured by public exhibition. But 'Black-box' syndrome is a nightmare for exhibition designers and content developers: in the case of computer cabinets and integrated circuits there is no transparency of function to speak of and this stresses established techniques of display and interpretation. Software poses a near-intractable challenge for traditional object-centred museums and it is unclear upon whom, and on what institution, custodial responsibility falls. The impermanence of storage media is incompatible with archaeological timescales. Whether software has meaning without the capability of running it is an open question. Yet running legacy software has formidable resource implications: the need for working historic machines or their physical surrogates, and/or expertise to migrate applications to contemporary platforms. We look to museums to preserve for posterity the material culture of these transformative technologies. There are formidable challenges. How are we to meet them?

Introduction

Information technology represents a substantial human endeavour. The intellectual, economic and material resources involved in production, distribution and use represent major social, cultural and technological movements. There is a prevailing sense that, given the scale and transformative influence of these technologies, their social and technical histories should be preserved.

The mandate of museums is to preserve a material record of change. So it is to museums and museum-like institutions that we turn as repositories and custodians of the technological narratives of our times. What privileges physical objects in preservational practice is the 18th-century concept of 'permanence of substance' -that objects are durable and unchanging and are therefore appropriate evidentiary sources for the preservation, in perpetuity, of meanings [START_REF] Swade | Virtual Objects: Threat or Salvation?[END_REF]. In the case of IT, it is to museums of science and industry, and to specialised computing museums, that we look to preserve a permanent record of technological change.

Material Culture

IT hardware artefacts fall within traditional collecting criteria, and there are well-established protocols and practices for their acquisition and preservation. In the case of computers, historical significance is framed in different ways. We collect 'world firsts', technological breakthroughs and major innovations -Fleming's diode, the first transistor, the first sub-micron integrated circuit. We collect physical examples that are typical of their kind or representative of widespread practice -PCs, smartphones, electronic calculators -mass produced artefacts where what is distinctive about them is, paradoxically, their uniformity. We collect objects with special provenance -an original lens from Colossus's optical tape reader, a console clock from a Ferranti Pegasus (a signature feature of several though not all Ferranti machines), the distinctive red logo from an Elliot 803, Alan Turing's pen -artefacts that have actual or mythical associations with legendary inventors, designers, makers or systems. We collect failures, real and virtual, that stumbled for whatever reason before viable realisation or, if realised, that failed commercially or simply were not favoured by the roulette of success (Apple Newton). We collect objects at the extremes of physical scale, performance and history -substantial chunks of mainframes; supercomputers (Cray-1, the Russian BESM-6), electromechanical race-track Totalisators [START_REF] Swade | Forgotten Machines: the need for a new master narrative[END_REF][START_REF] Rooney | Mathematics: how it shaped our world[END_REF]; and the Harwell Dekatron aka WITCH that dates from 1951 [START_REF] Dekatron | The National Museum of Computing[END_REF], respectively. We collect objects as cultural artefacts without any necessary technical distinction -the National Savings ERNIEs which generate Premium Bond winning numbers [START_REF] Rooney | Mathematics: how it shaped our world[END_REF][START_REF] Swade | The three ages of windfall fantasy[END_REF], acoustic hoods for daisy wheel printers, product packaging, and ephemera -trade literature, an Apple lapel badge [figure 1]. Curators, archivists and museum store managers are said to have two recurring nightmares. The first is that nothing will survive. The second is that everything will survive.

While the mandate to collect items of IT hardware is relatively unproblematic, in practice IT hardware presents dilemmas for curators and collections managers. Mainframes from the 'Big Iron' era are impractically large to collect in their entirety and there are few complete systems to be found in protective custody that include duplicated peripherals, console furniture and a full set of processor cabinets. It remains unclear whether there is a complete IBM System/360 to be found in a museum collection. Computing is not alone in this. The last remaining hot-metal printing press used in Fleet Street, the Woods press dating from the 1930s, measures nine metres tall, nine-and-a-half metres long and weighs 140-tonnes. The Press was acquired as part of the Science Museum's printing collection and is stored in an aircraft hangar outside London [6]. Locomotives and aircraft pose similar problems of physical scale. Faced with the improbable size of first-generation computing installations, and later, mainframes, curators resorted to what might be called 'artefactual synecdoche' -acquiring parts to stand for the whole. The struggle for increased speed and power was a perennial priority especially in the pre-chip era. Given the mismatch between physical scale and available storage space, curators and collectors chose the central processor or processor cabinets as placeholders for what was seen to be the essential significance of the whole, resonating perhaps with the idea of preserving only a brain as the pre-eminent organ of the human body. The same was largely true of memory and the wish to reflect the challenges of storage capacity. Magnificent examples of magnetic core memory, with the visually arresting layered arrays of minute ferrite beads, survive, torn, as it were, from their operational environments in a process we might call 'chainsaw acquisition' where only the severed part survives. [figure 2] The downside of reduction by selection was to lose an artefactual record of the operational environment and physical context -office furniture, for example, and enough kit, not always visually distinguished and often many times duplicated, to convey the physical scale of the original installation. In the case of Olivetti's computers, reduction by selection loses one of the signature features of Olivetti machines -cabinet design, floor layout, colour and overall appearance. To collect anything less than the machine in its entirety loses an historic convergence of design, technology, ergonomics and human machine interaction -an expression of Adriano Olivetti's personal philosophy about industry and society and the company culture of elegance and ergonomics in product design [START_REF] Mori | The Italian Computer: Olivetti's ELEA 9003 Was a Study in Elegant Ergonomic Design[END_REF]. A legacy of the prohibitive size of early systems is that none of the great first-generation machines survives complete, and some are barely commemorated through even partial relics -ENIAC, EDVAC, EDSAC, UNIVAC, SAGE, Whirlwind, and the Manchester 'Baby' (SSEM), LEO.

Another selective reduction was to acquire control consoles which straddled technological and cultural meanings. SAGE, a radar command and control air defence system, built in the US, in response to the threat of Soviet nuclear attack during the Cold War is an example. In cultural terms the sober grimness of its consoles, a small fraction of this vast system, conveys vigilance, the seriousness of the threat it was intended to neutralize, and the high stakes of Cold War tensions [figure 3]. Increased miniaturisation came to the rescue of despairing museum store managers. Starting with the mini-computer era of the 1960s, when complete machines were modestly sized and could be manageably stored, the problem of prohibitive space requirements for 'Big iron' acquisitions was for the most part self-resolving. For the first three generations of computers, machines and systems were defined localised entities that happily conformed to traditional categories of artefact. With the challenges of distributed processing, embedded devices and especially networks, the earlier era of collecting seems, in comparison, to be blithely innocent.

Exhibition and Interpretation

Maintaining a material of technological change through collections can legitimately be regarded as the core mandate of museums. Yet the social utility of collections is widely measured by public exhibition, interpretation and display. 'Inform/inspire, educate and entertain' is a tired but typical aspirational mission. In presentational terms the importance and transformative influence of early rail transport is conveniently symbolized by the massiveness of steam locomotives. In much the same way, up to the early 1960s, the fact that computer systems were big, conveyed significance and complexity, and there was a happy congruity between meanings and external form. As a free-standing object, function was largely opaque but this was masked by wonder at their size which supported the message of technological and historical significance. If it's big, it matters, is the perceptual equation. But from the early 1960s, developments in solid-state physics began to stress traditional exhibit presentation in a new and distinctive way. More and more was happening at component and device level, and there was less and less to see. What was of significance in an integrated circuit was invisible, whether live or not. 'Black-box syndrome' was the exhibitor's dread where external form and internal function, however innovative, agreed to amicable divorce. With diminished visual appeal, long-accepted display conventions were challenged in an unprecedented way. New techniques were called for to unpack meanings from small items with no functional transparency to speak of. While difficult, the problem of the chip, as museum artefact, is not intractable. Creative use of existing techniques -panel graphics, illustration, imaginative comparisons, screen-based layered interactive exposition -provide solutions. No such obvious relief is available for software.

5

Software in an Object-centred Culture

The acquisition, preservation and interpretation of software poses near-intractable practical and philosophical difficulties for museums. Traditional museums are part of an object-centred culture, and privileged in this culture are physical artefacts, their meaning, significance and their care. Items of hardware, the physical stuff of IT systems are, by the conventions of traditional curatorship, unproblematic candidates for acquisitionmonitors, keyboards, disk drives, system boxes, printers and the like. Software, a term in general use by the early 1960s, was defined negatively. The Oxford Dictionary of Computing defined software as 'a generic term for those components of a computer system that are intangible rather than physical' [START_REF] Software | Dictionary of Computing[END_REF]. Prentice Hall's Illustrated Dictionary of Computing severs any link with the physical by noting that that 'software is independent of the carrier used for transport' [START_REF] Nader | Software. Prentice Hall's illustrated dictionary of computing[END_REF]. We habitually refer to CD distribution disks, and previously to floppy disks, as 'software'. However, this fudges the distinction between the carrier, or medium of record, and the program. Strictly the software is a non-material logical abstraction, and the disk, PROM, mem-stick, punched cards, punched paper tape, are carriers [START_REF] Swade | Collecting Software: Preserving Information in an Object-Centred Culture[END_REF].

We do not collect polynomials or prime numbers, but we do collect mathematical instruments, physical models, mathematicians' workbooks, drafts of seminal publications, first editions and, in the case of prime numbers, hardcopy printouts. The mathematics gallery at the Science Museum, London completed in 2016 [START_REF] Rooney | Mathematics: how it shaped our world[END_REF] does not display number or mathematical operations, but computational machines and devices used in various arenas of human affairs, trade, war, finance, vital statistics, and structures i.e. ways in which number, an abstraction, has been physicalised for computational purposes. In this way abstract meanings are abducted by material artefacts. Similarly, when we 'collect software' we are not strictly collecting algorithmic logic but the physical media of creation representation, distribution, and storage -coding sheets, flow charts, written deliberation of designers and programmers, manuals, publicity literature, punched cards, and carriers in various forms -optical, magnetic, solid-state, paper. The distinction is not an entirely pedantic one. If software falls outside the mandate of object-centred museums then upon whom, and on what institution, does custodial responsibility for preservation fall?

Even when we collect, say, shrink-wrapped software products, we do so, at least partly, in their capacity as cultural artefacts -historical placeholders -rather than as archival records of a working product. The model for Museum preservation practice is essentially an archaeological one in which we retard physical degradation by passivating the physical environment -protection against physical trauma, low light, temperature and humidity control. The effectiveness of this model is limited when it comes to ensuring the data integrity of software carriers. Optical media and, a fortiori, magnetic media are notoriously impermanent, and manufacturers are loath to commit to figures for service lifetime. At best we are talking about decades, which is negligible in the archaeological timescales we envisage. Software is not alone in this. A curator of medicine might acquire a pack of Aspirin. There is significance in the branding, advertising graphics, tablet form, blister-pack press-through dispenser, logo and packaging -cultural content. But a pack of Aspirin would not be acquired and preserved as a permanent record of its chemical structure which it is accepted will degrade, and with it, its therapeutic potency. The inventoried Aspirin pack in a medical collection is not preserved as a pharmaceutical record of the product as it would be by retaining its chemical formula and/or the recipe for manufacture [START_REF] Swade | Collecting Software: Preserving Information in an Object-Centred Culture[END_REF]. Similarly, it is accepted that passivating the environment will not ensure the operational viability of software when stored on its native medium. In short, there is a basic incompatibility between life-expectancy of the medium-of-record and the long-term custodial aspirations of museums.

Software Preservation

A working party was set up by the Computer Conservation Society [11] in the early 1990s to address the issue of software preservation and take account of the impermanence of storage media and the resources required to ensure content renewal in perpetuity. The group disbanded after three months with the conclusion that the only credible way to preserve software systematically was to record the content as a bit stream on paper and entrust this printed record to archivists who have well-proven techniques for the indefinite preservation of paper media.

There is a variety of initiatives that have risen to meet the formidable challenges of software preservation. Perhaps the most broadly-scoped programme is that of the Computer History Museum (CHM), California, where the collecting emphasis is on source code -a form more revealing than most, of programmers' intent and approach [START_REF]Software Preservation[END_REF]. Enviably, the Museum supports a dedicated Software Curator, Al Kossow, who has developed protocols and practices to ensure the integrity of stored content including measures to prevent and detect corruption. In the UK David Holdsworth heads up the software conservation activity of the Computer Conservation Society, London. Here the mission is to preserve software in machine-readable form with a view to current or future capability to execute [START_REF] Holdsworth | History, Nostalgia and Software[END_REF]. There are countless software preservation initiatives by societies, by national libraries and archives, and through the efforts of enthusiasts some dedicated to a specific class of products, computer games, for example. All face a variety of common issues, principal amongst which are the limited life of storage media, the impermanence of contemporary hardware platforms, and the portability of virtual platforms for emulators and simulators.

Software Recovery

As with hardware, the historical significance of software was rarely evident at the time of its creation or implementation and, if it survived at all in whatever form, it did so for the most part by accident or, occasionally, through the agency of a conscientious practitioner. There are cases where the original medium survives intact but nothing is known of its contents. There are other cases where the original medium did not survive but there is a non-electronic record which may or not be complete. And yet other cases where software is known to have existed but nothing at all exists in material form. Only in the last of these cases can the software strictly be considered 'lost'. In each of the other cases software, taken as the logical algorithmic content, survives, whether or not in executable form. However, in all these cases, before the software can be preserved for future use, it needs first to be recovered, whether retrieved from the original medium, translated from a non-electronic medium, or reconstructed from scratch. An example of retrieval from an original medium is the restoration to working order of the Elliott-NRDC 401, an experimental vacuum-tube machine dating from 1952-3. Only one 401 was made and the restoration, begun in the early 1990s by the Computer Conservation Society, was obstructed by the absence of any documentation for the drum store -no data, metadata, or information on the file structure. The restoration team headed up by Tony Sale and Chris Burton built read heads, ran the drum and captured the bit stream on a PC where it could be inspected, analysed and decoded. That the data was readable from a 40-year old drum puts in a slightly more favourable light the notorious impermanence of magnetic media. Len Shustek at the Computer History Museum has recovered source code from degraded magnetic tapes from Whirlwind -this by digitising the streamed analog waveforms and using software to reconstruct the original data [START_REF] Shustek | Decode the analog waveform extracted from old computer mag tapes[END_REF][START_REF] Stachniak | Software Recovery and Beyond[END_REF].

There are more extreme examples involving retrieval from other than native media. David Holdsworth recounts the recovery of software for the KDF9 computer from lineprinter listings [START_REF] Holdsworth | Rescuing Software from Lineprinter Listings[END_REF]. The texts were copy-typed after OCR proved inadequate as a recovery tool for the 40-year old hardcopy. Two assembler language programs were recovered in this way and the code runs on an emulator.

8

Love Letters David Link's reconstruction of Christopher Strachey's program to generate love letters is an example of software recovery that combines computer archaeology and art [START_REF] Link | Machine Heart (das Herz der Maschine)[END_REF].

Strachey wrote his love letter generator for the Ferranti Mark 1 (1952) at Manchester. Nothing of the original storage medium appears to have survived. Link located the source code in the Bodleian Library in Oxford but found it impossible to decode the algorithmic structure or internal dynamics of the program. He concluded that the only way to analyse the program was to reconstruct it and run it live -this by first constructing a software emulator, and later a functional physical replica using contemporary parts including simulations of Williams-tube memory using WWII radar tubes. He rewrote a critical missing subroutine (the original was subsequently found) and the system formed the basis of an art installation which displayed continuously generated love letters on a screen, and also provided some interactivity with the visitor [START_REF] Roberts | Christopher Strachey's Nineteen-fifties Love Machine[END_REF]. It is clear from these examples that there are substantial, ingenious, and significant initiatives to recover and preserve software. It is also clear that such initiatives, which require high levels of specialised expertise, are not centralised or co-ordinated and do not have a shared mission. Perhaps most importantly, they do not have the long-term security conferred by statutory responsibility as is the case for state-funded national museums with its mandate defined in law, the UK government's 1983 Heritage Act, for example. This is not a criticism. Quite the reverse -let a thousand flowers bloom. The observation is intended simply to flag that if our concern is preservation in perpetuity then the long-term future of these remarkable efforts is not secure.

The creation of new institutions to preserve new informational media is not without precedent. The National Sound Archive (NSA) was founded in 1955 as the British Institute of Recorded Sound and was absorbed into the British Library in 1983. As part of the British Library the sound archive is state funded. The National Film Archive was founded in 1935. As part of the British Film Institute (BFI) the film archive is sponsored by the government's Department of Digital, Culture, Media and Sport (DCMS). Both archives are secure for as long as society continues to value these media as part of its cultural heritage. If it is time for institutionalised software preservation with the security of state funding, how do we articulate answers to the questions, what, and who is it for?

Functionality

Whether the preservation of software, without means to run it, is meaningful is a fraught question. Gerard Alberts argues that software is a dynamic artefact and not to preserve user experience and interaction with it is to lose essential elements of its meaning [START_REF] Alberts | Archaeology of the Amsterdam digital city; why digital data are dynamic and should be treated accordingly[END_REF].

Web archiving, which preserves 'snap shots' of static screen pages, has a very different outcome to preserving the experience of web-surfing in real time. David Holdsworth is of like mind in writing that 'a basic tenet of the CCS's software conservation activity is that software is only truly conserved when it can be run on current hardware and has the prospect of being run on hardware in the foreseeable future' [START_REF] Holdsworth | Rescuing Software from Lineprinter Listings[END_REF]. This is a judicious formulation in that it sanctions and encourages responsible preservation without making a contemporary hardware platform, or an operational equivalent, a prerequisite. We are enjoined to preserve, as a holding operation, before the material is lost and while specialised expertise survives. Two areas come to mind where running software might be thought to be indispensable to meaning. The first is computer games. Gaming has significantly driven innovation and it is questionable whether the preservation of only the physical paraphernalia of gaming can meaningfully replace the ability to run these programs live on contemporary platforms [START_REF] Guins | Game after: a cultural study of video game afterlife[END_REF]. A second example is that of art installations. Computer-enabled or computer-dependent art installations are largely meaningless unless run live. David Link's Poetry Machine and his self-regenerating book, Meditationes, are examples [START_REF] Link | Poetry Machine[END_REF][START_REF] Link | Poetry Machine[END_REF][START_REF] Link | Radar Angels: Art and Artificial Text Generation[END_REF][START_REF] Vega | Meditationes: A metaphysical laboratory by David Link[END_REF] [figure 4]. The demands of exhibition are usually time-bound and any given platform will last the duration before terminal obsolescence. Poetry Machine, which involves sophisticated software and interactivity was revived after twenty years by migrating the software to a Windows platform and refreshing the hardware -a non-trivial process even for Link, its original creator. Archiving an installation scarcely qualifies as meaningful preservation without the resources to renew it and without the security of continuity. A related context in which functionality impacts preservation is the art market where the installation is seen as product. If an installation is sold what is it one owns without the resources to maintain in perpetuity the original system, or migrate the software to a later platform? Paintings and sculpture are not plagued in this way. They do not require the mediation of complex technology to render them intelligible.

Platforms

There are countless initiatives to provide contemporary hardware platforms or functional facsimiles, primarily through the restoration to working order of surviving machines, or through the reconstruction of historic machines of which there are no surviving examples [START_REF] Swade | The Historical Utility of Reconstruction and Restoration[END_REF][START_REF] Swade | Historical Reconstructions[END_REF]. The hallmark of such initiatives is the expertise, ingenuity and commitment of largely volunteer effort. Teams at the Computer History Museum have successfully restored a DEC PDP-1 (1959) and an IBM 1401 (1959) [START_REF] Garner | Restoring and Demonstrating 1960s Vintage Computers at the Computer History Museum[END_REF]. At The National Museum of Computing (TNMoC) at Bletchley we find working restorations of the Harwell Dekatron ('WITCH') a hybrid relay-valve machine dating from 1951 [START_REF] Dekatron | The National Museum of Computing[END_REF], a Marconi TAC (1959), the discrete component germanium transistor Elliott 803 (early 1960s), an Elliott 903 (late 1960s), an IBM 1130 (1965), an ICL 2966 (1980s), ICL System 25 (1980s) and ICL DRS 6000 (1990), a Cray EL98 (1993), and a variety of classic personal computers [START_REF]Explore the Galleries, The National Museum of Computing[END_REF].

In instances where no original exists to restore or replicate, reconstructions have been resorted to. Prominent amongst these is the Manchester SSEM ('Baby') at the Science and Industry Museum in Manchester. At TNMoC there are several substantial working reconstructions of WWII machines [START_REF]Explore the Galleries, The National Museum of Computing[END_REF] -Colossus, Tunny, Heath Robinson, and the Turing-Welchman Bombe, as well as the post-war Cambridge EDSAC [START_REF]EDSAC -Electronic Delay Storage Automatic Calculator[END_REF].

Restorations and reconstructions provide contemporary hardware platforms, or functional equivalents, for running legacy software as well as new programs. Through these restorations and reconstructions, the fidelity of programming practices, performance, interactivity, and the user experience are preserved. These initiatives have other historiographical as well as social benefits: the practicalities of restoration or reconstruction draw one into a level of intimate detail with the machine that rarely occurs by other means; physical realisation almost invariably results in contingent or unexpected findings not foreseen by analysis or theory; operating the machines gives insights into contemporary ideas and practice and captures generational experience that would otherwise be lost; tacit knowledge is regenerated; documentary completeness is a contingent outcome; and the working system provides an authentic benchmark for simulation [START_REF] Swade | The Historical Utility of Reconstruction and Restoration[END_REF].

In addition to the historical and historiographical value of restorations and reconstructions there is the social capital they create i.e. their value to those who make up the project teams, to visitors who view and experience them on public display, and to museums and organisations that host these exhibits. The projects provide meaningful engagement within an organisational context for veteran experts to share expertise, exercise their craft, and extend their professional activities in historically and educationally meaningful ways. Demonstrating the machines has pedagogic value to users and visitors. Large machines on public display act as foci for visitor attention and provide a platform for storytelling through live demonstration and conventional exhibition graphics. Finally, large systems publicly displayed memorialise important episodes and practices for which they act as both placeholders and monuments that serve as generational bridges to an otherwise lost past.

The End of the Road

However successful are such efforts to extend the operational life of historic machines or to provide historically authentic reconstructed platforms, we have to accept the eventual demise of such systems if our aspirations are to extend to archaeological time spans. While degraded wiring can be replaced, and any part, vacuum tubes for example, can, in principle at least, be refactored as replacements, a point will arrive where the effort and cost will become prohibitive even if the relevant expertise can be migrated intergenerationally. It is anyway the case that any replacement programme compromises the physical integrity of the original artefact and, if the machine is unique, violates the central primacy of the original in the central tenets of museum practice. There are also political insecurities. The priorities of host institutions change. At the Science Museum, London, a Ferranti Pegasus (1959), a vacuum-tube computer with magnetic drum storage, was restored to working order by the Computer Conservation Society and successfully run and demonstrated from the early 1990s for over 25 years. There was no technical reason preventing further extending its operational life. Its maintenance and public demonstration were ended following a policy shift away from working exhibits, and the machine was moved off public display into storage.

The construction of an historic machine is essentially an act of physical replication. There is of course logical replication in the form of emulators and simulators. Emulators allow us to run contemporary software on a non-native platform and this offers liberation from the need for working original or non-original hardware. Simulation captures the operational persona of the machine in providing a virtual functional equivalent that allows a user to program, run, and interact with an operational facsimile. While the logic of virtual machines may be faithful to the original there are performance issues that are not automatically portable, real-time execution being one.

Emulation and simulation offer the promise of indefinite migration to new platforms and, as the languages used for the simulations become increasingly machine independent, indefinite migration from one generational platform to the next appears to offer a form of logical immortality. In practice, however, the independence of software from hardware is rarely absolute and there are interdependences that compromise the prospect of indefinite preservation by these means. There is also the question of the longevity of the languages that host the emulators. When these are superseded, we have a new migration problem.

Justification for Working Software

In a bid for state or institutional funding for software preservation a question that will inevitably be asked is, to what purpose i.e. what benefits would accrue from having an archive of software whether running or not? The tendency is to begin to stutter. The point is that we have little idea how these artefacts will be interrogated by those who follow. We cannot foresee what features may be of interest or significance. A researcher may be interested in the historic use of subroutines, the practice of altering instruction codes under program control, trawling early programs for forgotten techniques that could inventively be repurposed, algorithmic structures and precedents, the first use of nested looping, the first use of number as symbol rather than quantity, response and execution times, the speed of a compiler, latency . . . a gallimaufry of practical, philosophical, logical and technical inquiry. We do not ask this question of traditional museum objects nor of the specific utility of sound or film. It is accepted that these have cultural and historical value and are saved to be available for unforeseen and unforeseeable interrogation, and cultural appreciation. I have argued elsewhere [START_REF] Swade | Virtual Objects: Threat or Salvation?[END_REF] that what underpins the primacy of the original in museum culture is that only the original embodies predicates, properties and features the significance of which we cannot fully know nor anticipate. It is precisely this epistemic incompleteness that is responsible for the elevation of the original above object surrogates, replicas, or imitations. What underpins the value of the original, and is responsible at least in part for its mystique, is the sense that understanding is never complete, that knowledge is never total, that the original artefact embodies evidence the meanings which may not be accessible by other means, and whose significance cannot be foreseen. Software, as a digital object, is no different.

Exhibiting software poses daunting difficulties, and very few museum-like organisations have embraced the challenge. The deterrents are already familiar: abstractness, arcane complexity and the absence of anything physically meaningful upon which traditional object-based exhibition routinely relies. A bold exception is the exhibition Make Software: Change the World that opened in 2017 at the Computer History Museum [START_REF]Make Software: Change the World, Computer History Museum[END_REF]. The exhibition features seven exemplars of software products or classes of software identified as transformative: Photoshop, MP3, MRI, Car Crash Simulation, Wikipedia, Texting, and World of Warcraft i.e. applications regarded as 'game-changing' in computer-mediated virtual reality, compression, simulation, gaming, and crowdsourced knowledge. This treatment leapfrogs attempts to treat software as a traditional museum object, dumps otiose agonising about the relationship between software and hardware, and confidently abandons much else that has so far daunted and paralysed wishful exhibitors. The subtext of the exhibition is that these are wondrous objects and they are created or enabled by software which is demonstrably powerful, important and ingenious. This treatment uses widely known outcomes to raise interest in the generative process. This turns on its head the traditional approach that favours focusing on the generative process (programming, compiling, software engineering . . .) in an attempt to provide some kind of explanatory account for the outcome.

Summary and Conclusions

Non-working hardware devices and systems fall within the traditional collecting mandates of object-centred museums which have well-established preservation practices and protocols. Exhibiting these as static objects is amenable to existing interpretative display techniques. In the case of smaller devices, especially solid-state devices, opacity of function, uniformity, and physical size stretch conventional techniques of interpretation, but not unmanageably so.

Working historic machines and systems are heavily concentrated in the independent sector. Private collections and independent smaller museums are the primary hosts of restoration and reconstruction activity that heavily relies on the expertise, interest and commitment of volunteers. There tends to be little co-ordination between these groups for acquisition, exhibition or restoration activity, and security of support, not to mention their artefactual holdings, are perpetually at risk. Prominent in the independent sector are subject-specialist museums dedicated to computing. These include the Computer History Museum, California, the Heinz Nixdorf MuseumsForum (HNF) in Paderborn, Germany, The National Museum of Computing (TNMoC) at Bletchley. With company funding, philanthropic and sponsorship support these collections are relatively secure for the time being. There are many regional museums with local focus, small departmental collections in universities, and small museums funded by universities, where financial security is partly assured by their organisational hosts. Overall, the future of smaller private collections is precarious especially when viewed in the context of archaeological time scales.

Running historic software requires functional intactness in the form of bit-perfect records as well as contemporary hardware or a physical or virtual functional equivalent.

Software preservation is a growing activity though still tends to be localised and fragmented. The levels of specific expertise for effective preservation through capture and emulation are high, and dedicated volunteers, many retirees, tend to be the drivers here. When it comes to storage methods, formats, and media there is little standardisation.

The resource implications of running software on contemporary platforms, or on functional equivalents, act as formidable deterrents for traditional museums. High levels of expertise are required in skills areas that have high market value outside the museum sector. Organisational commitment is required to ensure continuity, and social, technical and historical utility is difficult to justify through cost-benefit arguments. The resources required to sustain an indefinite programme of copying, renewal, and integrity-checking are non-trivial and few organisations have yet been willing to commit to preservation programmes of this kind.

The dream is for an independently resourced organisation with a mandate to proactively acquire software, preserve executable versions in perpetuity, to maintain working contemporary hardware platforms, develop emulators and simulators, and migrate this material to new generational platforms on an indefinite basis, this to provide a gene pool of software evolution. Systematic pro-active collecting of this kind is rare even as an agenda for object-based state-funded museums. What tends to survive are artefacts seen to be important enough for people to care about. Advocacy for much of this collecting comes from outside the museum sector, from enthusiasts, practitioners, specialists and professionals. What survives for the most part is through the happenstance of history. Software, so far, is no exception.

Fig. 1 .

 1 Fig. 1. Apple lapel, badge (1982). Picture credit: Doron Swade

Fig. 2 .

 2 Fig. 2. Magnetic Core Memory (Digico 1972) ) (Inset: detail). Picture credit: Doron Swade

Fig. 3 .

 3 Fig. 3. SAGE Console (1960) Picture credit: Computer History Museum

Fig. 4 .

 4 Fig. 4. Poetry Machine by David Link. Picture credit: David Link