Elisa Parra

Lara H Moleiro

Ivan López-Montero

Antonio Cruz

Francisco Monroy

Jesús Pérez-Gil

A COMBINED ACTION OF PULMONARY SURFACTANT PROTEINS SP-B AND SP-C MODULATES PERMEABILITY AND DYNAMICS OF PHOSPHOLIPID MEMBRANES

Keywords: LIPID-PROTEIN INTERACTIONS, MEMBRANE PERMEABILITY, MEMBRANE

Proteins SP-B and SP-C are essential to promote formation of surface active films at the respiratory interface, but their mechanism of action is still under investigation. We have analyzed the effect of the proteins on the accessibility of native, quasi-native and model surfactant membranes to incorporation of fluorescent probes Nile Red (permeable) and FM®1-43 (impermeable) to membranes. We have also analyzed the effect of single or combined proteins on membrane permeation by the soluble fluorescent dye calcein. Fluorescence of FM 1-43 was always higher in membranes containing SP-B and/or SP-C than in protein-depleted membranes, in contrast to Nile Red, very similar in all the materials tested. SP-B and SP-C promoted probe partition with markedly different kinetics. On the other hand, physiological proportions of SP-B and SP-C made giant oligolamellar vesicles to incorporate instantaneously FM 1-43 from the external medium into apparently most of the membranes. In contrast, oligolamellar pure lipid vesicles appeared as mainly labelled in the outermost membrane layer. Pure lipidic vesicles were impermeable to calcein, while it permeated through membranes containing SP-B and/or SP-C. Vesicles containing only SP-B were stable but prone to vesicle-vesicle interactions, while those containing only SP-C were extremely dynamic, undergoing frequent fluctuations and ruptures. Differential structural effects of proteins on vesicles were confirmed by electron microscopy. These results suggest that SP-B and SP-C have different contributions to inter-and intra-membrane lipid dynamics, and that their combined action could provide unique effects to modulate structure and dynamics of pulmonary surfactant membranes and films.

INTRODUCTION

Pulmonary surfactant, a membrane-based lipid-protein complex, is strictly required for breathing in pulmonated organisms. The presence of a surfactant layer at the respiratory surface is simultaneously responsible for biophysical stabilizing activities [START_REF] Serrano | Protein-lipid interactions and surface activity in the pulmonary surfactant system[END_REF] and innate defence mechanisms [START_REF] Wright | Immunoregulatory functions of surfactant proteins[END_REF]. The lack, deficiency or inactivation of the surfactant system is associated with severe respiratory disorders such as the Neonatal Respiratory Distress Syndrome (NRDS) in preterm babies [START_REF] Seeger | Alveolar surfactant and adult respiratory distress syndrome. Pathogenetic role and therapeutic prospects[END_REF] or the pulmonary dysfunction associated with Acute Respiratory Distress Syndrome (ARDS) in cases of lung injury [START_REF] Hallman | Surfactant in respiratory distress syndrome and lung injury[END_REF]. In general terms, the composition of surfactant consists of about 90% lipids and 8-10% specific surfactant proteins, including two families: SP-A and SP-D, hydrophilic in nature, and SP-B and SP-C, both hydrophobic and membrane-associated [START_REF] Goerke | Pulmonary surfactant: functions and molecular composition[END_REF]. The phospholipid fraction in surfactant is essentially responsible for its ability to reduce dramatically the surface tension at the air-liquid interface of the alveolar spaces. About 80% of surfactant by mass is composed of phosphatidylcholine (PC), about half of which is dipalmitoylphosphatidylcholine (DPPC) [START_REF] Veldhuizen | The role of lipids in pulmonary surfactant[END_REF]. The acidic phospholipids phosphatidylglycerol and phosphatidylinositol represent 8-15% of the total surfactant phospholipid pool. Cholesterol is the main neutral lipid in surfactant, accounting for up to 8-10% by mass. The presence of proteins, specifically SP-B and SP-C, is absolutely necessary for interfacial adsorption, film stability and re-spreading activities of surfactant along the successive compression-expansion breathing cycles [START_REF] Perez-Gil | Structure of pulmonary surfactant membranes and films: the role of proteins and lipid-protein interactions[END_REF]. SP-B is thought to be the most important protein for respiratory physiology, as its lack or deficiency is associated with a lethal respiratory failure [START_REF] Melton | SP-B deficiency causes respiratory failure in adult mice[END_REF]. It has been previously shown that SP-B induces aggregation, fusion and lysis of phospholipid vesicles [START_REF] Ryan | Mapping and analysis of the lytic and fusogenic domains of surfactant protein B[END_REF], and these activities have been associated with a putative role of SP-B to promote lipid transfer and membrane restructuration processes required to form the alveolar surface active films [START_REF] Cruz | Microstructure and dynamic surface properties of surfactant protein SP-B/dipalmitoylphosphatidylcholine interfacial films spread from lipid-protein bilayers[END_REF]. The role of SP-C in surfactant is not as crucial [START_REF] Glasser | Altered stability of pulmonary surfactant in SP-Cdeficient mice[END_REF] but SP-C has been involved in membrane-membrane and membrane-interface contacts [START_REF] Creuwels | Neutralization of the positive charges of surfactant protein C. Effects on structure and function[END_REF][START_REF] Ross | Kinetics of phospholipid insertion into monolayers containing the lung surfactant proteins SP-B or SP-C[END_REF]. The combined action of both hydrophobic proteins is considered as responsible for the proper organization of functional membrane arrays in surfactant complexes. The pulmonary surfactant system constitutes an excellent example of dynamic membrane polymorphism and how it controls some biological functions through specific lipid-lipid and lipid-protein interactions. Surfactant is assembled by alveolar type II pneumocytes in the form of tightly packed membranes stored in specialized organelles called lamellar bodies (LB) [14]. Once secreted, surfactant develops a membrane-based network that covers very rapidly and efficiently the whole alveolar surface. From surfactant assembly until its adsorption into the interface, several restructuration processes take place: membrane packing and unpacking in LBs, rearrangement of LB membranes into tubular myelin (TM), reorganization of surfactant membranes to form layers close to the air-water interface, and transfer of surface active lipid species from these complexes into the interface -and vice versa-during expansion-compression respiratory cycles (see review [START_REF] Perez-Gil | Structure of pulmonary surfactant membranes and films: the role of proteins and lipid-protein interactions[END_REF]). Surfactant proteins have a major role in facilitating all of these processes, but the elucidation of their precise molecular mechanism is a challenging task. Different studies have suggested that pulmonary surfactant structures are composed of particularly dynamic membranes [START_REF] Bernardino De La Serna | Segregated phases in pulmonary surfactant membranes do not show coexistence of lipid populations with differentiated dynamic properties[END_REF], and it is not clear whether those membranes are actually isolating different membrane or aqueous compartments. In the present study, we have analyzed the permeability and accessibility of native surfactant complexes and membranes reconstituted from different surfactant fractions to the incorporation of the membrane-sensitive fluorescent probes Nile Red and FM 1-43, able and unable, respectively, to permeate across phospholipid membranes. Particularly, we have evaluated the dependence

EXPERIMENTAL

Materials -Native pulmonary surfactant materials were obtained from bronchoalveolar lavage of porcine lungs as described previously [START_REF] Taeusch | Inactivation of pulmonary surfactant due to serum-inhibited adsorption and reversal by hydrophilic polymers: experimental[END_REF]. Organic extraction of purified surfactant and chromatographic separation in Sephadex LH-20 (Pharmacia, Uppsala, Sweden) allowed obtention of the hydrophobic protein fraction and the lipid surfactant fractions [START_REF] Bernardino De La Serna | Segregated phases in pulmonary surfactant membranes do not show coexistence of lipid populations with differentiated dynamic properties[END_REF][START_REF] Perez-Gil | Solubility of hydrophobic surfactant proteins in organic solvent/water mixtures. Structural studies on SP-B and SP-C in aqueous organic solvents and lipids[END_REF]. A subsequent chromatographic step in LH-60 yielded purification of SP-B and SP-C separately [START_REF] Perez-Gil | Solubility of hydrophobic surfactant proteins in organic solvent/water mixtures. Structural studies on SP-B and SP-C in aqueous organic solvents and lipids[END_REF]. In general terms we obtain a proportion of around 0.8% SP-B and 1.1% SP-C with respect to phospholipid mass in the surfactant organic extract. Total phospholipid concentration in the different samples was determined by phosphorus analysis upon phospholipid mineralization [START_REF] Rouser | Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots[END_REF]. Proteins and lipids were stored as chloroform/methanol 2:1 (v/v) solutions at -20ºC. Protein solutions were routinely checked for purity by SDS-PAGE and quantified by amino acid analysis. 1-palmitoyl-2-oleyl-snglycero-3-phosphocholine (POPC) was purchased from Avanti Polar Lipids (Alabaster, AL, U.S.). Fluorescent dyes Nile Red and FM 1-43 were from Molecular Probes Inc. and calcein from Sigma Aldrich. Chloroform and methanol solvents, HPLC grade, were from Scharlau (Barcelona, Spain).

Vesicle preparation -Stock suspensions of native pulmonary surfactant were in 5 mM Tris buffer, pH 7, containing 150 mM NaCl. Multilamellar membrane suspensions (MLVs) of surfactant organic extract, the different surfactant lipid fractions or POPC supplemented or not with proteins SP-B and/or SP-C were prepared as follows. The appropiate amounts of proteins and lipids in chloroform/methanol 2:1 were mixed and the samples were then evaporated to dryness under nitrogen and 2 hours in a vacuum chamber to form a thin film, which was later resuspended at 45ºC (for native materials) or 37ºC (for POPC samples) in the desired final volume of 5 mM Tris buffer, pH 7, containing 150 mM NaCl. Large unilamellar vesicles (LUVs) were prepared using a Mini-Extruder (Avanti Polar Lipids) with 10 mm diameter drain discs and 0,1 Pm diameter Nuclepore Track-Etch membranes (Whatman), passing the MLV suspension 11 times through the filters at 50ºC, in the case of native or quasi-native material, or 37ºC for POPC samples with or without proteins. Small unilamellar vesicles (SUVs) were obtained by sonication (UP200, Hierlscher) of MLV suspensions (0,6 cycles, 65% amplitude; 4 cycles of 2 minutes). LUV and SUV suspensions were used immediately.

Fluorescence spectroscopy with Nile Red and FM 1-43 -Fluorescent probes Nile Red or FM 1-43 were incorporated into the membrane suspensions at a final concentration of 3 and 2 PM, respectively, and incubated at room temperature protected from light for 30 minutes. To follow incorporation kinetics of FM 1-43, fluorescence emission was measured immediately after incorporation of the probe to the suspensions. Fluorescence emission spectra were registered in a spectrofluorimeter JASCO FP 6200 thermostated at 37ºC, using 5 nm excitation and 10 nm emission slitwidths. Nile Red fluorescence was monitored upon excitation at 548 nm and registering emission from 550 to 700 nm, while samples labelled by FM 1-43 were measured exciting at 479 nm and recording emission from 500 to 650 nm.

Electron microscopy -Droplets of a unilamellar suspension of POPC (1 mg/ml) with or without protein (full hydrophobic protein fraction, 1% SP-B or 1% SP-C by weight), prepared by extrusion, were placed on carbon-coated grids (Electron Microscopy Sciences, Hatfield, PA) for 4 minutes, stained with uranyl acetate for 40 seconds and observed under a JEOL JEM-1010 Transmission Electron Microscope.

Giant Vesicle Preparation -Giant Vesicles (GVs) made of POPC or POPC supplemented with 1 % w/w of SP-B, SP-C or both were prepared following a slightly modified electroformation protocol [19]. Briefly, 10Pl of 1mg/ml lipid or lipid/protein solution in chloroform were spread in a indium tin oxide (ITO) coated glass slide (Sigma- A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Aldrich). The fabrication chamber is composed by two conductor glass slides separated by a Teflon spacer of 1 mm. After organic solvent evaporation, the film was re-hydrated with a sucrose solution (300 mM). The chamber was then connected to an A.C. power supply (8Hz and 1.1 V) for 3 hours. Then, the frequency was decreased to 4 Hz for 30 minutes. The obtained vesicles had a spherical shape and a large proportion of oligolamellar vesicles were found. A volume of 25 microliters of the solution containing GVs was transferred from the fabrication chamber to the observation chamber containing 75 Pl of glucose solution (310 mM).

Visualization of permeability of GVs to Calcein and FM 1-43 -In order to test the membrane permeability of GVs with different composition, giant vesicles were transferred to a glucose (310 mM) solution containing 0.05 mM of FM 1-43 . In some experiments, the liposomes were incubated first in glucose containing 1 mM of the bulk fluorescent marker calcein (Sigma-Aldrich, 494 nm excitation, 517 nm emission) and then diluted to glucose/FM 1-43 . Calcein and FM 1-43 fluorescence was then monitored under a fluorescence microscope (Nikon Eclipse TE2000 using a 100W mercury lamp and FITC and Texas Red filter sets) and recorded with a cooled CCD camera (Nikon DS-1QM, 14 fps, 1 Mpixel). All GV suspensions were observed freshly made, preferently within the same day of preparation, although no significant changes were observed in permeability or morphology of the vesicles observed up to 48 hours after lipid hydration and vesicle preparation.

Reproducibility -When possible, data figures represent mean ± standard error after averaging at least 3 different experiments, with at least two and often three entirely different surfactant and protein batches. In other cases, such as in GUV images, illustrative experiments are shown after repeating at least three experiments that exhibited consistent behaviour.

RESULTS

To assess the effect of the presence of hydrophobic surfactant proteins SP-B and/or SP-C on the permeability of phospholipids membranes we have taken advantage of the fluorescent emission of two extrinsic probes. Nile Red is a phenoxazone dye that fluoresces intensely in organic solvents and membrane environments, but is completely quenched in aqueous media [START_REF] Greenspan | Nile red: a selective fluorescent stain for intracellular lipid droplets[END_REF]. The emission properties of this probe depend strongly on the relative hydrophobicity of the surrounding environment. Phospholipid bilayers are highly permeable to Nile Red, so in principle it should label every single membrane in the samples. On the other hand, FM 1-43 is a styryl dye which is water soluble and also exhibits high affinity for lipid environments, although it doesn't translocate through membranes; consequently, it only labels the external leaflet of lipid vesicles. In addition, this probe only emits fluorescence once incorporated into membranes, as water molecules strongly quench its fluorescent emission [START_REF] Betz | Imaging exocytosis and endocytosis[END_REF]. Figure 1 compares the emission fluorescence intensity of Nile Red and FM 1-43 as a function of their incorporation into increasing concentrations of different lipid and lipid/protein suspensions, including native pulmonary surfactant complexes (NS) and multilamellar vesicles reconstituted from the whole surfactant organic extract (OE), the protein-free lipid fraction of surfactant (LF), surfactant lipid fraction depleted of cholesterol (LF'Chol) or the lipid fraction depleted of cholesterol but supplemented with the hydrophobic protein fraction of surfactant (LF'Chol+PF). For comparison, the fluorescence of the probes in increasing equivalent concentrations of multilamellar vesicles of POPC has been also determined. The effect of the presence of the different membrane materials on the fluorescence of the two probes is completely different. The fluorescence emission of Nile Red shows similar dependence on lipid concentration, within the experimental error, for the different materials tested up to concentrations around 200 Pg/ml (left panel in Figure 1), indicating that all these materials exhibit similar accessibility to this membrane-permeable probe. For higher lipid amounts, some differences in the emission intensity of Nile Red become relevant, probably as a consequence of local differences in the polarity of the environment sensed by the probe. The probe shows higher emission in materials containing cholesterol and other neutral lipids (NS, OE and LF) than in membranes prepared from fractions depleted of cholesterol and, specially, in pure POPC membranes. This is probably a consequence of the well-known effect of cholesterol to seal membranes and reduce their level of hydration [START_REF] Arrais | Bilayer polarity and its thermal dependency in the l(o) and l(d) phases of binary phosphatidylcholine/cholesterol mixtures[END_REF][START_REF] Theunissen | Membrane properties of oxysterols. Interfacial orientation, influence on membrane permeability and redistribution between membranes[END_REF]. The environment sensed by Nile Red in cholesterol-containing surfactant membranes would then be more dehydrated, and therefore more hydrophobic, than that in cholesterol-free bilayers, leading to a higher intensity of fluorescence emission. There are no apparent effects of the protein content on the fluorescence of this probe. In contrast to Nile Red, the fluorescence emission of FM 1-43 is remarkably different in the different membranes studied (right panel of Figure 1). The probe showed around two-fold higher fluorescence in protein-containing membranes, i.e., those from native surfactant, its organic extract or its lipid fraction depleted of cholesterol but supplemented with SP-B and SP-C, than in those made of protein-free surfactant lipids. The fluorescence of the probe in surfactant lipid membranes was also substantially higher than measured in POPC bilayers. The presence of proteins SP-B and SP-C seems therefore important to provide lipid membranes with full accessibility to membrane-impermeable probes such as FM 1-43. Interestingly, Nile Red fluorescence increases rather linearly with phospholipid concentration while FM 1-43 fluorescence seems somehow to plateau, which could reflect differences in partition of the two probes between different surfactant lipid phases [START_REF] Bernardino De La Serna | Segregated phases in pulmonary surfactant membranes do not show coexistence of lipid populations with differentiated dynamic properties[END_REF]24], and therefore different levels of probe accumulation. The differences in accessibility to FM 1-43 of the different membrane suspensions are probably manifested due to their multilamellar character. The outermost impermeable membranes would impede exposure of inner membranes to the probe. In Figure 2 we have compared probe accessibility of native surfactant membranes, membranes reconstituted from the whole surfactant organic extract, and model POPC bilayers when preparing suspensions with different lamellarity. As it could be expected, FM 1-43 produced higher fluorescence emission in unilamellar (LUV or SUV) vesicles than in multilamellar suspensions of POPC (right panel of Figure 2), as a consequence of the larger surface of membranes exposed to the externally added probe. In contrast, there are no apparent differences in the fluorescence emission of the probe partitioning in unilamellar or multilamellar suspensions of either native surfactant or its organic extract. Again this result points to a potential role of surfactant proteins to facilitate accessibility of the membrane-impermeable probe across different membranes.

To test whether the mere presence of SP-B and/or SP-C would be enough to facilitate partition of FM 1-43 across membranes, we have tested the effect of introducing surfactant proteins on the differences in probe accessibility when comparing unilamellar and multilamellar suspensions of simple POPC vesicles. Figure 3 compares fluorescence of FM 1-43 added to increasing concentrations of unilamellar or multilamellar suspensions of pure POPC in the absence or in the presence of hydrophobic surfactant proteins. Also, we have compared POPC membranes supplemented with the whole protein fraction of surfactant (SP-B + SP-C at equivalent lipid-to-protein ratio to that obtained in the LH-20 chromatography) with membranes containing only 1% of purified SP-B or SP-C. Figure 3 (left panel) shows that the fluorescent emission of FM 1-43 is 4-fold higher in all protein-containing POPC MLVs than in pure phospholipid membranes, with small but consistent differences between the different protein-containing samples. The order of accessibility of membranes to the probe was always with respect to the protein content SP-B > SP-B+SP-C > SP-C. In contrast, the differences between protein-containing and protein-free POPC membranes with respect to the fluorescence of FM 1-43 were much reduced in unilamellar POPC suspensions (right panel of Figure 3). To get further insight the differences between the effect of SP-B and SP-C to facilitate accessibility to FM 1-43 across multilamellar membrane arrays, we have compared in Figure 4 the kinetics of incorporation of the probe to different amounts of POPC multilamellar suspensions containing 0.25, 1.0 or 2.0% (protein to lipid by weight) of either purified SP-B or SP-C. Physiological proportions of these proteins are thought to be around 1% in native surfactant, but we wanted to test much lower subphysiological proportions to maximize kinetic differences. The kinetic of incorporation of FM 1-43 into the POPC membranes was significantly different when comparing the effect of SP-B and SP-C. In all the SP-Bcontaining membranes tested, the fluorescence emission intensity of the probe reached the maximum in less than 30 seconds since the addition of the dye, while in SP-C-containing samples, the emission increased gradually along the first 30 minutes, especially in the samples containing the lowest proportion of protein. Figure 5 compares the full dependence of maximal emission of FM 1-43 into POPC MLVs versus the protein density in the membranes for both SP-B and SP-C. Below 0.5% protein to lipid ratio (w/w), SP-B produced always higher fluorescence, hence better partition of the probe across membranes, than SP-C. The lower proportion of SP-B tested, 0.1% protein to lipid by weight, already produced maximal incorporation of FM 1-43 into POPC membranes, suggesting that SP-B is extremely efficient in facilitating permeability of this membrane-impermeable probe across bilayers. The different behaviour of SP-B and SP-C with respect to the kinetics of incorporation of FM 1-43 across membranes suggests that intrinsic differences must exist between the mode and extent of perturbation by the two proteins of the structure and the permeability barrier of membranes. To get further insight the effect of the two proteins on the structure of the model membranes used in the present study, we have examined by electron microscopy the ultrastructure of POPC LUVs prepared in the absence or in the presence of the whole hydrophobic surfactant protein fraction or 1% by weight of purified SP-B or SP-C. Figure 6 shows representative images obtained by negative staining and transmission electron microscopy of these lipid and lipid/protein samples. Pure POPC suspensions contained a relatively homogeneous population of vesicles with homogeneous size, and presumably unilamellar character (image A in Figure 6), as well as the suspension of POPC supplemented with the mixture of SP-B and SP-C (image B). The POPC/SP-B suspension presented a high complexity, since it was totally made up by large, apparently multilamellar, membrane complexes with a diameter in the micrometer scale, in spite that they were originally extruded through 100 nm pores. The vesicles prepared with POPC plus 1% SP-C showed a higher level of aggregation than pure POPC vesicles, but it still consisted of unilamellar and oligolamellar vesicles with a more or less spherical shape. In order to achieve a direct visualization of the effect of surfactant proteins on the membrane labelling by FM 1-43, in a more homogeneous and controlled membrane structural context, a series of fluorescence microscopy experiments have been carried out in which we have monitored the ability of the probe to label the different membranes in oligolamellar giant vesicles of POPC in the absence or presence of hydrophobic surfactant proteins. The large size of GVs prepared by electroformation allows their visualization under an optical microscope and the simultaneous examination of fluorescent probe partition and membrane macroscopic morphology. Figure 7 compares morphology and FM 1-43 labelling of pure POPC oligolamellar GVs and GVs formed from POPC supplemented with the hydrophobic surfactant protein fraction (PF) obtained after the LH-20 chromatography, at proportions mimicking lipid/protein ratios in surfactant organic extract. Two representative GVs of each sample are shown, exhibiting a clear oligolamellar structure under the optical microscope operated under phase contrast. Upon incubation with FM 1-43, pure POPC oligolamellar GVs were labelled only in the outermost layer (see images B and D in Figure 7). In contrast, exposure to the probe produced almost instantaneous labelling of every membrane in POPC/PF vesicles (see images F and H in Figure 7). Similar experiments were carried out with GVs made of POPC supplemented with 1% by weight of either purified SP-B or SP-C. Surprisingly, the morphology of these giant liposomes containing only one of the surfactant proteins was substantially different to that of liposomes containing no protein or the full hydrophobic protein fraction. No clearly oligolamellar GVs could be found as made of the POPC/SP-B mixture. As illustrated in Figure 8, suspensions of POPC/SP-B vesicles were entirely composed of apparently unilamellar giant liposomes, with different sizes and a strong trend towards aggregation (see images A-D in Figure 8), which were fully labelled by FM 1-43 . Suspensions of POPC/SP-C vesicles contained a large number of oligolamellar liposomes, but most of them became very unstable when observed under the microscope, showing a high tendency to distortion, rupture and reorganization. Figure 9 shows sequential frames taken from a video recording, illustrating the rupture of two giant oligolamellar vesicles: at first, the shape of both vesicles looked very irregular and their membranes started fluctuating; then, some membrane regions protruded and formed evaginations, which finally released smaller vesicles to the medium before becoming again spherical and stable. The complete recording is available as Additional Material (http://bbm1.ucm.es/biomil/video/Video_Parra_JBC_1000_5MB.avi). We never saw such dynamical behaviour in pure lipidic vesicles or in liposomes containing SP-B. To analyze further the effect of surfactant proteins on the permeability of membranes to polar molecules, we tested the ability of the fluorescent dye calcein to permeate through POPC liposomes in the absence or presence of the proteins. Figure 10 illustrates how pure POPC GVs can be incubated for several hours in a calcein solution without internalization of the dye. Vesicles appeared as dark spheres when observed under the blue light used to excite calcein (see image B in Figure 10). In contrast, incubation with calcein of vesicles of POPC supplemented with the hydrophobic surfactant protein fraction lead to the entrance of the dye. Image E in Figure 10 illustrates how calcein had entered into protein-containing POPC vesicles, showing even higher fluorescence that the liposome external medium. This indicates that equilibration of calcein concentration across the membrane takes some time once the concentration of the dye in the solution has been diluted down. Calcein could also permeate through POPC liposomes containing only SP-B or SP-C, but with an apparently different pattern than observed in liposomes containing the two proteins together. In the presence of either of the hydrophobic surfactant proteins no difference in fluorescence emission could be seen between the different compartments (see images H and K in Figure 10), indicating that calcein fluorescence equilibrated rapidly between the internal and external liposome compartments. Interestingly, images of calcein fluorescence in liposomes containing SP-B showed large segments of the membrane that were much darker than the aqueous calcein solution, as if calcein would be highly excluded from perimembranal regions.

DISCUSSION

The ability of proteins SP-B and SP-C to introduce significant perturbations of structure and dynamics in surfactant phospholipid membranes has been largely documented. These perturbations have been mainly interpreted in the context of the role of the proteins to promote the structural transformations associated with pulmonary surfactant biogenesis, secretion and adsorption into the air-liquid interface. However, the intrinsic properties imparted by the particular lipid and protein composition to native-like pulmonary surfactant membranes have not been completely described. Little is known, for instance, with respect to the symmetric or asymmetric character of these membranes as they are assembled, or their permeability to polar solutes. We have tested the accessibility of membranes with different native-like or model lipid and protein composition to the membrane-fluorescent probe FM 1-43. This probe has been extensively used as a marker for lamellar body exocytosis, in order to obtain a direct visualization of pulmonary surfactant secretion in alveolar type II cells [START_REF] Haller | Dynamics of surfactant release in alveolar type II cells[END_REF]. FM 1-43 stains intensely the whole lamellar body content immediately after the opening of the exocytotic fusion pore. This suggests the existence of a direct topological connection between all the lamellar body membranes and/or the inter-lamellar compartments. Our starting hypothesis was that the assemblies in which hydrophobic proteins SP-B and SP-C participate are involved in the free diffusion of probes like FM 1-43 across surfactant membranes once surfactant is released from type II pneumocytes. The present data confirm that the hydrophobic proteins SP-B and SP-C are responsible to make phospholipid membranes highly permeable to polar molecules as it had been largely suspected. The ability of these proteins to alter the permeability barrier of phospholipid membranes had been reported, but mainly as a consequence of their interaction with membranes upon injection as concentrated solutions in organic solvents [START_REF] Chang | Analysis of binding and membrane destabilization of phospholipid membranes by surfactant apoprotein B[END_REF][START_REF] Cruz | Different modes of interaction of pulmonary surfactant protein SP-B in phosphatidylcholine bilayers[END_REF][START_REF] Shiffer | Interactions of the low molecular weight group of surfactant-associated proteins (SP 5-18) with pulmonary surfactant lipids[END_REF]. In the present work, we demonstrate that i) native surfactant membranes are indeed intrinsically permeable, ii) proteins SP-B and SP-C are responsible of the permeability properties of surfactant structures, and iii) hydrophobic surfactant proteins are by themselves able to make a simple phospholipid membrane highly permeable to both membrane and soluble probes. This could be consistent with the assembly in the membranes of some sort of proteinaceous or proteolipid pores, because the mere introduction of the hydrophobic surfactant proteins into a single lipid model membrane is enough to permeabilize it. Permeabilization of membranes by surfactant proteins produces as a consequence a rapid equilibration of molecules, which are intrinsically impermeable through pure lipid membranes, among the different compartments. We cannot discard that membrane-permeabilizing protein assemblies are not also promoting membrane-membrane contacts that would facilitate even further the rapid movement of polar and non-polar molecules through the different membranes and compartments. The effect of the proteins to facilitate lipid dynamics in large multilamellar arrays has been widely reported [START_REF] Bernardino De La Serna | Segregated phases in pulmonary surfactant membranes do not show coexistence of lipid populations with differentiated dynamic properties[END_REF][START_REF] Perez-Gil | Interactions of hydrophobic lung surfactant proteins SP-B and SP-C with dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol bilayers studied by electron spin resonance spectroscopy[END_REF]. The present results introduce the effects on membrane permeability as an additional factor on surfactant dynamics. Several evidences had already suggested a potential effect of surfactant proteins on membrane permeability. SP-B has been reported to promote association and fusion of phospholipids membranes [START_REF] Chang | Analysis of binding and membrane destabilization of phospholipid membranes by surfactant apoprotein B[END_REF][START_REF] Poulain | Kinetics of phospholipid membrane fusion induced by surfactant apoproteins A and B[END_REF]. This SP-B-promoted fusion of lipid vesicles was described as "leaky", meaning that the protein-promoted merging of membrane compartments was always associated with leakage of at least part of the vesicle contents into the outer spaces. Our results suggest that leakage could not only occur at the fusion sites, but that membrane fusion and the subsequent transfer of the protein into the different lipid structures could end in the complete permeabilization of all the membranes. This permeabilization could be also behind the reported ability of hydrophobic surfactant proteins to permeabilize membranes to ions [START_REF] Oelberg | Pulmonary surfactant proteins insert cationpermeable channels in planar bilayers[END_REF], a property that could not be explained and has not A c c e p t e d M a n u s c r i p t been characterized any further. Also, we and others had noticed the problems to encapsulate polar solutes into membrane vesicles made from pulmonary surfactant, a relevant technology in the context of drug-delivering strategies, but that may be more difficult to achieve than anticipated in the light of the present results. The difference in the kinetics of membrane probe equilibration is consistent with SP-B and SP-C promoting membrane permeability by different mechanisms. The almost instantaneous permeation of probes through phospholipid membranes containing minimal amounts of SP-B could be indicative of the protein forming true pores. The sequence of SP-B is homologous to saposins, a family of membraneassociated proteins that includes several recognized cytolysins and membrane-pore forming structures [START_REF] Leippe | Ancient weapons: the three-dimensional structure of amoebapore A[END_REF][33][START_REF] Patthy | Homology of the precursor of pulmonary surfactant-associated protein SP-B with prosaposin and sulfated glycoprotein 1[END_REF]. We propose that pore-like oligomeric SP-B assemblies could be at the same time responsible to establish membrane-membrane contacts and to facilitate rapid transfer of lipids and polar molecules between the contacted membrane compartments. In contrast, the kinetics of probe permeation by SP-C is much slower than that promoted by SP-B, and much more dependent on a high enough proportion of protein into the membranes. We propose that, in contrast to SP-B, membrane permeabilization by SP-C could be rather dependent on the deep perturbation of the membrane core by the protein. These profound perturbations could be also the cause of the extraordinarily dynamic behaviour imparted by SP-C into the membrane morphology, as we have observed in the GUVs fluctuations. The ability of SP-C to introduce a dynamic contribution into phospholipid membranes has been already outlined, in which the N-terminal, relatively polar, segment of the protein seems to have a significant participation [35,[START_REF] Plasencia | The Nterminal segment of pulmonary surfactant lipopeptide SP-C has intrinsic propensity to interact with and perturb phospholipid bilayers[END_REF]. The present results strongly suggest that the combination of SP-B and SP-C impart special properties to membranes, which are clearly distinct to the effects of each individual protein by itself. Surfactant membranes are the most stable in the simultaneous presence of both SP-B and SP-C, while the individual proteins seem to have apparently opposite destabilizing effects. SP-B alone promotes transformation of single vesicles into large, complex, presumably multilamellar, membrane structures. This ability of the protein to promote association and eventually fusion of membranes, with the important participation of proteinprotein interactions, has been in fact widely documented [START_REF] Ryan | Mapping and analysis of the lytic and fusogenic domains of surfactant protein B[END_REF][START_REF] Cruz | Microstructure and dynamic surface properties of surfactant protein SP-B/dipalmitoylphosphatidylcholine interfacial films spread from lipid-protein bilayers[END_REF][START_REF] Chang | Analysis of binding and membrane destabilization of phospholipid membranes by surfactant apoprotein B[END_REF][START_REF] Poulain | Kinetics of phospholipid membrane fusion induced by surfactant apoproteins A and B[END_REF][START_REF] Poulain | Effects of surfactant apolipoproteins on liposome structure: implications for tubular myelin formation[END_REF]. SP-C, by its side, seems to strongly destabilize bilayers, promoting their disintegration and dispersion. This activity could be behind the ability of the protein to promote insertion of phospholipids into the interface [38,[START_REF] Perez-Gil | Interfacial adsorption of simple lipid mixtures combined with hydrophobic surfactant protein from pig lung[END_REF]. However, the incorporation of SP-B/SP-C complexes, with the precise protein stoichiometry obtained from the natural surfactant mixtures, maintains apparently the morphology of the lipid-protein membranes, at least in the context of the models studied here, suggesting that SP-B and SP-C might modulate each other. An obvious possibility is that the two proteins participate in common macromolecular protein complexes in the context of surfactant membranes. The differences in permeability induced upon introduction of single or combined proteins into giant liposomes are very illustrative. In the presence of SP-B/SP-C complexes, lipid membranes are permeable to calcein, which can permeate through pores of presumably limited size, considering that dilution of external calcein does not produce a concomitant immediate dilution of calcein from the internal liposome compartments. Equilibration of inner and outer calcein concentrations is much faster in GUVs containing either SP-B or SP-C, suggesting that the permeation structures assembled by a single protein permit a much rapid flow of the probe. Although recent results certainly suggest that a concerted action of the two proteins facilitates breathing dynamics in vivo [START_REF] Almlen | Surfactant proteins B and C are both necessary for alveolar stability at end expiration in premature rabbits with respiratory distress syndrome[END_REF], we and others have were unable to find evidences for specific SP-B/SP-C interactions [35]. In a recent study in which we have compared functional properties of systems prepared by reconstituting defined fractions of native surfactant, we have found that some differences exist between the behaviour of lipid/protein complexes prepared from the original mixtures and the behaviour of complexes reunited from isolated components [START_REF] Schurch | Combined and independent action of proteins SP-B and SP-C in the surface behavior and mechanical stability of pulmonary surfactant films[END_REF]. It is possible that proper SP-B/SP-C complexes are poorly re-stablished once the proteins are re-mixed, perhaps because some unknown cofactors were lost, or because the proper conformation of the proteins is altered, during the purification process. Our present data suggest that much care has to be taken to dissect the potential role and the structure-function relationships of surfactant proteins upon reconstitution of simplified systems that start from fully purified SP-B and SP-C. An unbalance in the appropriate SP-B/SP-C ratio or an inefficient re-assembly of potential multiprotein complexes, may end in lipid-protein membranes with structural and functional properties differing much of those exhibited by native surfactant membranes. The most successful clinical surfactant preparations currently in use for the treatment of respiratory pathologies are derived from extracts of animal-derived surfactants [START_REF] Blanco | Biochemical and pharmacological differences between preparations of exogenous natural surfactant used to treat Respiratory Distress Syndrome: role of the different components in an efficient pulmonary surfactant[END_REF]. These natural formulations probably preserve a fair proportion of the original surfactant protein complexes. Design and production of a new generation of therapeutic surfactants, based on the reconstitution of synthetic or recombinant human versions of surfactant proteins, will require a proper knowledge of the role of protein complexes and the way these complexes can be correctly established. An important question opened refers to the physiological meaning of the intrinsic permeable character of surfactant membranes. It is possible that the permeability to polar solutes imparted to membranes by the surfactant protein complexes is only a consequence of the architecture of the machinery that ensures a rapid and efficient flow of surface active lipid species along the membranous assemblies, all the way up to the interface. However, it is also possible that an efficient and unrestricted movement of polar molecules through the membrane structures developed by surfactant at the thin water layer lining the alveoli is also important for alveolar homeostasis. It has been recently proposed that surfactant might form a sort of "bi-continuous" membrane-based phase at the respiratory surface [START_REF] Perez-Gil | Structure of pulmonary surfactant membranes and films: the role of proteins and lipid-protein interactions[END_REF]43]. A fully interconnected network of membranes could be important to facilitate a rapid diffusion of lipids towards the interface, where they must form the surface active film that reduces surface tension and stabilizes the lung. However, the continuity of all the aqueous compartments could be equally important to ensure proper equilibrium of water and ions and to facilitate free diffusion of soluble components such as many of those in charge of maintaining the sterility of the alveolar surface. Surface active proteins and peptides, such as those currently under scrutiny as potential additive in artificial surfactants [START_REF] Mingarro | Synthetic pulmonary surfactant preparations: new developments and future trends[END_REF], could be active enough to ensure interfacial transfer of lipids and formation of surfactant films but still be inefficient to form a fully functional bi-continuous membrane network. The elucidation of the molecular architecture and the determinants of potential surfactant protein complexes is therefore a must to improve the possibilities to develop new enhanced surfactant-based therapies. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

LEGEND TO FIGURES

Biochemical

  Journal Immediate Publication. Published on 16 Jun 2011 as manuscript BJ20110681 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110681A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  of the accessibility of membranes and the environment sensed by the probes on the lipid and protein composition. Samples characterized in this study include complexes of native surfactant purified from porcine lungs and membranes reconstituted from whole surfactant organic extract or from partial fractions obtained by size-exclusion chromatography, which were compared to model lipid membranes. The effect of SP-B and SP-C on the permeability of model membranes has been also analyzed by fluorescence microscopy, through the reconstitution of POPC giant vesicles (GVs) in the absence or presence of the hydrophobic protein fraction of surfactant or purified SP-B or SP-C. Biochemical Journal Immediate Publication. Published on 16 Jun 2011 as manuscript BJ20110681 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110681A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 16 Jun 2011 as manuscript BJ20110681 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110681 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 16 Jun 2011 as manuscript BJ20110681 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110681A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 16 Jun 2011 as manuscript BJ20110681 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110681A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 16 Jun 2011 as manuscript BJ20110681 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110681

Biochemical

  Journal Immediate Publication. Published on 16 Jun 2011 as manuscript BJ20110681 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110681A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Fig. 1 .

 1 Fig. 1. Dependence of Nile Red and FM 1-43 (1 Pg/ml) fluorescent emission on phospholipid concentration in different materials. All values have been normalized, being F 0 the maximum emission of each probe corresponding to the highest phospholipid concentration tested: 700 Pg/ml in the case of Nile Red and 350 Pg/ml for FM 1-43 .

Fig. 2 :

 2 Fig.2: Comparison between unilamellar and multilamellar systems for native pulmonary surfactant, organic extract and POPC. The reference value F 0 is specific for each material, corresponding to the emission of FM 1-43 in the multilamellar sample with the highest lipid concentration tested.

Fig. 3 :

 3 Fig.3: Comparison between unilamellar and multilamellar systems for model membranes of POPC and membranes of POPC supplemented with the hydrophobic protein fraction, 1% (w/w) SP-B and 1% (w/w) SP-C. The reference value F 0 is specific for each material, corresponding to the emission of FM 1-43 in the pure POPC sample with the highest lipid concentration tested.

Fig. 4 :

 4 Fig. 4: Kinetic studies of the incorporation of FM 1-43 to multilamellar vesicles of POPC supplemented with different percentages (w/w P/L) of SP-B and SP-C. Results correspond to three different phospholipids concentrations: 50 (z), 200 (V) and 400 Pg/ml (). Kinetic studies of the incorporation of FM 1-43 to multilamellar vesicles of POPC supplemented with different percentages (by weight of phospholipids) of SP-B and SP-C. Results are shown in comparison with the control sample, MLV of pure POPC.

Fig. 5 :

 5 Fig. 5: Fluorescence emission of the probe FM 1-43 in POPC multilamelar vesicles (200 Pg/ml) as a function of protein density in membranes.

Fig. 6 :

 6 Fig. 6: Electron microscopy images of vesicle suspensions prepared by extrusion, which correspond to different lipid-protein composition: POPC (image A), POPC supplemented with the hydrophobic protein fraction of pulmonary surfactant (B), with 1% SP-B (C) and 1% SP-C (D) by weight of phospholipids. Samples were prepared by negative staining.

Fig. 7 :

 7 Fig. 7: Giant oligolamellar vesicles of POPC (A-D) and POPC supplemented with the hydrophobic protein fraction of pulmonary surfactant (E-H). A, C, E and G are transmission images; B, D, F and H, fluorescent images. All the giant vesicles are labelled with FM 1-43 . Scale bars correspond to 10Pm.

Fig. 8 :

 8 Fig. 8: Giant oligolamellar vesicles of POPC supplemented with 1% (w/w) SP-B (images A-D) and 1% (w/w) SP-C (E-H). A, C, E and G are transmission images; B, D, F and H, fluorescent images. Giant vesicles are labelled with FM 1-43 . Scale bars: 10Pm.

Fig. 9 :

 9 Fig.9: Image sequence extracted from a video recording. Giant vesicles of POPC supplemented with 1% (w/w) SP-C became very unstable and dynamic when observed under the microscope; even exocytotic-like processes were seen. The complete recording is available on Additional Material section. Scale bar: 20 Pm.

Fig. 10 :

 10 Fig. 10: Giant vesicles of POPC (first row), POPC supplemented with the hydrophobic protein fraction of pulmonary surfactant (second row), with 1% (w/w) SP-B (third row) and

  1% (w/w) SP-C (fourth row). The first column corresponds to transmission images; the second and the third columns, to fluorescent images obtained with FITC and Texas Red filter sets, respectively. Giant vesicles were incubated in a calcein solution for several hours, then diluted in a glucose solution and finally labelled with FM 1-43 prior to the observation. See Experimental section for details. Biochemical Journal Immediate Publication. Published on 16 Jun 2011 as manuscript BJ20110681 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110681A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 Biochemical

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© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

ACKNOWLEDGEMENTS

This research has been supported by grants from the Spanish Ministry of Science (BIO2009-09694, FIS2009-14650-C02-01 and CONSOLIDER-INGENIO 2010 CSD2007-00010), Community of Madrid (S0505/MAT/0283) and Universidad Complutense.


A Sanna

T G Karayiannis
email: tassos.karayiannis@brunel.ac.uk

D B R Kenning

C Hutter

K Sefiane

A J Walton

I Golobič

E Pavlovič

R A Nelson

STEPS TOWARDS THE DEVELOPMENT OF AN EXPERIMENTALLY VERIFIED SIMULATION OF POOL NUCLEATE BOILING ON A SILICON WAFER WITH ARTIFICIAL SITES

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Most applications and studies of nucleate boiling heat transfer have been for metallic surfaces, on which the spatial and size distributions of nucleation sites can only be controlled in a general way during manufacture. Applications of boiling to the cooling of devices micro-fabricated on silicon open the way to the creation of large ACCEPTED MANUSCRIPT arrays of closely-spaced nucleation sites of specified micro-geometry. Theoretical methods are required to optimise the design of the arrays in a rational way that takes account of the interactions between the sites. Interactions occur by a number of thermo-hydrodynamic mechanisms in the liquid-vapour region that may be coupled to interactions by conduction in the heated wall. Three-dimensional numerical simulations of the combined fluid-solid regions are currently limited to interactions between a few sites, e.g. [START_REF] Mukherjee | Study of Lateral Merger of Vapor Bubbles During Nucleate Pool Boiling[END_REF], [START_REF] Dhir | Mechanistic Prediction of Nucleate Boiling Heat Transfer -Achievable or a Hopeless Task?[END_REF], and have a minimum spatial resolution that is usually larger than the radius of typical nucleation sites. Hybrid mechanistic simulations, reviewed by [START_REF] Kenning | Mechanistic Models for Pool Nucleate Boiling Heat Transfer: Input and Validation[END_REF], replace the accurate simulation of events in the fluid region by simplified physical models, correlations and networking rules, e.g. for site activation, bubble growth and departure, seeding of unstable sites and local heat transfer. Nelson and colleagues at Los Alamos National Laboratory developed a hybrid model to investigate the influence of wall properties on interactions by conduction between hundreds of sites, [START_REF] Pasamehmetoglu | Cavity-to-Cavity Interaction in Nucleate Boiling: the Effect of Heat Conduction within the Heater[END_REF]. The transient conduction equation was solved numerically on a 3-D rectangular grid representing the wall, with nucleation sites having different activation superheats placed at selected grid nodes at the wall-fluid interface. Bubble-related events in the fluid were represented by very simple sub-models. These models were later modified to simulate boiling at high heat flux with vapour chimneys through a liquid macrolayer between the wall and a hovering bubble, [START_REF] Sadasivan | Nonlinear Aspects of High Heat Flux Nucleate Boiling[END_REF]. Golobič et al. (1996a,b) at Ljubljana University developed a version of the low-flux model, in which sites could be placed at any position and the computational grid was refined temporarily in a cylindrical region under every growing bubble. The bubble growth sub-model was a simplified version of the [START_REF] Stephan | A New Model for Nucleate Boiling Heat Transfer[END_REF] model for evaporation at a triple contact line, now regularly employed in 3-D numerical simulations of bubble growth, as in [START_REF] Dhir | Mechanistic Prediction of Nucleate Boiling Heat Transfer -Achievable or a Hopeless Task?[END_REF]. Only interactions transmitted through the wall were included in the model.

The validation of a multi-site model against experimental data for boiling on metal surfaces is difficult because there are few sufficiently detailed data sets to provide input information about site positions and characteristics in addition to data to test the model, and because the potentially chaotic nature of boiling (Nelson et al., 1996, Mosdorf andShoji, 2004) requires statistical methods, [START_REF] Kenning | Mechanistic Models for Pool Nucleate Boiling Heat Transfer: Input and Validation[END_REF]. These issues were explored by comparing the Ljubljana model with liquid crystal measurements of the spatio-temporal temperature fields on the back of a thin stainless steel plate during the boiling of water, [START_REF] Golobič | Comparison of a Mechanistic Model for Nucleate Boiling with Experimental Spatio-temporal Data[END_REF], during which bubbles were detected by computerised image analysis at 1505 locations, most of which were concentrated in 16 dense clusters. The poor agreement between the data and the model may have been caused by a combination of shortcomings in the data and the very simple sub-models used in the simulation. Sub-models may be improved by comparisons with experimental data and fully-3D simulations for isolated sites. Golobič et al. (2007a,b) measured wall temperature fields with higher spatial and temporal resolution by IR thermography during isolated bubble growth and bubble coalescence on very thin metal foils. The observations suggested that the contact line evaporation model for bubble growth was not appropriate for these particular conditions. Well before the advent of the triple contact line model, different modes of bubble growth were noted by [START_REF] Chesters | Modes of bubble growth in the slow-formation regime of nucleate pool boiling[END_REF], Fig. 1, illustrated here by the differences in bubble growth and detachment during boiling of water from natural sites on a thin metal foil (Golobič et al., 2007b), Fig. 2(a), and from micro-machined sites on a thick silicon disk (Mosdorf and Shoji, 2004), Fig. 2(b). The internal geometry of nucleation sites influences bubble production, [START_REF] Shoji | Nucleation Site Interaction in Pool Nucleate Boiling -Serial Experiments Using Artificial Boiling Surfaces[END_REF].

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Micro-fabricated sites on silicon provide much greater control over site positions and characteristics than can be achieved with natural sites on metal surfaces. Shoji and co-workers at Tokyo University used this approach for an exhaustive investigation of the thermo-hydrodynamic interactions between sites, e.g. [START_REF] Zhang | Nucleation Site Interaction in Pool Boiling on the Artificial Surface[END_REF], [START_REF] Chatpun | Experimental Study on Characteristics of Nucleate Pool Boiling by the Effects of Cavity Arrangement[END_REF], [START_REF] Shoji | Nucleation Site Interaction in Pool Nucleate Boiling -Serial Experiments Using Artificial Boiling Surfaces[END_REF]. Experiments were conducted on a silicon wafer (thickness 0.2 mm, diameter 15 mm) with cylindrical cavities (diameter 10 µm, depth 80 µm) arranged as single or twin cavities with different spacing S (1, 2, 3, 4, 6 and 8 mm). The boiling liquid was saturated water and the silicon wafer was heated uniformly from below by laser irradiation. Temperature fluctuations on the rear of the wafer were measured along lines through the centres of cavities with an IR camera in line-scanning mode (spatial resolution 120 µm, temperature resolution 0.08 K and time resolution 3 ms). Three types of interaction were identified: thermal interaction between nucleation sites, hydrodynamic interaction between bubbles (longrange, transmitted through the fluid) and horizontal and declining bubble coalescence (lateral, short-range). Their ranges of influence on average bubble departure frequency are shown in Fig. 3 in terms of the ratio of the spacing parameter S to a characteristic bubble dimension D b . Recently [START_REF] Sato | Experimental Study on Fundamental-Microscopic Mechanism of Boiling by using MEMS Technique (Examination from Aspect of Time Series Chaos Analysis[END_REF], used a similar experimental setup with a single site or triple in-line sites at S = 1, 2, 3, 4 mm that produced bubbles with departure diameters in the range 2.5 -3 mm. They performed chaos analysis of the time series for bubble growth and wall temperature at the single and central sites by constructing return maps, using delay times much smaller than the bubble growth time. They concluded that bubble growth at a single site was nearly deterministic but that the frequent coalescence between bubbles at triple cavities with S ≤ 3mm made bubble diameter deterministically unpredictable over a long time period. The attractors of wall temperature for interacting sites had some structure indicative of chaotic complexity, rather than random variation. Interaction disappeared for S ≥ 4 mm.

There were some differences between the findings of [START_REF] Shoji | Nucleation Site Interaction in Pool Nucleate Boiling -Serial Experiments Using Artificial Boiling Surfaces[END_REF] and those of Gjerkěs andGolobič (2001, 2003) for long-range interactions between natural sites on a metal foil and a site controlled by localized heating by a focussed laser, using different measures of intensity. Golobič et al. (2007a,b) later measured with a high-speed, high resolution IR camera the two-dimensional temperature fields under isolated and coalescing sites on metal foils.

An alternative approach is to micro-fabricate on a transparent, electrically-insulating substrate a closelyspaced array of electrical resistance heaters, each with its own control system so it can be run either at constant temperature or at constant heat flux. A design by [START_REF] Rule | Heat Transfer Behavior on Small Horizontal Heaters During Pool Boiling of FC-72[END_REF] was improved, as described in [START_REF] Demiray | Microscale Heat Transfer Measurements During Pool Boiling of FC-72[END_REF] and [START_REF] Myers | Time and Space Resolved Wall Temperature and Heat Flux Measurements During Nucleate Boiling with Constant Heat Flux Boundary Conditions[END_REF] to give a boiling surface formed of 96 closely-spaced platinum resistance heater elements deposited on a quartz wafer. Simultaneous high-speed images of bubbles in boiling FC-72 were taken through the semi-transparent heaters. [START_REF] Cheng | Heat transfer effects of coalescence of bubbles from various site distributions[END_REF] investigated coalescence with a similar array, initiating nucleation by pulse heating of individual elements. This is a way of avoiding the difficulties of hysteresis and irregular activation at natural and artificial cavity sites.

It is clear that experimental studies of bubble growth, detachment and interaction ranging from detailed studies of heat transfer patterns around individual nucleation sites to studies of the networked interactions of a large population of sites covering a large area lead to conflicting requirements for instrumentation and data analysis. The translation of experimental measurements into simple rules for the physics sub-models in hybrid models for numerical simulation is not straightforward and the hybrid models must be sufficiently flexible to accommodate different modes of bubble growth.

OBJECTIVES

This paper reports progress on a project to develop the Ljubljana code into a design tool for arrays of micromachined nucleation sites to achieve high rates of cooling of silicon chips by pool boiling. The developments will include improvements to the physical sub-models and to numerical procedures to increase computational speed and remove some current restrictions on physical modelling, together with more formal Verification and Validation of the code than has been attempted previously. The first part of the paper describes the structure of the Ljubljana model and code, identifies aspects requiring development and gives some examples of work done so far.

The development of the physical sub-models will depend on experiments at Edinburgh University on boiling of FC-72, and later water, on arrays of sites and micro-thermometers micro-fabricated on silicon. The

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experiments will also provide data for validation of the design code. Further information will be drawn from a separate investigation at Ljubljana University of pool boiling on thin metal foils, employing high-speed IR thermography to measure 2-D wall temperature variations, Golobič et al. (2007a,b).

The philosophy for design of the experiments is summarized in the second part of the paper.

THE LJUBLJANA MODEL

The code solves the 3-D transient conduction equation for the temperature distribution T (x,y,z;t) in a rectangular slab representing the heated wall of thickness δ, with 1. specified edge boundary conditions, e.g. constant temperature, adiabatic or connection to unheated wall, 2. specified volumetric heat source (ohmic heating of metal foil) and/or 3. specified heat flux distribution (x,y;t) on rear surface z = 0 (thin-film heaters on silicon), 4. specified positions and properties of potential nucleation sites on front surface z = δ, 5. specified heat flux distribution (x,y;t) on z = δ (local thin-film heaters to trigger nucleation sites on silicon), 6. specified heat transfer coefficient distribution h(x,y;t) on z = δ, supplied by approximate physics modules for bubble nucleation, growth, departure and coalescence, single phase convection and hydrodynamic interactions that replace accurate simulation of the fluid region.

The transient conduction equation is solved by a simple explicit method, with consequent limitations on numerical accuracy and stability. The basic computational mesh is temporarily refined in a cylindrical region round an active site during bubble growth from nucleation to detachment. The original version of the code, [START_REF] Golobič | Comparison of a Mechanistic Model for Nucleate Boiling with Experimental Spatio-temporal Data[END_REF], employed an unstructured basic mesh, which was appropriate for very irregularly-spaced natural sites on a metal foil. This has been replaced by a rectangular mesh that is suitable for more regular arrays of artificial sites, saving some of the time required for mesh generation.

Originally, the mesh was refined in a region of fixed radius corresponding to the maximum contact radius, which occurred at detachment and which was specified at each site. In order to avoid overlapping of refined regions, nucleation of a new bubble had to be prohibited at sites close to a growing bubble, thus artificially preventing coalescence and in some circumstances forcing bubbles to grow alternately at a pair of sites. This constraint has been partially removed by improvements to the meshing procedure and the bubble growth model, so that the radius of the refined region grows in a series of steps to follow the contact radius of the bubble as it expands and then contracts in the approach to detachment, Fig. 4, and the introduction of an approximate model for coalescence, described below. The minimum distance between two sites allowed to be active at the same time was equal to the sum of their maximum contact radii, increased by 20% to allow at least one row of Cartesian unrefined cells between the refined regions. With the new procedure, combined with the variation of the apparent contact angle described below, the minimum distance can be reduced by more than 50% during the early stages of bubble growth. The procedure will be further improved to eliminate a loss of accuracy during mesh replacement due to the present impossibility of replacement of one circular mesh distribution by another without first merging cells into the original Cartesian grid. These improvements significantly increase the computing time and introduce small errors in calculations of bubble growth and temperature at the nucleation sites.

The code has been restructured by Nelson at LANL for parallel computation: running time has been strongly reduced; formal verification will be performed.

The primary outputs from the code are the bubble radius r bd,i , the contact radius r c,i , the temperature T i and records of activity for each nucleation site and the instantaneous temperature T(x,y;t) and heat flux q(x,y;t) distributions on the front surface z = δ, which are used in the physics sub-models.

The current criterion for nucleation is that bubble growth commences when the local wall superheat rises to an activation superheat (corresponding to an equivalent critical radius) that has to be inserted as input datum for each potential nucleation site. Immediate nucleation of one bubble following the detachment of a preceding bubble is permitted but the thermo-fluid consequences of vertical coalescence of bubbles from the same site are This criterion is appropriate for nucleation at a critical radius within a cavity. It will be extended in due course to include the effect of the external temperature gradient in the liquid due to the local wall heat flux. Other influences that will be examined are hydrodynamic disturbances due to bubble growth at adjacent sites and seeding of unstable sites by vapour from a stable site in close proximity.

Approximate models have to be defined for the shape of the bubble, the various heat flows into it and its detachment. The original version of the code employed very simple models. The bubble was assumed to grow as a truncated sphere with a constant apparent contact angle φ. Heat for evaporation was supplied only through the base of the bubble; heat transfer from the bulk liquid through the dome was neglected. Heat transfer through the base of the bubble was based on the [START_REF] Stephan | A New Model for Nucleate Boiling Heat Transfer[END_REF] contact line concept. The contact area was divided into two concentric zones:

(i) a central circle covered by an adsorbed liquid film with a very low heat transfer coefficient;

(ii) an outer ring with a high heat transfer coefficient, representing the heat flow at the triple contact line. The width of the ring was much greater than the sub-micron length scale proposed in models of the contact zone and the heat transfer coefficient was reduced accordingly. The values of the heat transfer parameters were chosen to match growth rates with experimental observations. The effects of interfacial non-equilibrium at high heat fluxes were assumed to be included in the heat transfer coefficient, so that the vapour was at the saturation temperature.

The model for (i) was modified in the [START_REF] Golobič | Comparison of a Mechanistic Model for Nucleate Boiling with Experimental Spatio-temporal Data[END_REF] version of the code to allow for the possible presence of liquid drops trapped in surface roughness in the adsorbed film region, by having a somewhat higher heat transfer coefficient at the nucleation site, decreasing linearly towards the triple contact line. Detachment occurred at a specified volume expressed as the equivalent radius of a sphere, with values obtained from correlations or experimental observations for individual sites.

Recent experiments with thin metal foils, Golobič et al. (2007a,b), have shown that heat transfer may be maintained over the entire contact area, with the highest heat transfer coefficient at its centre, decreasing radially without a maximum at the apparent contact line. The code has been modified so that any radial distribution of heat transfer coefficient may be specified; choice of the correct model depends on experiments. All the examples in this paper have been simulated using a zero heat flux in the inner area, a very high heat transfer coefficient for the ring containing the triple contact line and an enhanced natural convection heat transfer coefficient (not contributing to the bubble growth) for the ring outside the triple contact line region. The latter accounts the effect of movements of colder liquid from the region far from the bubble; example of this phenomenon can be observed in Golobič et al. (2007a,b) that also measured an enhancement of the heat flux in a narrow ring just outside the expanding contact area in the early stages of bubble growth. When the contact area contracted prior to detachment, the heat flux measured at its periphery was reduced, instead of enhanced. Since contraction of the contact area is now included in the code, further investigation is required to determine how this effect may be represented in the hybrid model.

In its original form, the code applied a uniform and single phase heat transfer coefficient slightly dependent on temperature in the wall region outside the bubble contact area. Its value was taken from a standard correlation for natural convection on a horizontal plate. In addition, the code contained provision for enhanced single phase heat transfer in a circular area of influence immediately following bubble detachment at maximum contact radius, calculated by the "wall quenching" model of transient conduction to liquid at the bulk temperature. This feature of the code is not employed at present, because it greatly overestimated the heat transfer measured for conditions of rapid bubble growth followed by a long waiting time in experiments on thin metal foils, Golobič et al. (2007a,b). Further research is required to determine whether it may be appropriate in other conditions, e.g. when streams of bubbles are produced with small waiting times or in boiling at large subcooling.

In the current project, these modelling assumptions are being developed to improve their physical realism, usually at the cost of higher simulation time and introduction of more parameters that have to be specified in the computer code. The following improvements have now been implemented:

Heat transfer to the bubble dome has been added, estimated by

( ( ( ( ) ) ) ) 2 1 - -- - ⋅ ⋅ ⋅ ⋅ ∆ ∆ ∆ ∆ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ = = = = t T k A Q l D πα πα πα πα ψ ψ ψ ψ & (1)
where A is the surface area of the dome at time t after nucleation, ∆T is a specified fraction (usually 0.5) of the activation superheat of the nucleation site, and ψ is a parameter to fix the relative importance of the heat flux at the dome. During the following simulation, ψ has been set equal to 1.

The original assumption of constant apparent contact angle resulted in sudden detachment when the contact area was a maximum. The first method used to model detachment at zero contact area was to reduce the contact angle linearly with time after the bubble reached a defined fraction (e.g. 0.6) of the specified detachment radius. The sub-model required a manual determination of the correct rate of decrease to match original detachment

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conditions when the bubble radius reaches its maximum value. The linear dependence on time has been replaced by a linear decrease with the inverse of the bubble volume, as shown in Fig. 5a,b. This matches approximately the bubble evolution during boiling on a thin metal foil, Fig. 2a. A comparison of the use of constant and variable contact angles for a bubble detachment radius of 1.75 mm in water on a thin (25 µm) titanium foil, with an equivalent heat flux of 50 kWm -2 is shown in Fig. 6. Detachment with a zero apparent contact angle leads to an increase in growth time of approximately 10%, and a decrease of waiting time. This is caused by the different temperature distribution around the nucleation site after detachment, as shown in Fig. 7. For the constant contact angle, a deep trough in the temperature profile, caused by previous cooling at the triple contact line, reduces the lateral heat flow towards the centre of the site, leading to a slower recovery. By contrast, for the variable contact angle simulation, the trough is at the centre, and the effect of lateral conduction, not relevant over a large scale for a thin foil as discussed later, becomes important for a short distance. The temperature profile and bubble period strongly depend on the contact angle rate of decrease and on the value of the fraction of the specified detachment radius at which the decrease is specified to start.

A second detachment model, in which a conical extension of the truncated sphere is introduced to match a detachment shape approximating to Fig. 2b, is under development.

At present, the only hydrodynamically transmitted interaction between bubbles introduced into the code is a rudimentary model for coalescence. If two bubbles grow in close proximity, their domes will come into contact while their contact areas are still separate. Two situations are considered, both of which were observed in Golobič et al. (2007b):

(a) If the bubbles have similar size, Fig. 8a, they are assumed to continue growing as two independent isolated bubbles, but a dedicated heat transfer coefficient (h C ) is defined for the region between them. In reality, immediately after coalescence, the bubble assumes the shape of a horizontally elongated bubble.

(b) If the sizes are sensibly different, Fig. 8b, the smaller bubble is assumed to be rapidly absorbed by the larger bubble, which undergoes a sudden increase in volume. A larger departure radius must then be specified for the combined bubble. Examples of the application of this model are given in a conference paper, [START_REF] Sanna | Simulation and experimental investigation of pool boiling on a silicon wafer with artificial nucleation sites[END_REF].

PRELIMINARY RESULTS

Two simulations have been run to evaluate the influence of the different wall characteristics for experiments in Ljubljana and Edinburgh Universities, using respectively a titanium foil 25 µm thick with natural cavities and a silicon wafer 0.38 mm thick with micro-machined cavities. Table 1 summarizes the characteristic of the two plates. Saturated water at atmospheric pressure has been used for both simulations, although initial experiments

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in Edinburgh employ FC-72. All other inputs to the code are the same (bubble departure radius, activation temperature, apparent contact angle, heat transfer coefficients). The disposition of the nucleation sites has been chosen to avoid coalescence but to allow the possibility of thermal interaction. In order to reduce computing time, the number of sites has been kept small and a low level of grid refinement has been used. Consequently, the grid values in plots of the radial distributions of temperature and heat flux are joined by straight lines.

The geometry used for the simulations was a 24x24 mm solid plate with seven potential active sites located at the centre in triangular symmetry at a spacing of 4 mm. Fig. 9 shows the mesh distribution when all the sites are active with each dot representing the centre of a cell mesh. The site activation temperature is 115.5 ºC. The bubble departure diameter is 3.5 mm, with an initial apparent constant angle of 32º. The contact angle reduction model has been used and the uniform volumetric heat source corresponds to an input heat flux of 50 kWm -2 . The bottom of the plate is adiabatic and its edges are kept at a constant temperature of 125 ºC, approximately equal to the equilibrium temperature in the case of pure natural convection cooling.

Comparison between simulations for Ti and Si plates

Representative distributions of wall surface temperatures on a line passing through sites 5 -1 -2 are shown in Figs. 10 and 11 for titanium and silicon respectively. The restricted edge effects and the large areas of uniform temperature outside the area influenced by the cluster of sites in Fig. 10 indicate that lateral conduction is very small because of the small thickness of the titanium foil. The temperature between the sites is nearly the same as the undisturbed temperature, so there is hardly any thermal interaction between the sites. Each site has to remove only the heat supplied in its immediate vicinity. Because of the slight differences in conditions, bubbles at the different sites grow out of phase but over a long period of simulation their growth characteristics are similar, Table 2. Because of the low thermal capacity of the titanium foil, there are large variations in the local temperature, falling to within 4 K of the saturation temperature under the bubbles. By contrast, everywhere on the thicker silicon wafer in Fig. 11 is influenced by lateral conduction. There are large flows of heat to the edges and towards the cluster of sites, which must therefore remove more heat than the sites on the titanium foil for the same uniform heat input. The total volumetric production of vapour is therefore increased and, because the bubble departure volume is fixed in the simulations, the computed bubble periods are reduced. There is a strong thermal interaction between the sites on silicon and the outer sites receive more heat by lateral conduction than the central site. The local fluctuations in temperature are reduced by the much larger thermal capacity of the silicon wafer and the higher transient lateral conduction. The computed bubble periods, growth and waiting 2. The absence of thermal interaction on the titanium foil results in similar times at all the sites. On silicon, all the sites have similar growth times that are sensibly shorter than on titanium, because the wall superheat is less depressed. The waiting times at the outer sites are negligible, which is consistent with experimental observations of boiling on silicon. The waiting time at the central site is marginally longer, because it receives less heat by lateral conduction than the outer sites.

For both materials, there is a rapid initial reduction in the wall temperature at the nucleation site when the contact area is very small, because of the assumed very high heat transfer coefficient at the position of the triple contact line. As this region of high heat transfer coefficient moves outwards with the contact line, the reduction in temperature also moves outwards. There is a gradual recovery in the wall superheat at the nucleation site, as it is subjected to a much lower heat transfer coefficient. On the titanium foil, the wall superheat falls initially to less than 7 ºC, consequently limiting the heat flux, Fig. 12. The changes in superheat are smaller on the silicon wafer, with a reduction of 2-3 ºC, because of its much larger thermal capacity, combined with lateral conduction, Fig. 13. After the contact area reaches its maximum size and the contact line starts to move back towards the nucleation site, the region of high heat transfer coefficient moves back with it but the heat flux is strongly dependent on the pre-cooling of the wall during the previous expansion. There is a large effect for the titanium foil, Fig. 14. The inward-moving peak of high heat flux is followed by a narrow trough of reduced heat flux as liquid with a low heat transfer coefficient floods back into the pre-cooled contact area. This trough reproduces qualitatively the observations of Golobič et al. (2007a,b) for boiling on metal foils, noted earlier. The temperature and heat flux remain much higher on the silicon wafer, Fig. 15.

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As the peak in heat transfer coefficient and therefore in heat flux returns towards the nucleation site, just before detachment, the superheat there falls again, Figs. 16 and 17. The time taken for the superheat to recover to the activation superheat required to trigger the next bubble depends on the value of this second minimum in the superheat, the thermal capacity of the wall and the imbalance between heat input by generation and lateral conduction and heat removal by single-phase convection.

The model assumes that growth is driven by the heat flux through the contact area and also by transient conduction from the superheated liquid to the dome of the bubble. This second contribution is not affected by the cooling of the wall so it becomes relatively more important for bubble growth on the titanium foil than on the silicon wafer, as shown in Figs. 18,19.

Comments

These preliminary examples have employed an established model for bubble growth driven partly by intense evaporation at a moving triple-contact line. The radial distributions of heat flux calculated by the model are different in nature from those observed by Golobič et al. (2007a,b) for boiling on a 25 µm thick titanium foil, despite the apparent success in modelling a local trough in the heat flux during contraction of the contact area, but much more work is required to compare a variety of modelling assumptions with local measurements on thin metal foils and on thicker silicon wafers. The code has the necessary flexibility to do this and the examples have demonstrated its usefulness in exploring the influence of wall conditions on thermal interactions between bubble nucleation sites. Significant variations in local wall temperature may occur during boiling on a silicon wafer, even at the low heat flux of 50 kWm -2 used in the examples. The waiting time between bubbles, important in determining the cooling capability of a nucleation site, is large on the titanium foil but very small on the silicon wafer. In other circumstances, it may depend delicately on the interaction between the mechanism of heat transfer in the contact area of the bubble and the properties of the heated wall.

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continuously, with no waiting time, as shown in Fig. 23. There was a small decrease in the rate of growth, which approximately coincided with the maximum apparent contact radius. This may have been a consequence of the contact line receding over the pre-cooled wall as the bubble started to lift off. The bubble shape during detachment was similar to that for bubbles in water on silicon (Fig. 2b) rather than the more rounded shape for water on a titanium foil (Fig. 2a).

The next version of the silicon test section will have an integrated heater on the back and micro-sensors below the artificial nucleation sites, as shown schematically in Fig. 24.

CONCLUSION

This paper has reported on the early stages of a project to develop a numerical simulation of pool boiling as a design tool for arrays of micro-fabricated bubble nucleation sites to achieve the removal of high heat fluxes from a silicon chip. The simulation is based on numerical codes developed first at Los Alamos National Laboratory and then at Ljubljana University, employing a hybrid model in which 3-D analysis of conduction in the wall is combined with simple models for physical processes in the liquid-vapour region. Improvements to the

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bubble growth model with local refinement of the numerical mesh are described in the paper. These open the way to the inclusion of a model for the lateral coalescence of bubbles. The code has been restructured for parallel computing to meet the need for faster computation of more complicated models.

The simplified treatment of events in the liquid-vapour region necessarily relies heavily on experimental input. This information relies on very localised measurements taken during boiling on thin metal foils with natural nucleation sites in current experiments with high-speed IR thermography and from a newly commissioned rig employing a silicon wafer with micro-machined cavities and integrated micro-sensors.

The numerical hybrid code has the flexibility to incorporate the experimental findings as they become available. Its capability to simulate the different wall conditions in the titanium foil and silicon wafer has been demonstrated by two examples. The simulations suggest that there are significant changes in the wall temperature during bubble growth and detachment, even on a material of high thermal conductivity like silicon.

Figure 1 :Figure 2 :

 12 Figure1: Bubble growth modes, after[START_REF] Chesters | Modes of bubble growth in the slow-formation regime of nucleate pool boiling[END_REF]

Figure 3 :

 3 Figure 3: Site interactions, Shoji et al. (2005).

Figure 4 :

 4 Figure 4: Mesh refinement scheme

FigureFigure 6 :Figure

 6 Figure 5 a, b: Apparent contact angle models (a) constant, (b) variable a b

Figure 9 :

 9 Figure 9: Mesh distribution

Figure 12 :

 12 Figure 12: Heat flux and temperature distributions for titanium foil -contact area increasing

Figure 16 :

 16 Figure 16: Bubble growth and temperature time-trends for the titanium foil

Figure 17 :

 17 Figure 17: Bubble growth and temperature time-trends for the silicon wafer

FigureFigure 24 :

 24 Figure 23: Bubble growth at 11.7 kWm -2

Table 1 :

 1 Characteristic of the plate

Table 2 .

 2 Bubble periods and growth times for titanium and silicon plates

ACKNOWLEDGEMENT

This work is supported by EPSRC Grants EP/C532805, EP/C532813/1. The authors are grateful for the help of Mr. G. Cummins and Dr. J. T. M. Stevenson at the Scottish Microelectronics Centre (SMC) Edinburgh in manufacturing the silicon devices.

EXPERIMENTAL PROGRAMME

The experimental setup at Edinburgh is shown schematically in Fig. 20. The boiling chamber has four borosilicate glass windows, which allow optical access from the top, the bottom and two sides. A siliconeinsulated resistance heater is attached to the walls to reduce heat losses. The stainless steel rig is designed to operate over a range of pressures from atmospheric to 5 bars and a temperature up to 160 °C. The pressure is controlled by the cooling liquid flow through the condenser coil regulated by a control valve. The boiling liquid is initially heated up with a flow heater. Two filters in the pipe system remove any particles contained in the boiling liquid and all materials and seals are chosen to prevent any contamination. All electrical connections pass through vacuum-tight seals to the computer data acquisition system. The refrigerant FC-72 was chosen as the working fluid for the preliminary tests, because being a dielectric it eliminates electrical insulation issues associated with water. The boiling temperature of FC-72 is about 56 °C at atmospheric pressure (3M product information, 2000).

Preliminary boiling tests were performed with FC-72 boiling at atmospheric pressure on a 76.2 mm diameter silicon wafer with a thickness 0.38 mm. On the surface there were forty five 2 µm diameter cylindrical cavities. These were approximately 22 µm deep having been etched using the deep reactive ion etch (DRIE) Bosch process, Fig. 21. Heating with a flux density up to 15.5 kWm -2 was provided over a diameter of 50.8 mm by a kapton-insulated resistance heater between the back of the wafer and a PTFE table. Back-lit images of boiling were recorded by a high speed digital camera operating at 1000 Hz. At fluxes of 14.4 kWm -2 nucleation was not confined to the cylindrical cavities and it was difficult to observe the activity on all cavities, Fig. 22.

Bubble formation was recorded at a single stable site at 11.7 kWm -2 with similar bubbles being formed