domain: sde chim

text:
Rababe Es-Soufi

Mohammed L'bachir El Kbiach

Tomader Errabii

Rabah Saidi

Alain Badoc

Ludovic Chaveriat

Patrick Martin

Ahmed Lamarti

Biology and Physiology of Colletotrichum acutatum Strains Causing Strawberry's Anthracnose

Keywords: Anthracnose, Strawberry, Colletotrichum acutatum, Environmental Factors, Pathogenicity

Seven Colletotrichum acutatum strains isolated from strawberries were cultivated on various culture media and tested in vitro and in vivo on Fragaria x ananassa for anthracnose symptoms. PDA caused an optimum growth of all isolates, MEA, ML and Strawberry allowed a good but not optimal growth. Czapeck, Sabouraud and the organic medium from potato gave the lowest growth rate of all isolates. PDA allowed a good sporulation of isolates follow-up by strawberry, MEA; in contrast, ML, Czapeck and Sabouraud gave a low sporulation. The fungal development is maximal at 25˚C and 27˚C for all the isolates studied. No growth was observed at 5˚C and 37˚C. The studied strains developed at all pH values. They didn't develop at 0.6, 0.65 and 0.7 aw but mycelial growth was perfect at 1 and 0.95 aw. The in vitro test of the pathogenicity caused by C. acutatum strains on strawberry's leaves showed an increasing percentage of infection with time and different infection rates among C. acutatum strains, strain Ca6 having a pathogenic power very high compared to the other isolates. After in vivo inoculation and incubation of the seedlings, all isolates caused severe symptoms related to anthracnose on leaflets and petioles of the studied strawberry plants.

Introduction

Strawberry plant, Fragaria x ananassa, is one of the most widespread horticultural crop in the world. In Morocco, it was first introduced in the fifties, but its culture started toward the end of the seventies in two irrigated perimeters of the Loukkos and Souss-Massa. Ten years later the culture extended to the perimeter of the Gharb and then the area of Souss [START_REF] Mouden | Mycoflore de quelques variétés du fraisier (Fragaria ananassa L.), cultivées dans la région du Gharb et le Loukkos (Maroc)[END_REF]. The climate in these regions allows the growth of strawberry in greenhouses but commercial fields are ideal for the development of a large number of diseases like anthracnose. The latter is regarded as an economically important disease affecting different hosts in the world caused by different species of Colletotrichum [START_REF] Buddie | Molecular Characterization of Colletotrichum Strains Derived from Strawberry[END_REF] [START_REF] Debode | Quantitative Detection and Monitoring of Colletotrichum acutatum in Strawberry Leaves Using Real-Time PCR[END_REF]. Three species have been reported as causal agents of strawberry's anthracnose: C. acutatum J. H. Simmonds, C. gloeosporioides (Penz.) Penz. & Sacc., and C. fragariae A. N.

Brooks [START_REF] Gunnell | Taxonomy and Morphology of Colletotrichum Species Pathogenic to Strawberry[END_REF] [START_REF] Denoyes-Rothan | Genetic Diversity and Pathogenic Variability among Isolates of Colletotrichum Species from Strawberry[END_REF].

C. gloeosporioides and C. acutatum are distributed on a large number of hosts in the world, whereas C. fragariae has a range of hosts very close [START_REF] Mackenzie | Host Range and Genetic Relatedness of Colletotrichum acutatum Isolates from Fruit Crops and Leatherleaf Fern in Florida[END_REF]. Anthracnose causes up to 80% of death in nurseries and more than 50% loss of performance in the strawberry fields [START_REF] Sreenivasaprasad | Genotypic and Phenotypic Diversity in Colletotrichum acutatum, a Cosmopolitan Pathogen Causing Anthracnose on a Wide Range of Hosts[END_REF]. It has been defined as one of the most serious diseases in the commercial production of strawberry fruit. Colletotrichum acutatum is the most frequently species reported of the genus. It is today known as especially destructive on strawberry fruit [START_REF] Garrido | Phylogenetic Relationships and Genome Organization of Colletotrichum acutatum Causing Anthracnose in Strawberry[END_REF]. It causes mainly black spots on fruit and can also attack crowns, roots and leaves [START_REF] Freeman | Identification of Colletotrichum Species Responsible for Anthracnose and Root Necrosis of Strawberry in Israel[END_REF] [10] [START_REF] Smith | Morphological, Cultural, and Pathogenic Variation among Colletotrichum Species Isolated from Strawberry[END_REF].

Environmental factors such as relative humidity, pH and temperature have been reported to have a profound influence on the virulence of a variety of fungi.

The optimum temperatures for growth were often found between 25˚C and 30˚C; because of high temperatures the mycelial growth become weak and, in some cases, the mortality of fungi can occur. Several studies have been conducted on the effects of environmental factors on the growth of C. acutatum [START_REF] Wilson | Influence of Temperature and Wetness Duration on Infection of Immature and Mature Strawberry Fruit by Col-R. Es-Soufi et al[END_REF].

In this context, the aim of this study was to examine the behavior of seven isolates of Colletotrichum acutatum by changing the medium composition, temperature, pH and water activity and to study the disease severity caused by these isolates on Fragaria x ananassa in vitro and in vivo.

Materials and Methods

Fungal Material

Seven isolates of Colletotrichum acutatum (Ca1, Ca2, Ca3, Ca4, Ca5, Ca6 and Their identification has been carried out by macroscopic and microscopic observations with the help of determination keys [START_REF] Smith | Morphological, Cultural, and Pathogenic Variation among Colletotrichum Species Isolated from Strawberry[END_REF] [13].

Study of the Pathogenicity of the Isolates of Colletotrichum acutatum

Plant Material

Fourteen strawberry plants (Fragaria x ananassa (Weston)) Duchesne ex Rozier cultivar Camarosa (Figure 1) were conducted at the Laboratory of the Plant Biotechnology in Faculty of Sciences of Tetouan in pots containing black sandy soils from the origin field of the plants. The plants were watered daily.

Preparation of the Inoculum

Conidial suspensions of the isolates were obtained by adding 10 ml sterile distilled water on Petri dishes containing 10 to 15 day culture of C. acutatum on PDA, and gently rubbing the colonies using a sterile Pasteur pipette. At the end of spore release, the suspension of spores was filtered using sterile gauze nylon, pore size 100 μm. Concentration was determined using a Malassez cell.

In Vitro Leaves Inoculation

Young and healthy leaflets were harvested, carefully washed with distilled water and disinfected with a solution of sodium hypochlorite (2% w/v) for five minutes followed by three washes of 15 minutes in sterile distilled water. Leaves have been dried under an air stream in laminar flow hood. They have been filed on sterile Petri dishes (four leaflets per box) containing a double layer of sterile filter paper previously soaked in sterile distilled water. Central lesions were incised with a sterile scalpel in each leaflet [START_REF] Hmouni | Etude de la résistance de Botrytis cinerea aux benzimidazoles, dicarboximides et dithiocarbamates dans les cultures abritées de tomate de la région du Gharb (Maroc)[END_REF]. Three Petri dishes per strain were used.

On each lesion 30 µl of C. acutatum's suspension (10 4 spores/ml) was added (Figure 2); Petri dishes were incubated in dark at ambient temperature. As a blank, leaflets were inoculated by 30 µl sterile distilled water. The test was repeated three times.

Growth and Sporulation of the Isolates of Colletotrichum acutatum on Culture Media

The isolates of Colletotrichum acutatum have been grown on four mixed culture media (Potato Dextrose Agar (PDA), Yeast Malt (ML), Sabouraud and Malt Extract Agar (MEA)), a semi-synthetic (Czapek) and two organic culture media

Effect of Temperature on Mycelial Growth, Germination and Sporulation of the Isolates of Colletotrichum acutatum

Mycelial discs, from older cultures of ten days, were placed in Petri dishes of 70 mm containing the culture medium PDA. These plates were incubated in the dark at different temperatures (5˚C, 18˚C, 23˚C, 25˚C, 27˚C, 30˚C and 37˚C).

Effect of pH on Mycelial Growth, Germination and Sporulation of the Isolates of Colletotrichum acutatum

The effect of pH on the development of Colletotrichum acutatum strains was tested at pH 4, 4.5, 5, 5.5, 6, 6.5, 7.5 and 8. The culture media PDA has been stamped, according to the desired pH, by different buffers (Table 1) [START_REF] Es-Soufi | Assessment of the Effect of Environmental Factors on the Antagonism of Bacillus amyloliquefaciens and Trichoderma harzianum to Colletotrichum acutatum[END_REF]. Using a pH-meter, the pH was adjusted by addition of HCl and NaOH 1N [START_REF] Es-Soufi | Assessment of the Effect of Environmental Factors on the Antagonism of Bacillus amyloliquefaciens and Trichoderma harzianum to Colletotrichum acutatum[END_REF]. Plates were incubated at 27˚C in the dark.

Influence of the Water Activity on the Development of Seven Isolates of Colletotrichum acutatum

The In each test, three Petri dishes were used and the experiment was repeated three times.

Statistical Analysis

Before you begin to format your paper, Isolates development rates have been subjected to analysis of variance (ANOVA) using STATISTICA software for Windows V.6. The statistical significance of the results was determined by performing a test of Duncan's multiple range (p < 0.05). other isolates. After 11-day incubation, strain Ca6 reached up to 95% infection, all inoculated leaves being infected (Figure 3 and Figure 4). All leaflets inoculated with isolates showed symptoms typical of anthracnose symptoms with black spots at the surface of the leaflets (Figure 4).

Results

Pathogenicity of Isolates of Colletotrichum acutatum

In Vivo

After the inoculation and incubation of the seedlings, all the isolates cause severe symptoms typical from anthracnose on leaflets and petioles. The severities of the disease for each isolate increase in the course of time. Isolate Ca6 is the most aggressive among other isolates on leaflets and petioles (Figures 567).

Effect of Culture Medium

All culture media have enabled the mycelial growth of Colletotrichum acutatum (Figure 8) with different means of development (Table 2). PDA remains the culture medium the most favorable for the mycelial growth of Colletotrichum acutatum, with a maximum enlargement of all isolates. MEA, ML and Strawber- ry have allowed a good growth but not optimal. Czapeck and Sabouraud have given an average growth of all isolates.

PDA has allowed a good sporulation of isolates follow-up by Strawberry, MEA while ML, Czapeck and Sabouraud have allowed low sporulation (Table 2).

Effect of Temperature

All isolates didn't develop at 5˚C and 37˚C (Figures 9101112). Mycelial growth is low at 30˚C, average at 18˚C, maximum at 25˚C and 27˚C for all the isolates studied (Figure 10).

The seven isolates sporulate weakly at 18˚C and 3˚C. Sporulation is maximal at 25˚C and 27˚C and averages 23˚C for all the isolates studied (Figure 11). The conidia do not germinate at 5˚C and 37˚C. Conidial germination of all species studied is medium at 18˚C and 30˚C and maximal at 23˚C, 25˚C and 27˚C (Figure 12).

Effect of pH

All isolates studied developed at all pH values (Figures 13141516). The mycelial growth is maximum at pH 5 and 5.5.

Effect of the Water Activity

All isolates do not develop at aw 0.6, 0.65 and 0.7. The mycelial growth is perfect at aw 1 and 0.95, average at aw 0.85 and 0.90, and low at aw 0.75 and 0.8 (Figure 17 and Figure 18). Sporulation is perfect to aw 1, average at aw (0.9 and 0.95) and low at aw (0.75, 0.8 and 0.85) (Figure 19). The germination is maximal at aw 1, average at aw (0.9 and 0.95) and low at aw (0.75, 0.8 and 0.85) (Figure 20).

Discussion and Conclusion

Colletotrichum acutatum strains cause symptoms related to the anthracnose in vitro and in vivo but the severity of the disease varies from an isolate to another, isolate Ca6 representing a high level of aggressiveness among the strains studied; the percent of pathogenicity has been affected to 92% after 11 days of the in vitro inoculation and 100% after 20 days of the in vivo inoculation. Peres et al., [START_REF] Peres | Lifestyles of Colletotrichum acutatum[END_REF] have described that the symptoms caused by C. acutatum are mainly necrosis including burns on various types of tissues of the host such as leaves, petioles on a wide range of hosts. By comparing the pathogenicity of C. acutatum and C. fragariae on fruit, petioles and roots of Fragaria x ananassa, Tanaka et al., [START_REF] Tanaka | Caracterizaçãopatogênica de Colletotrichum acutatum e C. fragariaeassociados à antracnose do morangueiro[END_REF] have found that C. acutatum is less aggressive than C. fragariae against this host. Same results were found by Smith and Black [START_REF] Smith | Morphological, Cultural, and Pathogenic Variation among Colletotrichum Species Isolated from Strawberry[END_REF] and McInnes et al., [START_REF] Mcinnes | Disease-Free Plants for Management of Strawberry Anthracnose Crown Rot[END_REF] who have noted the association of C. acutatum with lesions of the rhizomes. C. acutatum from strawberry can parasitize and cause diseases on other hosts [19] [20] [21] or, alternatively, survive on other cultures and on weeds without producing symptoms. Smith and Black [START_REF] Smith | Morphological, Cultural, and Pathogenic Variation among Colletotrichum Species Isolated from Strawberry[END_REF], Peres et al., [START_REF] Peres | Lifestyles of Colletotrichum acutatum[END_REF] and Hyde et al., [START_REF] Hyde | Colletotrichum-Names in Current Use[END_REF] have found that their studied Colletotrichum acutatum isolates haven't caused leaf lesions on wounded inoculated leaves unlike two other Colletotrichum species whereas, in our study, all the isolates tested caused lesions on the strawberry leaves in vitro. C. acutatum isolated from almond and peach were demonstrated to be pathogenic on wounded and nonwounded fruit [START_REF] Adaskaveg | Characterization of Colletotrichum acutatum Isolates Causing Anthracnose of Almond and Peach in California[END_REF]; artificial inoculations demonstrated that fruits of all host species except for the banana were susceptible to C. acutatum isolates from strawberry [START_REF] Peres | Identification and Characterization of Colletotrichum spp. Affecting Fruit after Harvest in Brazil[END_REF]. Miles et al., [START_REF] Miles | The Effect of Environmental Factors on Infection of Blueberry Fruit by Colletotrichum acutatum[END_REF], Grahovac et al., [START_REF] Grahovac | Morphological and Ecological Features as Differentiation Criteria for Colletotrichum Species[END_REF] and Fernando et al., [START_REF] Fernando | Factors Affecting Spore Production, Germination and Viability of Colletotrichum acutatum Isolates from Hevea brasiliensis[END_REF] have found that Colletotrichum acutatum has an optimal development at 25˚C ± 2˚C. These results are consistent with other studies that have evaluated the effect of the temperature on species of Colletotrichum from different hosts [START_REF] Gunnell | Taxonomy and Morphology of Colletotrichum Species Pathogenic to Strawberry[END_REF] [START_REF] Wilson | Influence of Temperature and Wetness Duration on Infection of Immature and Mature Strawberry Fruit by Col-R. Es-Soufi et al[END_REF] [13] [30] [START_REF] Adaskaveg | Characterization of Colletotrichum acutatum Isolates Causing Anthracnose of Almond and Peach in California[END_REF]. The comparison of the effect of temperature between C. acutatum strains isolated from avocado, banana, guava, papaya, mango and passion fruit shows that the optimum temperature for their development is 28˚C. None of the strains has grown at 8˚C and only the isolates from avocado, papaya and banana have developed at 36˚C [START_REF] Peres | Identification and Characterization of Colletotrichum spp. Affecting Fruit after Harvest in Brazil[END_REF]. Optimum germination of C. acutatum isolated from coffee has occurred at 21˚C -29˚C [START_REF] Kenny | Germination and Growth of Colletotrichum acutatum and Colletotrichum gloeosporioides Isolates from Coffee in Papua New Guinea and Their Pathogenicity to Coffee Berries[END_REF] and its mycelial growth was maximal at 21˚C [START_REF] Kenny | Germination and Growth of Colletotrichum acutatum and Colletotrichum gloeosporioides Isolates from Coffee in Papua New Guinea and Their Pathogenicity to Coffee Berries[END_REF] [32] [START_REF] Waller | Characterization of Coffee Berry Disease Pathogen, Colletotrichum kahawae sp[END_REF].

The environmental pH plays an important role in the growth and differentiation of microorganisms. In the present work, pH variation has no remarkable effect on mycelial growth and germination of the seven strains studied: the mycelial growth, sporulation and germination of all strains reach an optimum at pH 5, 5.5, 6 and 6.5. Colletotrichum musae, the causal agent to anthracnose of banana tree, has an optimal development at pH 4.5 [START_REF] De Costa | Effect of Exogenous pH on Development and Growth of Colletotrichum musae and Development of Anthracnose in Different Banana Cultivars in Sri Lanka[END_REF].

The water activity has a remarkable effect on the mycelial growth, which is optimal at 0.95 and 1, medium at 0.75 -0.90 and null between 0.60 and 0.70: C. acutatum, the agent of anthracnose of strawberry, requires water in order to de- velop.

The anthracnose caused by several species of the genus Colletotrichum and especially C. acutatum represents one of the major fungal diseases of Fragaria x ananassa. The disease manifests itself by small circular spots which merge to form large elliptical spots on fruit and leaves. Therefore the control against this disease for cultures of strawberry plants, devoid of phytopathogenic agents, is necessary in order to produce healthy fruit to meet the consumer's requirement.

  Ca7) have been isolated from strawberry plants which has been collected from strawberry's fields of Loukkos (Larache, Morocco) naturally affected by anthracnose, purified in Laboratory of Plant Biotechnology, Faculty of Sciences, Tetouan. They have been cultivated on PDA (Potato Dextrose Agar) medium for 7 to 10 days at 25˚C in the dark, and successive subculturing were made up within a total purification of strains. R. Es-Soufi et al. DOI: 10.4236/as.2018.98068 976 Agricultural Sciences

Figure 1 .

 1 Figure 1. Strawberry plant (cultivar Camarosa).

4. 1 . 1 .Figure 3 .

 113 Figure 3. Pathogenicity degree of seven isolates of Colletotrichum acutatum on the leaflets of strawberry in vitro.

Figure 4 .

 4 Figure 4. Development of the degree of the pathogenicity of the strain Ca6 on leaves: (a) inoculated leaflet by the suspension at the day of inoculation; (b) infected leaflets after 3 days; (c) infected leaflets after 8 days; (d) infected leaflets after 11 days of incubation.

Figure 5 .Figure 6 .

 56 Figure 5. Symptoms of anthracnose caused by strains Ca6 on petioles and leaves of strawberry "Camarosa" compared with blank (c).

Figure 7 .

 7 Figure 7. Pathogenicity degree of seven isolates of Colletotrichum acutatum on the leaflets and petioles of strawberry "Camarossa" grown in vivo after 20 day-incubation.

RFigure 8 .

 8 Figure 8. Effect of culture media: (a) PDA; (b) ML; (c) MEA; (d) Sabouraud; (e) Czapeck; (f) Potato and (g) Strawberry on the mycelial growth of seven isolates of Colletotrichum acutatum after 10 days of incubation.

Figure 9 .

 9 Figure 9. Influence of the temperature at: (a) 5˚C; (b) 18˚C; (c) 23˚C; (d) 25˚C; (e) 27˚C and (f) 37˚C on the mycelial growth, on PDA of seven isolates of Colletotrichum acutatum after 10 days of incubation.

Figure 10 .Figure 11 .

 1011 Figure 10. Influence of the temperature on the mycelial growth (mm), on PDA of seven isolates of Colletotrichum acutatum after 10 days of incubation.

Figure 12 .

 12 Figure 12. Influence of the temperature on percent of germination of seven isolates of Colletotrichum acutatum.

Figure 13 .

 13 Figure 13. Influence of pH: (a) 4; (b) 4.5; (c) 5; (d) 5.5; (e) 6; (f) 6.5; (g) 7.5 and (h) 8 on the mycelial growth on the PDA of Ca6.

Figure 14 .

 14 Figure 14. Influence of pH on the mycelial growth (mm) on PDA of seven isolates of Colletotrichum acutatum.

Figure 15 .Figure 16 .

 1516 Figure 15. Influence of pH on sporulation (×10 4 spores/ml) of seven Colletotrichum acutatum strains.

Figure 17 .

 17 Figure 17. Influence of aw (a) 0.60; (b) 0.65; (c) 0.70; (d) 0.75; (e) 0.80; (f) 0.85; (g) 0.90; (h) 0.95 and (i) 1 on the mycelial growth of a Colletotrichum acutatum strain after 5 days of incubation.

Figure 18 .

 18 Figure 18. Influence of water activity on the mycelial growth (mm) of seven Colletotrichum acutatum strains after 10 days of incubation.

Figure 19 .

 19 Figure 19. Influence of water activity on sporulation (×10 4 spores/ml) of seven Colletotrichum acutatum strains.

Figure 20 .

 20 Figure 20. Influence of water activity on germination of seven Colletotrichum acutatum strains.

Table 1 .

 1 Different values and buffers of used pH. a suspension of 10 4 spores/ml was spread on the surface of Petri dishes containing 0.5 g agar/100 ml distilled water. The counting of the germinated spores was carried out on a total of 100 spores after 24-hour incubation at 25˚C.

	pH	Common	Buffer Chemical	Molecular weight	20 mM (g/l)
	4 4.5	Trizma (TrisHCl)	2-amino-2-(hydroxymethyl)-1,3-propanediol, hydrochloride	157.59	3.152
	5
	5.5	MES	2-(N-morpholino)ethanesulfonic acid	213.25	4.265
	6
	6.5	PIPES	piperazine-N,N'-bis (2-ethanesulfonic acid)	302.4	6.048
	7.5	MOPS	3-(N-morpholino)propanesulfonic acid	209.3	4.186
	8	BICINE	N,N-bis(2-hydroxyethyl)glycine	163.2	3.264

water activity (aw) represents the availability in open water for the biochemical reactions for the development of microorganisms. Different values of activity of the water have been tested (1; 0.95; 0.90; 0.85; 0.80; 0.75; 0.70; 0.65 and 0.60) by the addition of glycerol in the culture medium PDA [15] [16], which will attach a part of the water and make it unusable to microorganisms. 2.7. The Studied Factors Were Tested on 2.7.1. The Mycelial Growth For each medium, three Petri dishes were inoculated in their center by mycelial discs of 5mm diameter. After ten-day incubation at 25˚C in the dark, the growth R. Es-Soufi et al. DOI: 10.4236/as.2018.

Table 2 .

 2

	Culture medium	Mycelial growth (mm)	Sporulation (×10 4 )
	Mixed medium
	Potato Dextrose Agar (PDA)	70.00 ± 0.00 a	34.44 ± 0.86 a
	Yeast Malt (ML)	66.31 ± 0.43 ab	19.03 ± 1.12 bc
	Malt Extract Agar (MEA)	65.30 ± 0.93 ab	1.10 ± 0.29 d
	Sabouraud	49.14 ± 0.47 bc	1.02 ± 0.18 d
	Semi-synthetic medium
	Czapeck	55.67 ± 1.09 c	1.50 ± 0.32 d
	Organic medium
	Strawberry	61.76 ± 0.98 b	26.78 ± 1.12 b
	Potato	52.60 ± 0.43 bc	10.59 ± 0.62 c

Effect of seven culture media on the mycelial growth (mm) after 10-day incubation and sporulation of Colletotrichum acutatum.

Averages of a column with the same letter are not significantly different between them at the threshold of 5%. R. Es-Soufi et al. DOI: 10.4236/as.2018.98068 984 Agricultural Sciences

  Fungi generally require different pH and temperature conditions during the course of their development. These two factors influence the stages of their life cycle. The development of Colletotrichum acutatum changes with the environmental factors studied (culture medium composition, temperature, pH and aw).

			R. Es-Soufi et al.
		Mycelial growth and sporulation of the strains are perfect in the culture me-
		dium PDA, followed by MEA and Strawberry. For ML, mycelial growth was
		good but the sporulation was very low as in the other culture media. PDA has
		been used as the base culture medium for the isolation, purification and growth
		of Colletotrichum species causal agents of grown strawberry's anthracnose [5]
		[11] [24] [25] [26]. Variation of temperature also affects the development of C.
		acutatum strains; 5˚C and 37˚C cause no development of the fungus. Germina-
		tion of strain Ca4 is perfect at other temperatures; germination of Ca1, Ca2, Ca5,
		Ca6 and Ca7 is maximal at 23˚C, 25˚C, 27˚C and 30˚C. Sporulation of all iso-
		lates is too low at 18˚C and 30˚C, average at 23˚C and maximum at 25˚C and
		27˚C. Mycelial growth is maximum at 25˚C and 27˚C, average at 18˚C and 30˚C,
		and no growth is observed at 5˚C and 37˚C.
	DOI: 10.4236/as.2018.98068	987	Agricultural Sciences

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.


Michiel Postema

Philippe Marmottant

Charles T Lanc Ée

Sascha Hilgenfeldt

Nico De Jong

Ultrasound-induced Microbubble Coalescence

Keywords: Microbubble Coalescence Microbubble coalescence, ultrasound contrast agent, film drainage, high-speed photography

We study the interaction of ultrasound contrast agent bubbles coated with a layer of lipids, driven by 0.5 MHz ultrasound. High-speed photography on the sub-microsecond timescale reveals that some bubbles bounce off each other, while other show very fast coalescence during bubble expansion. This fast coalescence cannot be explained by dissipation-limited film drainage rates. We conclude that the lipid shell ruptures upon expansion, exposing clean free bubble interfaces that support plug flow profiles in the film and inertia-limited drainage whose timescales match those of the observed coalescence.

Introduction

Ultrasound contrast agent (UCA) is a suspension of encapsulated gas bubbles.

The bubbles have diameters ranging from 1 to 10 µm. They oscillate upon insonification, generating a characteristic acoustic response. Contrast agents are widely used in medical diagnostics [START_REF] Burns | Intravenous contrast agent for ultrasound Doppler: In vivo measurement of small tumor vessel dose-response[END_REF][START_REF] Goldberg | Ultrasound Contrast Agents. Basic principles and clinical applications[END_REF].

Observing ultrasound insonified UCA with a high-speed camera is a promising method for analyzing microbubble oscillation and destruction behavior (Takeuchi, 1999;[START_REF] De Jong | Optical imaging of contrast agent microbubbles in an ultrasound field with a 100-MHz camera[END_REF][START_REF] Chomas | Threshold of fragmentation for ultrasonic contrast[END_REF][START_REF] Kudo | Study on mechanism of cell damage caused by microbubbles exposed to ultrasound[END_REF][START_REF] Postema | Simulations and measurements of optical images of insonified ultrasound contrast microbubbles[END_REF]. Expanding UCA microbubble coalescence, observed with a high-speed camera during one cycle of ultrasound, has been previously reported by us [START_REF] Postema | Simulations and measurements of optical images of insonified ultrasound contrast microbubbles[END_REF]. Colliding bubble coalescence has been studied with a camera by [START_REF] Kok | Dynamics of a pair of gas bubbles moving through liquid. Part II. Experiment[END_REF] and [START_REF] Van Wijngaarden | The mean rise velocity of pairwise-interacting bubbles in liquid[END_REF]. In the same year, [START_REF] Kumaran | The rate of coalescence in a suspension of high Reynolds number, low Weber number bubbles[END_REF] computed how bubbles may repeatedly collide with each other.

To understand microbubble coalescence, one needs to comprehend the drainage of the liquid separating the bubble surfaces. [START_REF] Reynolds | On the theory of lubrication and its application to Mr. Beauchamp Tower's experiments, including an experimental determination of the viscosity of olive oil[END_REF] noted that the viscosity of a liquid can be determined by pressing two flat plates together, squeezing the liquid out, and measuring the drainage velocity. Thus, he formulated an equation for the drainage velocity of a fluid between rigid surfaces.

General theories on the coalescence of colliding bubbles and droplets that are based on liquid film drainage, were put forward by [START_REF] Marrucci | A theory of coalescence[END_REF], [START_REF] Dimitrov | Hydrodynamics of thin liquid films. On the rate of thinning of microscopic films with deformable interfaces[END_REF], [START_REF] Ivanov | Generalized equations of thin films hydrodynamics and their application to the calculations of the thinning rate of films with non-deformed surfaces[END_REF], Lin & Slattery (1982a), [START_REF] Chesters | Bubble coalescence in pure liquids[END_REF], Duineveld (1994a), and [START_REF] Klaseboer | Film drainage between colliding drops at constant approach velocity: experiments and modeling[END_REF]. Literature overviews on film drainage and bubble coalescence were given by [START_REF] Kralchevsky | Thin liquid film physics[END_REF], [START_REF] Narsimhan | Structure, drainage, and coalescence of foams and concentrated emulsions[END_REF], and [START_REF] Dhainaut | Literature study on observations and experiments on coalescence and breakup of bubbles and drops[END_REF].

Theories on droplet coalescence find applications in fuel ignition research and aerosol studies, whereas the research on bubble coalescence focuses on thin film physics and foam stability [START_REF] Kralchevsky | Thin liquid film physics[END_REF][START_REF] Narsimhan | Structure, drainage, and coalescence of foams and concentrated emulsions[END_REF]. This paper explores ultrasound-induced coalescence of microbubbles.

Controlled coalescence has potential applications in the clinical field.

Theories on bubble coalescence are generally based on the collision of unencapsulated bubbles or droplets, approaching each other at constant velocity.

During expansion, microbubbles may also come into contact with each other, resulting in coalescence or bounce. We discriminate the following stages in the coalescence mechanism, optically observed in Fig. 1 and schematically represented in Fig. 2. These stages are similar to those presented for colliding bubbles and droplets in [START_REF] Dhainaut | Literature study on observations and experiments on coalescence and breakup of bubbles and drops[END_REF]. First, two bubbles approach collision while expanding (Fig. 2a). Prior to contact, there may be a flattening of the adjacent bubble surfaces, trapping liquid in between (Fig. 1a, Fig. 2b). This trapped liquid drains (Fig. 1b, Fig. 2c) until the separation reaches a critical thickness (Fig. 2d). An instability mechanism (Fig. 2d, magnified) results in rupture of the separation (Fig. 2e) and the formation of a merged bubble (Fig. 1c). After coalescence the resulting bubble will have an ellipsoidal shape (Fig. 1d, Fig. 2f). Owing to surface tension, it will relax to a spherical shape. When the contact time is less than the time needed for film drainage, the bubbles bounce off each other [START_REF] Chaudhari | Coalescence of gas bubbles in liquids[END_REF]. We define bubble coalescence as the fusing of two or more bubbles into a single bubble. The process begins with the flattening of the bubble surfaces and is considered finished when the resulting bubble has a spherical shape.

In this paper, we give a description of the coalescence of expanding microbubbles, based on optical observations and theoretical modeling. First, we present theories for expanding bubble coalescence, based on film drainage theory.

Then, we present experimental results, obtained by recording optical images of insonified UCA. Finally, theories and results are compared and discussed.

Theory

Flattening of the interface

Flattening of the opposing bubble surfaces occurs because the liquid inertia overcomes the capillary pressure, as described in earlier work on colliding bubbles with constant volumes. For colliding bubbles, flattening happens if the bubble system has a Weber number We 0.5 [START_REF] Chesters | Bubble coalescence in pure liquids[END_REF]Duineveld, 1994b). The Weber number for two colliding 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 R m , ( 1
)
where u is the relative approach velocity of the bubble walls, ρ is the fluid density, σ is the surface tension, and R m is the mean bubble radius for which holds:

2 R m = 1 R 1 + 1 R 2 . ( 2
)
We propose to extend the Weber number criterium to approaching walls of expanding bubbles. Then, for bubbles with a constant center-to-center distance, u = Ṙ1 + Ṙ2 . In our results, Weber numbers are found to be We 1 because of the rapid bubble expansions, with maximal radius increases of several m s -1 . If the Weber number is low, 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 the high Weber number regime, coalescence is determined by a second step, after flattening: film drainage.

Film drainage

We investigate microbubble coalescence by computing the film drainage for no-slip (rigid) bubble surfaces resulting in a laminar flow, and for mobile (free) bubbles surfaces resulting in a plug flow.

Let us consider two bubbles with radii R 1 and R 2 , and internal pressures p 1 and p 2 , respectively, assumed spherical everywhere with the exception of a flattened interface that separates them through a liquid film of thickness h (cf. Fig. 3). The drainage rate of the liquid film depends on the difference (p + Π) between the film pressure p f and the liquid ambient pressure p 0 . Here, p is the difference in hydrodynamic pressure, while Π is the disjoining pressure in the film. We estimate the pressure in the film by the mean of pressures p 1 and p 2 , since the parallel film surfaces lead to equal pressure differences towards both bubbles:

p + Π = p f -p 0 = 1 2 (p 1 + p 2 ) -p 0 = = σ 1 R 1 + 1 R 2 ≡ p LY , ( 3
)
where p LY is the Laplace-Young film pressure [START_REF] Isenberg | The science of soap films and soap bubbles[END_REF], and σ is the surface tension. The disjoining pressure begins to slow down film thinning when h drops below 0.1 µm, and becomes the dominant pressure term (usually owing to Van der Waals forces) when h thins to about 10 nm [START_REF] Marrucci | A theory of coalescence[END_REF][START_REF] Chaudhari | Coalescence of gas bubbles in liquids[END_REF][START_REF] Bergeron | Forces and structure in thin liquid soap films[END_REF]. The eventual coalescence is very fast compared to the film drainage time scales considered later. Therefore, we neglect Π and take p equal to the Laplace-Young pressure for the films observed. As such, the pressure gradient determining the drainage velocity is independent of the ambient pressure.

We choose an r-z coordinate system such that the film is symmetric around the plane z = 0 and the line r = 0, and that its boundaries are located at z = ± h 2 and r = R f . The Laplace-Young pressure gradient drives liquid out of the film. The radial velocity of the liquid is described by a combination of a plug flow (present without any resistance to flow) and a laminar flow profile (in z) of Poiseuille-type induced by resistance at the film interfaces [START_REF] Klaseboer | Film drainage between colliding drops at constant approach velocity: experiments and modeling[END_REF][START_REF] Young | A Brief Introduction to Fluid Mechanics[END_REF]. The drainage of the liquid film can be parameterized by functions of these two contributions [START_REF] Bazhlekov | The effect of the dispersed to continuous-phase viscosity ratio on film drainage between interacting drops[END_REF][START_REF] Klaseboer | Film drainage between colliding drops at constant approach velocity: experiments and modeling[END_REF][START_REF] Hagesaether | Coalescence and break-up of drops and bubbles[END_REF]. We will study the two limiting cases of bubbles with no-slip interfaces and bubbles with free interfaces.

No-slip interfaces

In the presence of surfactant at sufficient surface concentration, the interfaces can be considered immobile (no-slip) (Lin & Slattery, 1982b,a;[START_REF] Chen | Effects of London-van der Waals forces on the thinning of a dimpled liquid film as a small drop or bubble approaches a horizontal solid plane[END_REF][START_REF] Van Wijngaarden | The mean rise velocity of pairwise-interacting bubbles in liquid[END_REF]. In the case of no-slip interfaces, the interfacial tangential velocity is zero, so the plug flow contribution is zero [START_REF] Klaseboer | Film drainage between colliding drops at constant approach velocity: experiments and modeling[END_REF].

The film drainage velocity for rigid radial surfaces (disks) is given by the Reynolds equation [START_REF] Reynolds | On the theory of lubrication and its application to Mr. Beauchamp Tower's experiments, including an experimental determination of the viscosity of olive oil[END_REF][START_REF] Sheludko | Thin liquid films[END_REF][START_REF] Kralchevsky | Thin liquid film physics[END_REF]:

- ∂h ∂t = 2 p h 3 3 η R 2 f , ( 4
)
where η is the viscosity of the liquid, and R f is the radius of the film surface.

The drainage time, τ d , between the initial film thickness h i and the critical film thickness h c can be determined by integration of eq. ( 4):

hc h i - dh h 3 = τ d 0 2 p 3 η R 2 f dt .
(5)

By taking p and R f constant over time, * we obtain:

τ d = 3 η R 2 f 4 p h 2 c 1 - h 2 c h 2 i . ( 6
)
If h 2 c h 2 i the drainage time can be approximated by

τ d ≈ 3 η R 2 f 4 p h 2 c . ( 7
)
Figure 4 shows two no-slip drainage time curves as a function of equivalent * Flattening takes place when:

Ṙ1 + Ṙ2 ∂h ∂t ,
whereas the flat film drainage happens in the next stage, when

Ṙ1 ≈ Ṙ2 ≈ 0.
Thus, during drainage, we may take p and R f constant over time.

η = 0.001 Pa s ρ = 998 kg m 3 σ = 0.072 N m -1 2 R e = 1 R 1 + 1 R 2 p = 2σ R e h c = 10 nm or 100 nm h i h c ≈ 100
Table 1: Parameters used in the drainage equations.

bubble radius, using the parameters from Table 1. Clearly, even for h c = 100 nm, the drainage takes several microseconds for radii greater than 2 µm.

Even if the surfactant is very mobile, the interfaces have been observed to have a significant Poiseuille dissipation [START_REF] Koehler | Foam drainage on the microscale. II. Imaging flow through single Plateau borders[END_REF]. Drainage timescales for interfaces covered with mobile surfactant will therefore not be dramatically smaller than those indicated here for no-slip boundary conditions.

Free interfaces

In the case of free interfaces, the Poiseuille contribution to the drainage flow becomes negligible [START_REF] Van Wijngaarden | The mean rise velocity of pairwise-interacting bubbles in liquid[END_REF][START_REF] Klaseboer | Film drainage between colliding drops at constant approach velocity: experiments and modeling[END_REF], and the drainage is inertial. The film drainage velocity for free radial surfaces is given by the equation [START_REF] Kirkpatrick | The influence of approach velocity on bubble coalescence[END_REF][START_REF] Hagesaether | Coalescence and break-up of drops and bubbles[END_REF]:

- ∂h ∂t = 8 p ρ h R f . ( 8
)
Note that the viscous term is absent. Similarly to the no-slip case, making the same quasi-static assumptions with regards to p and R f , the drainage time can be approximated by

τ d ≈ R f ρ 8 p log h i h c . ( 9
)
Figure 4 also shows two drainage time curves for free interfaces. These drainage times are much smaller than those for the no-slip situation, and depend only logarithmically on both the initial and the critical film thickness.

Film rupture

The disjoining pressure induces rupture by amplifying surface perturbations.

These are initialized by either thermal fluctuations or by capillary waves [START_REF] Sharma | Critical thickness and lifetimes of foams and emulsions: role of surface wave-induced thinning[END_REF]. For thermal perturbations of a gas bubble in the micrometer range, the initial perturbation will be on the order of kT σ , where k is Boltzmann's constant, and T is the absolute temperature, in our situation approximately 300 K. Hence, the initial thermal perturbation is lower than 1 nm.

A film gradually thins to a critical thickness at which it either ruptures due to a local instability or at which it attains an equilibrium thickness. The mechanism of thin film rupture has been reviewed by [START_REF] Sheludko | Thin liquid films[END_REF]. [START_REF] Angarska | Detection of the hydrophobic surface force in foam films by measurements of the critical thickness of film rupture[END_REF] measured these thicknesses in films, dependent of surfactant concentration and film radius. They found critical thicknesses in the range 20 nm < h c < 40 nm for film radii 60 µm < R f < 160 µm.

For our film radii (R f < 10 µm), we may assume critical thicknesses around 10 nm, knowing that below 10 nm Van der Waals forces become very strong and rapid rupture of the film (and thus coalescence) ensues. Because of the weak dependence on film thickness, predictions from eq. ( 9) for coalescence time scales can be quite accurate even without precise knowledge of h i and h c .

Experimental setup

Our experimental setup for imaging insonified contrast bubbles is as previously described by [START_REF] Postema | Simulations and measurements of optical images of insonified ultrasound contrast microbubbles[END_REF]. We investigated coalescence events of an experimental UCA (Bracco Research SA, Geneva, Switzerland). According to manufacturer specifications, these bubbles are covered with a monolayer phospholipid shell and range in diameter from 1 to 6 µm with a median of 2 µm. Undiluted UCA (5 ml of a 0.9% NaCl dilution, added to a 25 mg vial) was inserted through a cellulose capillary tube using either a syringe pressed by hand or a gravity fed or pumped infusion. This tube was positioned in the acoustic focus area. Since the capillary tube slightly moved within the acoustic focus area between experiments, the exact phase of the ultrasound wave in an image frame is not known. We performed 482 experiments at high acoustic amplitudes with the experimental UCA. We recorded 133 optical image sequences where microbubble coalescence was observed. Bubble sizes and distances were measured manually or by using a segmentation method described by [START_REF] Postema | Simulations and measurements of optical images of insonified ultrasound contrast microbubbles[END_REF].

Results

The observed phenomena are classified as follows: coalescence, bounce, multiple coalescence, and combined coalescence and fragmentation. In the following, we discuss representative image sequences typical for each process.

Coalescence

Figure 5(i) shows an example of coalescence. Figure 5(i)a shows three microbubbles with diameters (1) 2.5 µm, (2) 2.5 µm, and (3) 2.0 µm. After ultrasound arrival, microbubbles 2 and 3 have apparently coalesced (Fig. 5(i)b).

The remaining bubble, with a diameter of 5.0 µm, is separated from microbubble 1, which is seen to have expanded to 3.8 µm, with a center-to-center distance d 0 = 4.8 µm. The center of bubble 1 has shifted slightly to the upper right.

The thickness of the liquid film separating the bubble shells is approximately h = 1.1 µm, and the film radius is 1.7 µm. In Fig. 5(i)c the liquid film appears to have drained while the bubbles expanded, but, a separation is still visible. This boundary appears to have disappeared in Fig. 5(i)d, † leaving a pear-shaped bubble that turns ellipsoidal (Fig. 5(i)e) when contracting. Figure 5(i)f,g,h shows that the coalesced bubble expands uniformly. Because of the 3 Mfps frame rate of the † If the line of sight is not perpendicular to the film, but at a tilt, the projection of the film boundaries might be obfuscated.

image sequences, we conclude that the processes of drainage and rupture for the interposed film in Fig. 5(i)b take between 0.33 µs and 0.66 µs.

Bounce

Figure 5(ii) shows an example of bounce, i.e. bubbles approaching and flattening, but not coalescing. Figure 5(ii)a shows two microbubbles with diameters (1)

12.4 µm, and (2) 18.0 µm, much larger than those in Fig. 5(i)a. After ultrasound arrival, the bubble surfaces have flattened (Fig. 5(ii)b). The bubble centers have shifted towards each other. The film radius is R f = 4.8 µm. The film thickness is approximately h = 1.8 µm. In the remaining frames the bubbles expand and contract, but coalescence does not occur.

Multiple coalescence

Figure 5(iii) shows an example of multiple coalescence. In Fig. 5(iii)b an agglomerate of 8 touching microbubbles can be seen. Microbubbles 1 and 3 appear to be slightly above the focal plane [START_REF] Postema | Simulations and measurements of optical images of insonified ultrasound contrast microbubbles[END_REF]. As the bubbles expand, they coalesce into one heart-shaped bubble and one ellipsoidal bubble (Fig. 5(iii)c,d). In the contraction phase, the heart-shaped bubble takes on an ellipsoidal shape (Fig. 5(iii)e).

Combined coalescence and fragmentation

We observed repeated coalescence and fragmentation behavior in 12 events.

Discussion

The variables obtained from Fig. 5(i)b are summarized in the first column of Table 2. For h i we took the film thickness h measured in this frame. If we substitute the parameters from Tables 1 and2 into equation ( 9), we find a film drainage time τ d = 0.35 µs for free interfaces, consistent with the observed rapid coalescence 2. For h i we took the film thickness h measured in Fig. 5(ii)b. If we use the parameters from the Table 1 and substitute the obtained parameters into equation ( 9) for free interfaces, τ d = 2.0 µs. This is consistent with the absence of coalescence in Fig. 5(ii). Within 1 µs, the film can only drain to a minimum thickness h m = 0.13 µm. For no-slip interfaces (7), even for a critical thickness as great as h c = 150 nm, we find a film drainage time τ d = 39 µs, much longer than any timescale relevant to the observations.

The difference in drainage time scales between Fig. 5(i) and Fig. 5(ii) has a natural explanation in the different sizes of the bubbles involved in the process. As smaller films drain much faster, the relatively small bubbles of Fig. 5(i) coalesce over the experimental timescales, while the larger bubbles of Fig. 5(ii) are stable.

The results presented in Fig. 1 and Fig. 5 show that the bubbles have expanded to more than ten-fold their initial surface areas before coalescing. The UCA shell consists of a lipid monolayer that, under the conditions of our experiments, is in a solid state. It behaves like an elastic membrane that ruptures under relatively small strain [START_REF] Zhou | Mechanisms of membrane rupture: from cracks to pores[END_REF]. By the time of coalescence, therefore, the shell has ruptured, leaving newly formed clean free interfaces. Such interfaces will be on both sides of the film, and no surface dissipation can be expected here, so that the flow has to be (very near to) a plug flow, determined by a balance of inertia and driving pressure. This confirms that the interfaces may be assumed free and eq. ( 9) is applicable. We demonstrated previously with high-speed optical images that the UCA microbubbles used may expand to several times their original sizes, particularly at high acoustic pressures [START_REF] Postema | Simulations and measurements of optical images of insonified ultrasound contrast microbubbles[END_REF]. The existence of stable holes of stretched lipid has been observed by [START_REF] Stine | Evolution of foam structures in Langmuir monolayers of pentadecanoid acid[END_REF].

Our observations are incompatible with any drainage mechanism that involves resistance in either the bulk or the interfaces. The observed coalescence times agree with those computed for free interfaces, confirming that the expanded (snapped) lipid shells behave as if their surfaces are truly stress-free. The stretching and rupture of lipid membranes is currently under investigation at the Physics of Fluids group of the University of Twente.

Fragmentation may occur when the bubble interfaces are unstable, which is the case when the bubble is contracting and (inward) decelerating, i.e., when [START_REF] Plesset | On the stability of the spherical shape of a vapor cavity in a liquid[END_REF][START_REF] Brennen | Fission of collapsing cavitation bubbles[END_REF]. Also, the formation of a re-entrant jet from a collapsing bubble has been related to the bubble shattering into fragments when the jet impacts the other side of the bubble

Ṙ < 0 ∧ R > 0 (
surface. An optical image sequence of jet occurrence in a contrast microbubble was demonstrated by Postema et al. (2002b). Apparently water was projected through the freely flowing microbubble. Although we clearly observed jets in two events, we did not observe fragmentation in the same events (Postema et al., 2002a). We recently summarized the behavior of insonified UCA microbubbles (Postema et al., 2004b) including fragmentation. The number of fragments has been associated with the dominant spherical harmonic oscillation mode [START_REF] Brennen | Fission of collapsing cavitation bubbles[END_REF].

Irregular shapes of oscillating bubbles, like those shown in Fig. 5(iii) and

(iv), were interpreted as modes of shape instability of a single bubble before (Chomas et al., 1999a,b;[START_REF] May | Acoustic fragmentation of therapeutic contrast agents designed for localized drug delivery[END_REF][START_REF] May | Dynamics and fragmentation of thick-shelled microbubbles[END_REF], but may also be accounted for by coalescence of bubbles or bubble fragments.

After coalescence, the resulting bubble will have acoustic properties different from those of the original bubbles, especially if its size is comparable to the size resonant with the ultrasonic driving. 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., 2004a), controlled microbubble coalescence may be applicable. By the drainage timescales explained above, using the right ultrasound frequency could restrict coalescence to smaller bubbles, conveniently limiting the potentially dangerous coalescence of larger-radius contrast agents.

One of the effects of secondary radiation forces is that they lead to mutual attraction of similar-sized bubbles over multiple cycles. This may account for the translations observed in Fig. 5. Owing to secondary radiation forces, clusters of bubbles may be formed. By inducing coalescence of such groups of bubbles, and thus creating emboli, the perfusion of tumor vascularization may be reduced.

If coalescence of a lipid-shelled microbubble and a cell membrane can be induced, this will imply a promising technique in targeted drug delivery [START_REF] Van Wamel | Effects of diagnostic ultrasound parameters on molecular uptake and cell viability[END_REF][START_REF] Marmottant | Controlled vesicle deformation and lysis by single oscillating bubbles[END_REF].

Conclusions

Ultrasound-induced microbubble coalescence is the fusion of two or more microbubbles when subjected to an ultrasound field. Contrast agent microbubble coalescence has been observed frequently in an experimental setup. We showed that a coalescence mechanism for colliding bubbles also applies for expanding bubbles.

As adjacent bubbles expand, the following stages can be distinctly observed:

flattening of the adjacent bubble surfaces prior to contact, drainage of the interposed liquid film toward a critical thickness, rupture of the liquid film, and formation of a single bubble. The time interval from flattening to coalescence was observed to take less than 1 µs for ultrasound contrast agent microbubbles in the micron diameter range.

This fast coalescence cannot be explained by dissipation-limited film drainage rates. We conclude that the lipid shell ruptures upon expansion, exposing clean free bubble interfaces that support plug flow profiles in the film and inertia-limited drainage of which the timescales match those of the observed coalescence. At a given frequency, small bubbles are more prone to undergo coalescence than larger bubbles because of the smaller drainage timescales involved.

Ultrasound-induced coalescence has potential clinical applications in harmonic imaging, noninvasive blood pressure measurements, and targeted drug delivery.

List of Figures

  In short: a V389-SU 500 kHz single-element transducer (Panametrics Inc., Waltham, MA) was mounted into a water-filled container, spherically focused at the focal plane of the optical system. The optical images were recorded through a microscope with an Imacon 468 fast framing camera (DRS Hadland, Ltd., Tring, UK), capable of taking 8 frames per experiment. It operated at a frame rate of 3 million frames per second (Mfps), corresponding to interframe times of 0.33 µs. Exposure times ranged from 10 ns to 70 ns. The frames presented in this paper correspond to 30 × 30 µm 2 areas, except for Figure 1(i). The first frame was typically captured prior to arrival of the ultrasound wave at the focal area. Seven frames were taken during ultrasound insonification, spanning a full ultrasound cycle of 2 µs. Contrast agent was insonified by 10 cycles of 0.5 MHz ultrasound with high acoustic amplitudes, in the range 0.66-0.85 MPa.

Figure 5 (

 5 Figure 5(iv) demonstrates the fragmentation, coalescence, and re-fragmentation

Fig

  Fig. 5(i) Fig. 5(ii)

1Figure 1 :Figure 2 :Figure 4 :Figure 5 :

 1245 Figure 1: Optical images of stages of ultrasound-induced microbubble

Acknowledgments

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

The authors are greatly indebted to Andrea Prosperetti for many fruitful 20 discussions on coalescence theory.

List of Tables