Harnessing the Power of Nature: Monodisperse Pickering Emulsion Droplets and Yolk‐Shell Microcapsules Utilizing Bee Pollen Particles

The preparation of monodisperse Pickering emulsions currently can only be achieved using microfluidics or membrane emulsification, limiting its widespread applications in many fields. Here, by simply introducing naturally occurring pollen grains during mechanical emulsification, it becomes extremely feasible to fabricate uniform Pickering emulsions. The process of shearing and turbulence leads to a constant reduction in the size of the droplets of the dispersed phase. The robust exine of the pollen grains in the continuous phase enables them to retain their structural integrity during emulsification, resulting in the formation of a single pollen‐in‐water‐in‐oil structure. Meanwhile, the intrinsic monodispersity of the pollen grains allows for the resulting emulsions to be similarly uniform, and they can be easily collected through centrifugation. As a result, this novel methodology achieves the effective formulation of monodisperse Pickering emulsions in an unprecedented way. Furthermore, the utilization of sporopollenin exine capsules (SECs) as a replacement for pollen, in conjunction with sol–gel interfacial engineering, enables the attainment of “yolk‐shell” dual‐shell microcapsules. This approach not only effectively addresses the issue of cargo leakage in SECs but also imparts exceptional uniformity and stability to the microcapsules.


Introduction
Conventional emulsion preparation usually involves the mixing of oil and water phases, followed by processes such as agitation, DOI: 10.1002/adfm.202316510vortexing, or shear homogenization to form a colloidal system of water-in-oil (w/o) or oil-in-water (o/w).The shear forces exerted during this procedure are inhomogeneous, resulting in a broad range of droplet sizes and the failure to achieve a uniform emulsion. [1,2]][9][10][11] The utilization of microfluidic chip designs and the advancements in photolithographic technology have led to the widespread adoption of the microfluidic method as the main method for producing monodisperse emulsions. [12,13]The approach involves the introduction of two or more liquid phases into microchannels through injection, with subsequent regulation of the flow rate and relative proportions of the fluids to form a mixing region within the channels.Subsequently, using the principles of microfluidic mechanics such as inertial forces and interfacial tension, the liquid droplets are subjected to fragmentation within the channels.This process leads to the generation of smaller droplets and the formation of monodisperse emulsions with a narrow size distribution. [14,15][18] An alternative technique employed in the fabrication of monodisperse emulsions is Shirasu Porous Glass (SPG) membrane emulsification, which also utilizes microchannels to achieve size control of the emulsion droplets. [19,20]Ma and Ngai successfully extended the technique of membrane emulsification to particle-stabilized emulsions, thereby attaining the ability to adjust droplet size through the utilization of SPG membranes of variable pore size. [21,22]In principle, the implementation of membrane emulsification has the potential to increase the throughput of emulsions.However, this method may compromise a certain level of uniformity as a result of the stringent necessity for accurate regulation of the pore size distribution within the membranes.
Recently, Wang et al. presented a novel method for the largescale preparation of double emulsions avoiding the need for conventional microfluidic approaches. [23]In essence, a w/o emulsion was made and subsequently emulsified into another aqueous phase to accomplish secondary emulsification.By utilizing the density difference and vortex-shearing effect, they were able to control the formation of water-in-oil-in-water (w/o/w) double emulsions containing a single droplet inside.During vortex shearing, the injected w/o emulsion was sheared and dispersed into small oil droplets.These small droplets, undergoing continuous shear, actually served as soft templates that limited the minimum droplet size they could reach.As a result, a double emulsion structure with a morphology of a single internal droplet was formed.It was found that the size and uniformity of the original emulsion droplets played a crucial role in the formation of the double emulsion.Hence, the potential for generating droplets with a uniform distribution may be possible by replacing the soft template with uniform particles and employing shear emulsification.
Therefore, our attention is directed toward the natural endowment of pollen grains.Pollen is an essential component of plant biology and is generated by the male gametes of plants. [24][27] Moreover, the size distribution of pollen varies from a few micrometers to several hundred micrometers, which is comparable to the size range observed in emulsions. [28,29]Herein, we utilized the inherent uniformity of natural pollen grains in shearing aqueous pollen dispersions into w/o Pickering emulsion droplets.The robust mechanical properties of pollen particles allow them to withstand high-speed shearing while staying intact in the aqueous phase, ultimately resulting in the formation of pollen-in-water-in-oil Pickering emulsions.Through a simple process of centrifugal separation, w/o Pickering emulsions containing individual pollen particles with droplet sizes akin to those of the pollen particles themselves are easily obtained, exhibiting a high level of monodispersity.A significant development in our work is the successful integration of sporopollenin exine capsules (SECs) as the core template in order to design novel yolk-shell microcapsules.The microcapsules possess a double-layered structure comprising pollen exine as the inner layer and a silica shell as the outer layer, enabling doublelayered encapsulation and protection of active substances as well as demonstrating exceptional uniformity and sustainability.

Results and Discussion
As depicted in Figure 1a, after washing with a mixture of ethanol and water for several times, the pollen grains brought back by bees can be homogenously distributed in the aqueous phase.At the same time, hydrophobic silica nanoparticles are introduced into the silicone oil phase D5 to serve as a particulate emulsifier in the formation of w/o emulsions. [30]In the emulsification process, the fragmentation of the water phase occurs resulting in the formation of numerous small droplets.These water droplets are promptly stabilized by hydrophobic silica nanoparticles (Figure S1, Supporting Information).However, the presence of pollen grains can impede the continued decrease in droplet size, resulting in the formation of droplets that include only one single pollen grain.Consequently, the resultant w/o Pickering emulsion has two distinct size dimensions of water droplets: minuscule water droplets (without pollen grains) and larger uniformly sized droplets that encapsulate a single pollen grain.The remarkable stability of Pickering emulsions can prevent the emulsion droplets from coalescence, thus these two dimensions of droplets can be readily separated by centrifugation in which the large water drops sediment and the smaller ones remain in the supernatant.This process yields a uniform distribution of water-in-oil Pickering emulsion drops (subnatant), with droplet sizes slightly exceeding those of pollen particles.Hydrophobic silica nanoparticles are commonly used to stabilize extremely small w/o Pickering emulsion droplets, and a stable Pickering emulsion with droplet sizes below 5 μm can be obtained through high shear emulsification as shown in Figure 1b.It is noteworthy that the introduction of pollen particles (camellia pollen here) into the aqueous phase leads to a phenomenon where the water around the pollen particles exhibits resistance to detachment when subjected to shear forces.As a consequence, larger droplet structures referred to as "pollen-in-water-in-oil" are formed, as seen in Figure 1c.The robust shell of pollen particles offers superior mechanical strength preventing it from being destroyed during shear, and their incredibly uniform size acts as a core template for monodisperse droplets.So, monodisperse pollen-in-water-inoil Pickering emulsion drops can be collected by centrifugation at the bottom of the centrifuge tubes.As seen in Figure 1c,d, this Pickering emulsion distributes pollen particles uniformly throughout the dispersed phase and can achieve the encapsulation of only one pollen particle in each droplet by optimizing the emulsification parameters (Figure 1e).[33] We found however that o/w Pickering emulsions stabilized by pollen grains were obtained in the absence of hydrophobic silica nanoparticles (Figure S2, Supporting Information).Thus a second particulate stabilizer (here hydrophobic silica nanoparticles) had to be added in order to stabilize primarily w/o emulsions.
With an average particle size of 30-40 μm (Figure S3, Supporting Information), camellia pollen grains resemble an elevated triangular cone (Figure 1f,j).The inherent fluorescence properties of natural pollen also allow us to clearly see the microstructure of the emulsion under confocal microscopy. [34]When activated by lasers with wavelengths of 405 and 488 nm, respectively, pollen grains can display strong blue and green fluorescence.As a result, the presence of water adhering to the surface of pollen grains can be easily seen by staining the aqueous phase with sodium fluorescein.The CLSM images in Figure 1g-i provide good vi-sual evidence of the distribution of the small water droplets and a single pollen-in-water droplet.In addition, centrifugation has been demonstrated as a successful method for the separation of larger pollen-in-water droplets from their smaller counterparts (see Figure 1k-m).
Moreover, by employing Nile Red it becomes possible to label hydrophobic silica nanoparticles so facilitating a visual illustration of the adsorption of these silica particles at the interface between water and oil.This is illustrated by the outermost purple band in Figure 2. The observation provides more evidence of the existence of a water-in-oil emulsion containing a single pollen grain in each drop.Nevertheless, it is noticeable that the droplet shapes formed using this technique deviate from spherical due to the influence of the morphology of the pollen particles.
To attain uniform-sized droplets, precise control of the pollen particle distribution within each droplet is crucial, with the optimal scenario being the presence of just one pollen particle per droplet.Hence, effective regulation and optimization of emulsification conditions are fundamental for shaping the emulsion structure.It is generally observed that higher shear homogenization speeds yield smaller droplets.However, the inclusion of pollen particles in this study posed a partial hindrance to droplet shearing.By applying more vigorous shearing, the dispersed pollen particles are expected to be separated more efficiently, thus facilitating the formation of droplets containing a solitary pollen particle.Conversely, slower shearing speeds may result in droplets containing several pollen particles.As the results show, at a shear speed of 10 000 rpm, although some water droplets containing a single pollen particle were formed, the majority of the droplets encompassed two or more pollen particles profoundly impacting the uniformity in droplet size (49.5 ± 12.5 μm) as seen in Figures 3a and S4 (Supporting Information).At a speed of 15 000 rpm, some droplets still contain 2-3 pollen particles (Figure 3b).Further increasing the speed to 17 600 rpm considerably reduced the occurrence of droplets with two pollen particles as shown in Figure 3c.Observations made at a speed of 20 000 rpm revealed that all droplets contained exclusively Figure 3. a-c) Optical microscope images of w/o Pickering emulsions containing silica nanoparticles and camellia pollen grains after homogenization at 10 000, 15 000, and 17 600 rpm respectively.Oil:water volume ratio was 10:1, pollen concentration was 20%.d) Optical microscope image of w/o Pickering emulsion with camellia pollen grains, oil:water volume ratio was 10:1, 20 000 rpm, pollen concentration was 20%.e) Optical microscope image of w/o Pickering emulsion with camellia pollen grains, oil:water volume ratio was 5:1, 20 000 rpm, pollen concentration was 10%.f) Optical microscope image of w/o Pickering emulsion with camellia pollen grains, oil:water volume ratio was 10:1, 20 000 rpm, pollen concentration was 40%.
only one pollen particle, resulting in highly uniform droplet sizes (38.6 ± 2.1 μm) as shown in Figure 3d.
Furthermore, the volume ratio of oil to water has a certain influence on the structure of emulsions.When the oil:water volume ratio was 5:1, the system became too viscous and the emulsion drops tend to encapsulate multiple pollen grains with a diameter of 42.3 ± 8.8 μm as shown in Figure 3e.Therefore, a volume ratio of 10:1 or higher is chosen for emulsion preparation (Figure S5, Supporting Information).Also, the concentration of pollen grains in the aqueous phase remarkably influences the emulsion structure.At a pollen concentration of 10%, it is possible to obtain a monodisperse water-in-oil Pickering emulsion although the overall efficiency remains relatively low.Increasing the pollen content to 20% allows for the maintenance of excellent size uniformity and achieves higher preparation efficiency (Figure 3d).However, as shown in Figure 3f and Figure S6 (Supporting Information), when the pollen content is increased to 30-40%, a limited number of droplets possess two pollen particles yet the overall homogeneity remains acceptable.As a result, it is recommended to add <30% pollen to the aqueous phase for optimal outcome.The statistical results considering the effect of shearing speed, oil:water ratio, and pollen content are presented in Figure S7 (Supporting Information), and are consistent with optical microscopy observations.
There are various types of natural bee pollen grains of different shapes and sizes.Rapeseed flower pollen is a prevalent form of bee pollen that exhibits a near-spherical morphology, characterized by a mean diameter ≈26 μm as shown in Figures 4b and S3 (Supporting Information).To explore the universality of producing uniform Pickering emulsions utilizing pollen grains, we selected rapeseed flower pollen grains as depicted in Figure 4a.As expected, the utilization of this pollen can also result in the formation of emulsion droplets that encapsulate individual pollen particles after shear emulsification.Furthermore, it was observed that the morphology of the water-in-oil droplets within the emulsions exhibited a greater degree of sphericity compared with those containing camellia pollen grains (Figure 4c,d).This suggests that the inherent morphology of the pollen grains plays a key role in determining the shape of the resulting emulsion droplets.In the same way, CLSM characterization provided confirmation of the adsorption of silica nanoparticles at the oil-water interface, which ensures the stability of the resulting uniform w/o Pickering emulsions (Figure 4e-g).
Despite the presence of a rough and uneven surface, both camellia pollen and rapeseed pollen grains have a predominantly spherical curvature.Hence, we chose sunflower pollen with a mean diameter of ≈32 μm (Figure S3, Supporting Information) due to its surface features protruding spike-like antennae resembling the morphology of the coronavirus (Figure 5b,c).It is noteworthy to emphasize that the utilization of sunflower pollen grains is suitable with the methodology developed here (Figure 5a).The presence of spike-like protrusions on the surface of the grains did not have any adverse impact on the formation of the emulsion.As displayed in Figure 5d,e, during the process of shear emulsification and subsequent centrifugal separation, a distinct and discernible dark boundary was observed surrounding each individual sunflower pollen particle, indicating the newly formed water-oil interface.In addition, CLSM images provide a close-up view of the liquid-liquid interface that was stabilized with silica nanoparticles (Figure 5f-k).3D reconstruction of a droplet encapsulating the pollen through CLSM clearly shows that pollen particles are dispersed in the droplet, and there is an obvious annulus gap between the outer wall of the pollen and the water-oil interface (Figure 5l-q).However, several elongated spike-like antennae seem to breach the water-oil interface, thereby extending into the continuous phase and generating a unique structure.It is hypothesized that the presence of spike-like antennae creates a significant steric barrier against shearing and turbulence particularly when the radius of the water droplet is less than the radius of the pollen grain.Simultaneously, the water-in-oil droplets derived from sunflower pollen grains exhibit superior sphericity compared with the other two pollen types owing to the highly symmetrical characteristics of the pollen grains.All w/o emulsions remained stable to coalescence (no water phase separated) although they sedimented to the bottom of the vessel liberating oil above.Centrifugation was used as an accelerated test of the storage stability of the monodisperse emulsions.After centrifugation at 4000 g for 10 min, the statistics of emulsion droplet sizes before and after centrifugation were also determined and no significant change was observed (Figure S8, Supporting Information), demonstrating the high stability of the resultant emulsions.
Pollen is widely present in nature and is a natural carrier of genetic material.The pollen wall is composed of two layers: a tough exine layer primarily made of sporopollenin and a polysaccharide-based intine layer. [35,36]The internal cavity of natural pollen is mainly filled with cytoplasm and sporoplasmic material including clusters of biomolecules and organelles.Proteins and lipids present in pollen grains are known to be essential for pollen development, germination, and fertilization.It is believed that certain pollen components can interact with the human immune system resulting in the onset of pollen allergies. [37]o eliminate potential allergic reactions, the pollen is first defatted to remove proteins and lipids from the exine. [38]Acid treatment is then used to remove residual proteinaceous materials and sporoplasmic organelles within the pollen grains. [29,39,40][43][44][45][46] It has been demonstrated here that untreated pollen particles have the capability to be utilized in the formulation of uniform Pickering emulsions.Nevertheless, the pollen particles themselves account for a significant proportion of the interior volume of the emulsion droplets hence constraining the encapsulation efficiency.
Following the processes of defatting and protein removal, the surfaces of pollen grains display a significant proportion of micro-and nano-scale holes and apertures.These structural features have a profound impact on the efficacy of encapsulating active substances.Hence, it is of considerable significance to investigate the potential amalgamation of emulsion interface engineering concepts with pollen microcapsules, with the aim of fabricating a monodisperse microcapsule system utilizing the inherent characteristics of naturally-occurring pollen exine.In this study, sunflower pollen grains were selected as the subject for investigation.The original pollen particles were replaced by sporopollenin exine capsules (SECs) and subjected to shear emulsification with silica nanoparticles as illustrated in

Figures 6a and S9 (Supporting Information
).The findings indicated that it is possible to generate monodisperse w/o droplets containing single SECs.After emulsification, the continuous phase was supplemented with tetraethyl orthosilicate (TEOS) and the resulting uniform Pickering emulsion droplets were solidified into monodisperse microcapsules through an interfacial sol-gel reaction.After defatting and acid treatment, nanopores and microscale apertures are present on the initial SEC surface (Figure 6b-e), which significantly affects the loading efficiency of actives.In this system, in addition to providing protection for internal active substances by inclusion of SECs, the generated silica shell covering the SECs functions as a strong shield to further prevent the leakage of active material.Opti-cal microscope and SEM images clearly demonstrate that the silica shell effectively encloses the SECs with minimal protrusions observed (Figure 6f-i), and the SECs become entirely enveloped by the silica shell resulting in the formation of a "yolkshell" microcapsule structure with a dual layer comprising exine and silica.A detailed comparison can be seen in Figure S10 (Supporting Information) demonstrating the obvious silica shell formation.
As demonstrated in Figure 7a, the SECs can be identified as naturally occurring hollow microcapsules.Silica particles of 300 nm diameter were employed as representative cargo in our study, and they were successfully encapsulated within the hollow cavity of the SECs with the assistance of ultrasound through the apertures on the pollen exine (Figure 7b).However, this approach is susceptible to the occurrence of leakage of the cargo via the apertures.The CLSM observation revealed that a significant number of model particles within the SECs were seen to permeate into the bulk as shown in Figure 7c.In contrast, the utilization of yolk-shell SECs featuring a dual-layer configuration (Figure 7d) presents a viable solution to address the issue of cargo leakage.As displayed in Figure 7e, subsequent to the encapsulation of the model particles within SECs, the process of interfacial sol-gel and silica shell encapsulation effectively ensures their preservation within the entirety of the microcapsule.Despite the possibility of certain model particles being expelled through the apertures on the exine during vortexing, their passage is successfully blocked by the outermost silica shell (Figure 7f), thereby accomplishing successful and durable encapsulation of the model cargo.

Conclusion
Our study reveals that the inclusion of uniform bee pollen grains greatly facilitates the production of monodisperse w/o Pickering emulsion droplets.The method employed does not require welldesigned chips and channels commonly found in conventional microfluidics, nor does it rely on the costly membrane materials used in membrane emulsification.Pollen particles have a diverse array of shapes and size distributions that are comparable to the sizes of droplets typically found in emulsions.Coupled with the exceptional mechanical strength of pollen particles and their uniform size distribution within a given species, they serve as internal templates and support for the formation of monodisperse droplets under high shear emulsification.Moreover, the interfacial adsorption of silica nanoparticles at the oil-water interface prevents the coalescence of the monodisperse droplets during collection.Our research reveals that the w/o droplets prepared using sunflower pollen grains display a distinctively spherical and symmetrical structure, while the entire pollen grains were positioned at the central region of the water phase within the droplets.By substitution of pristine pollen grains with SECs, it was easy to fabricate monodisperse microcapsules with a "yolkshell" structure via the sol-gel reaction at the oil-water interface.The microcapsules are composed of an inner exine layer and an exterior silica shell, which significantly enhances the encapsulation effectiveness of active ingredients compared with raw SECs.Since pollen particles are derived from nature and can be combined with interface engineering, this provides a diverse and sustainable platform for fundamental research and applications in materials science.
Methods: Preparation of Monodisperse Pickering Emulsions: Bee pollen grains were simply washed with ethanol and water via centrifugation and initially dispersed in water (10-40%, w/v) by shaking.3% (w/v) of R974 hydrophobic silica nanoparticles were dispersed in D5 silicone oil using an ultrasonic probe (Elmasonic P 60H, 37 kHz).During the emulsification process, the aqueous dispersion was added dropwise into the oil phase at varied homogenizing speed to form w/o Pickering emulsions.Afterward, the obtained emulsions underwent multiple centrifugations to collect the monodisperse Pickering emulsion from the bottom layer.
Preparation of Yolk-Shell SECs: The fabrication of SECs includes three steps: i) Pollen grain washing -pollen grains collected by bees were washed with a mixture of ethanol-water in the volume ratio of 2:1.Typically sunflower pollen grains (20 g) were dispersed in 100 mL aqueous ethanolic solution with ultrasound (Elmasonic P 60H, 37 kHz) for 15 min for three times, followed by washing with 20 mL of acetone until the supernatant became clear; ii) defatting -the pollen grains were refluxed with 20 mL of acetone at 50 °C overnight and collected through vacuum filtration.Then the defatted pollen was transferred to a glass dish or beaker to be dried to a powder; iii) acid treatment -the defatted pollen (3.5 g) was added to 20 mL of 85% phosphoric acid and refluxed at 80 °C for 5 h.Finally, the pollen grains were washed with hot water three times, acetone two times, once with 2 m hydrochloric acid, deionized water three times, and dried to SECs as a powder.
Preparation of Monodisperse Yolk-Shell Dual-Layered Microcapsules: Replace the raw pollen grains with 2.5% (w/v) hollow sunflower SECs to prepare w/o Pickering emulsions.Then, TEOS was introduced into the oil phase to initiate the interfacial sol-gel reaction to form the silica shell.To enhance the growth of the silica shell, ammonia was added and the sol-gel reaction was carried out at 50 °C overnight.
Microencapsulation of Model Active in SECs and Yolk-Shell SECs: The model active (300 nm silica particles) was first labeled with green fluorescer by soaking the particles in an ethanolic solution of FSS and TEOS with stirring.Then, 2.5% (w/v) hollow sunflower SECs and 20% (w/v) model particles were mixed using ultrasound (Elmasonic P 60H, 37 kHz) for 10 min for passive loading, followed by centrifugation to remove the free silica nanoparticles.For encapsulation in yolk-shell microcapsules, the SECs with encapsulated silica nanoparticles are homogenized with D5 silicone oil using the same procedure as in the preparation of yolk-shell SECs.Then TEOS was introduced to produce the outer silica shell via the interfacial sol-gel reaction at 50 °C for 20 h.
Characterization: Silica particles, pollen grains, emulsions, and capsules were characterized by optical microscopy (Nikon Ni-U), confocal laser scanning microscopy (CLSM; Nikon AX equipped with Eclipse Ti2 body), and scanning electron microscopy (SEM; Hitachi S-4800).The pollen grains or SECs observed by CLSM were excited with lasers of 405, 488, and 561 nm and showed blue, green, and red/purple colors in different channels.The fluorescence of FSS and Nile Red were excited with lasers of 488 and 561 nm respectively.The contact angles were measured using a contact angle analyzer (Dataphysics OCA15EC).Size distributions of the three types of pollen were measured using a Malvern MasterSizer 3000 instrument (Malvern, UK).The size analysis of "pollen-in-water-inoil" Pickering emulsion droplets was calculated by Image J software from 100 droplets.

Figure 1 .
Figure 1.a) Schematic illustration of the fabrication of monodisperse water-in-oil Pickering emulsions utilizing bee pollen grains, created with Med-Peer (www.medpeer.cn).b,c) Optical microscope images of w/o Pickering emulsions without or with camellia pollen grains, respectively.d) Optical microscope image of monodisperse w/o Pickering emulsion with a single camellia pollen grain inside water drops after separation.e) Sketch of the model of monodisperse w/o Pickering emulsion droplet containing camellia pollen grain.f,j) SEM images of the camellia pollen grains after washing.g-i) 3D CLSM images of the primary w/o Pickering emulsion with camellia pollen grain in different fluorescence channels; inset images represent the cross-section of the emulsion droplet.k-m) CLSM images of the monodisperse w/o Pickering emulsion containing a single camellia pollen grain in each drop in different fluorescence channels.For emulsions, oil:water volume ratio was 20:1 homogenized at 20 000 rpm with a pollen concentration of 10%.

Figure 2 .
Figure 2. CLSM images of the monodisperse water-in-oil Pickering emulsion containing a single camellia pollen grain in each drop in a) differential interference contrast channel, b) purple channel, and c) overlapped channel.

Figure 4 .
Figure 4. a) Schematic illustration of the fabrication of monodisperse w/o Pickering emulsions utilizing rapeseed flower pollen grains.b) SEM image of rapeseed flower pollen grains after washing.c) Optical microscope image of w/o Pickering emulsion with rapeseed flower pollen grains.d) Optical microscope image of monodisperse w/o Pickering emulsion with single pollen grain inside each drop after separation.e-g) CLSM images of monodisperse w/o Pickering emulsion containing a single pollen grain within water drop in different confocal channels.Oil to water volume ratio was 10:1, homogenizing speed was 20 000 rpm, pollen concentration was 20%.

Figure 5 .
Figure 5. a) Schematic illustration of the fabrication of monodisperse w/o Pickering emulsions utilizing sunflower pollen grains, sunflower cartoon created with MedPeer (www.medpeer.cn).b) SEM image of sunflower pollen grains after washing.c) Optical microscope image of sunflower pollen grains after washing.d) Optical microscope image of w/o Pickering emulsion containing sunflower pollen grains.e) Optical microscope image of monodisperse w/o Pickering emulsion with single sunflower pollen grain inside each drop after separation; inset image is magnification of one droplet.f-k) CLSM images of monodisperse w/o Pickering emulsion containing a single sunflower pollen grain in each drop in different fluorescence channels at two magnifications.l-n) 3D CLSM front views of w/o Pickering emulsion drop with sunflower pollen grains in different fluorescence channels.o-q) 3D CLSM side views of w/o Pickering emulsion drop with sunflower pollen grains in different fluorescence channels.Oil to water volume ratio was 10:1, homogenizing speed was 20 000 rpm, pollen concentration was 20%.

Figure 6 .
Figure 6.a) Schematic illustration of the fabrication of monodisperse "yolk-shell" microcapsules utilizing SECs.b) Optical microscope image of the SECs.c) SEM image of the SECs.d,e) SEM images of a single SEC with open aperture.f) Optical microscope image of the monodisperse "yolk-shell" microcapsules.g) SEM image of the "yolk-shell" microcapsules.h,i) SEM images of a single "yolk-shell" microcapsule.

Figure 7 .
Figure 7. a) CLSM images of empty SECs.b) CLSM images of SECs after loading with 300 nm silica particles.c) CLSM images of silica-loaded SECs after vortexing.d) CLSM images of empty yolk-shell SECs.e) CLSM images of yolk-shell SECs after loading with 300 nm silica particles.f) CLSM images of silica-loaded yolk-shell SECs after vortexing.Silica particles with FSS labelling (green) were used as the model cargo.SECs were dispersed in water, yolk-shell SECs were dispersed in oil.