Amoebae Assemble Synthetic Spherical Particles to form

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Biological and Environmental Phenomena at the Interface

Amoebae Assemble Synthetic Spherical Particles to form Reproducible Constructs Pei Bian, Joseph Strano, Peiwen Zheng, Miriam Steinitz-Kannan, Stephen J. Clarson, Ramamurthi Kannan, and Thomas J McCarthy Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00333 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 23, 2019

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Amoebae Assemble Synthetic Spherical Particles to form Reproducible Constructs Pei Bian,*,† Joseph Strano, ‡ Peiwen Zheng,† Miriam Steinitz-Kannan,‡ Stephen J. Clarson, § Ramamurthi Kannan,‡ Thomas J. McCarthy*,† †

Polymer Science and Engineering Department, University of Massachusetts, Amherst,

Massachusetts 01003, USA. ‡

Department of Biological Sciences, Northern Kentucky University, Highland Heights,

Kentucky, 41099, USA. §

Department of Materials Science and Engineering, University of Cincinnati, Cincinnati, Ohio

45221, USA.

ABSTRACT: Difflugia are testate Amoebae that use particulate inorganic matter to build a protective shell (generally called a test or theca). Difflugia globulosa were grown both in culture containing only naturally occurring theca building materials and under conditions where synthetic particles were present as well. The presence of particles, monodisperse Stöber silica microspheres of 1, 3 and 6 µm diameter or 4 µm polystyrene spheres, dramatically increased the rate of Difflugia growth, and foreign microspheres became the overwhelmingly dominant

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construction material. Optical and electron microscopy of the 6 µm particle studies revealed that Difflugia construct spherical vase-shaped thecae with strikingly reproducible composition, morphology and size. Time-lapse photography revealed construction techniques and masonry skills as Difflugia herded particles together, trapped them using phagocytosis, and applied the particles with bio-cement from inside the developing theca. The reported observations identify taxonomy complications, biomicrofabrication possibilities, and a discrete environmental impact of synthetic particle pollutants.

INTRODUCTION Difflugia are single-celled testate Amoebozoa in the family Arcellinidae that construct “houses” (called tests or thecae) using mineral particles like sand grains, mud, clay, diatom frustules and shell fragments that are present in their surroundings. Their environments are widespread on earth and include soils, bogs, mosses, rivers, lakes and ponds.1-4 These species have been of keen interest to ecologists, soil scientists and paleontologists since the 19th century and have gained increased importance as a study group over the most recent two decades.4 More than 300 species and 200 subspecies, varieties or forms have been described as belonging to the genus Difflugia Leclerc 18155-7 and it has been suggested that taxon richness has been considerably underestimated.8-10 The taxonomy of this genus has historically been based on differences in shape, size and mineral composition of the thecae as determined primarily by light microscopy, however recent studies involving molecular (DNA) analysis have suggested a greater complexity than is revealed by shell composition and morphology, and that both phenotypic plasticity and cryptic diversity are significant.11-14 Shell morphology and composition can be related to growth


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location, to the external environment, and in particular, to the building materials available in those environments.15-20 These taxonomic problems make determination to species level by traditional methods extremely difficult, even for specialists. The lack of taxonomic expertise in this area has been noted4 and described as an impediment to research progress. Here we emphasize another issue that contributes to these problems: As testate amoebae assemble their thecae from microparticulate solids, human-derived pollutants of the appropriate length scale may affect theca construction and likely more importantly, this stage of their life cycle. Recent studies have identified particulate matter derived from extreme atmospheric pollution18 and a volcanic eruption19 in testate amoebae. Perhaps certain types of synthetic particles will favor the success of one species over others and lead to a change in community structure. Other issues, which are positive, potentially useful, and interesting, are whether these amoeba’s construction skills can be harnessed to prepare assembled structures that are useful in materials science and technology or whether they can be useful for bioremediation of microparticulate plastic pollutants. In this study, we added artificial building materials to culture media and found that Difflugia globulosa readily accept and incorporate these materials in their shells. The synthetic materials were micron-sized monodisperse spherical silica and polystyrene particles. The results suggest that the rate of Difflugia growth is increased by the addition of particles and that more symmetric and consistent theca structures are formed when monodisperse spheres are present. Time-lapse photographs were taken that give insight into the process of Difflugia shell construction. We note the 19th century studies21 of Verhorn who attempted to study theca growth of Diffugia urceolata using finely powdered dark blue and black glass.


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MATERIALS AND METHODS Difflugia were collected from stones beneath a small waterfall that drains a pond downstream from Sharon Lake and leads, via Sharon Creek and Mill Creek to the Ohio River (39o16’ 47.93” N; 84o 24’ 01.96” W). An algae biofilm was scraped with a brush into a bottle. Well-mixed samples of this biofilm were placed in square 100 mm x 100 mm x 15 mm plastic disposable petri dishes (culture plates), in sufficient depth to cover the bottom of the dishes. Suspensions of 0.1 g of microspheres (1 µm, 3 µm or 6 µm silica Stöber particles22 or 0.1 g of 4 µm polystyrene particles23 in 50 mL of deionized water) were added. Samples were maintained in a light chamber (Percival MFG. Co. Model MB-60B) at 28 °C under a light level of 500 lux provided by white fluorescent bulbs with a 16 h / 8 h light/dark photoperiod) for several weeks. Periodic observations were generally made daily and were made at least as often as every two days. Optical microscopy was performed with Nikon Optiphot and Olympus BX60 microscopes. SEM data were recorded using a JEOL NeoScope JCM-5000 on samples that were coated with a thin layer of gold.

RESULTS Difflugia globulosa24 was collected from a biofilm growing on rocks below a waterfall from a pond that leads, via creeks, to the Ohio River. The initial research goal was to culture diatoms that were abundant in these samples. Diatoms are single celled organisms that create elaborate silica skeletons (frustules) with a wide variety of structures. Our initial objective was to produce monodisperse diatoms from which monodisperse silica microparticles (frustules) with complex


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and multiple-length-scale morphology could be prepared. Optical microscopy revealed abundant diatoms and other algae, bacteria, and a variety of other protozoa and microscopic invertebrates, the typical biofilm community in this location. We emphasize that we did not observe Difflugia in initial microscopy studies, however we were not looking for it specifically. The biofilm was cultured in plates and aqueous suspensions of silica Stöber particles22 of 1, 3 and 6 µm diameter were added as a diatom nutrient (silicic acid source). Difflugia thecae became obvious and abundant in these samples within two days of when the cultures were prepared with Stöber particles; colonies developed that primarily appeared anchored to green algae that were present. Figure 1a-c shows optical micrographs of these colonies grown with 1, 3 and 6 µm diameter

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Figure 1. Optical Micrographs of Difflugia globulosa thecae, cultured in the presence of 1 µm silica (a), 3 µm silica (b), 6 µm silica (c) and 4 µm polystyrene (d) particles. Individual theca


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cultured with no synthetic particles present (e), 1 µm silica particles (f), 3 µm silica particles (g), and 6 µm silica particles (h, i, j).

silica particles, respectively. Our interest turned from monodisperse diatom preparation to the Difflugia and ~4 µm diameter polystyrene particles were prepared23 and used in culture as a control to test whether the shape or the surface chemistry of the Stöber particles was responsible for their selection. Fig. 1d shows Difflugia that were constructed with polystyrene particles. Cultures of the biofilm that were prepared without synthetic particles were re-examined and we note that it was difficult to find and identify complete Difflugia thecae; they were present in much fewer number than in cultures containing synthetic particles and we did not obtain a very good photograph for Fig. 1e. We recommend that the reader do an internet search of “Difflugia globulosa images” and compare dozens of much better images of native Difflugia globulosa with those in Fig. 1. Fig. 1e-g show optical micrographs of a wild theca (prepared with no added particles), one prepared with 1 µm particles, and one formed with 3 µm particles, respectively. Fig. 1h-j show different examples of theca formed with 6 µm Stöber particles. The optical microscopy studies with the 6 µm particles were most lucid, and good examples of thecae, that could be easily photographed, were numerous. This system was chosen for further electron microscopy studies. A drop of an 8-day old culture containing the 6 µm particles was applied on carbon conducting tape, allowed to dry in air, and was analyzed by scanning electron microscopy (SEM). A conducting layer of gold was applied with a sputter coater and images were obtained at a voltage of 10 kV. Figure 2a shows a region of this sample that contains ~30 relatively intact thecae. We note that they are remarkably similar in morphology (vase-like and nearly spherical) and dimension (~60 µm diameter). Also visible are numerous diatoms, diatom fragments, Stöber


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particles that were not incorporated in Difflugia shells, other debris, and dried fibrous algae that is considerably shrunken and less obvious than in the optical micrographs. The drying during sample preparation was not controlled in any way and we point out that the dehydration process from dilute (high surface tension) aqueous dispersion to high vacuum precipitate caused considerable damage to the native structure. An enlarged view of Fig. 2a (upper right) is shown

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Figure 2. Scanning electron micrographs of Difflugia globulosa thecae obtained from gold coated samples that were prepared by drying a drop of culture on conducting tape. (a) Low magnification image showing ~30 Difflugia theca that were cultured with 6 µm silica particles. (b) Enlarged view of the upper right of a. (c, d) Higher magnification images of two 6 µm silica particle - derived thecae detailing the pseudostome and back of two different thecae. Note that


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the interior wall of the theca is visible in c and is comprised primarily of close-packed particles. (e) A theca that was prepared with 3µm silica particles present.

as Fig. 2b and indicates that the openings (pseudostomes) of thecae are nearly perfectly circular and rather consistent, but not identical in diameter. Fig. 2c is shown to demonstrate that the spherical particles are present and apparently close-packed on the interior walls of the theca. Fig. 2d is the back of a shell that remained relatively intact during drying. Spherical particles and sections of bio-cement/particle composites dislodged from most theca during the SEM sample preparation. Figure 2c and 2d also indicate that other inorganic debris including small diatoms and diatom fragments that is smaller than the particles is incorporated in the Difflugia thecae in much lower volume concentration than silica spheres. Figure 2e is included to show that thecae of similar size and shape are formed when 3 µm Stöber particles are used in culture. This micrograph is the best of only a few obtained and shows the theca well-anchored to algae; we do not have reproducible data as for the case of the 6 µm studies. The 3 µm particle cultures were not studied in detail by SEM as initial experiments were less productive than the 6 µm studies, but we note that the 3 µm particles appear less well-ordered on the exterior of the theca in comparison with the 6 µm particles. Other micrographs are included as Supporting Information (Figs. S1 - S6). Several chance observations of Difflugia in action, collecting particles and constructing thecae, were made and a few were recorded as individual optical micrographs that could be put together in time lapse sequence. Figure 3a and 3b show a Difflugia using pseudopodia to collect and trap about ten 6 µm Stöber particles and a larger diatom. These two photographs were taken about 30 sec apart. Figure 3c-e show photographs that were taken over less than 1 min and exhibit the


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Figure 3. Diffugia globulosa collecting and ingesting 6 µm silica particles. The white scale bars are 10 µm in length. (a, b) These two photographs were recorded ~30 sec apart. Note that a larger diatom was also ingested. (c-e) These three photographs were recorded over ~60 sec.


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collection of particles and subsequent movement (to the left) of a Difflugia. We note that this event was not recognized during the microscopy experiment, however it was recorded in the corner of the field of view of a larger diatom that was being followed (Fig. S6). Figure 4 shows time lapse photographs of a Difflugia collecting 6 µm Stöber particles and bringing them inside a theca under construction. Figure 4a shows a Difflugia containing particles above its theca. Three minutes after the photograph shown in Fig. 4a was recorded, the Difflugia reaches out with pseudopodia and grasps the back of the theca (Fig. 4b). After an additional 3 min, it rotates the theca and adsorbs to it (Fig. 4c). Figures 4c-f span less than 1 min as the Difflugia almost completely enters the theca. Figures 4g-k span the subsequent minute as the Difflugia completes its entry. We highlight that this figure shows a partially built hemispherical theca that consists of nearly close-packed spherical particles embedded in bio-cement. The wall construction appears to have a very regular, and perhaps equilibrated, composite composition and structure.

DISCUSSION The observations described above lead to a few factual statements that can be made concerning one species of Difflugia isolated from a single environment. They also permit us to make multiple conjectures that both suggest and would require additional experimentation for verification, both for this species and this environment and most certainly for different species and environments. The research has thus produced multiple questions as well as a few answers. It is clear from Fig. 1a-d, which show abundant thecae present in spherical particle - containing cultures, in comparison to their relative absence in cultures that did not contain added particles, that 1-6 µm diameter particles enhance the rate of theca construction. It could be interpreted


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Figure 4. Time lapse photographs of a Difflugia delivering multiple 6 µm silica particles to the interior of a theca under construction. The white scale bars are 10 µm in length. (a) A particleladen Difflugia above its theca. (b) Difflugia grasps (attaches to) and turns the theca. (c-k) Difflugia enters the concave side of the developing theca and completely disappears from view. Images c-k were recorded in less than 120 sec indicating its rapid movement.


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from these experiments that Difflugia globulosa is difficult or impossible to culture using its native particle-containing environment as a medium, but that the addition of spherical particles contributes to their livelihood and permits their culture. It could also be interpreted that construction material which offers protection from predation is at least as important as nutrition for Difflugia to attain a large population. We assume, but have only thecae as confirmation, that the number of Difflugia globulosa increases in the presence of spherical particles. We have no evidence, however, to discount that single amoebae prepared all the thecae in each culture. Comparisons of Fig. 1d with Fig. 1a-c suggest that the composition, density and surface chemistry of the particles (silica or polystyrene) are not important. The size of the thecae remains constant at ~ 60 µm diameter, independent of particle size or composition. It is remarkable that Difflugia not only readily adapts (apparently hungrily) to the synthetic materials, but also to their perfectly spherical shape, which would not be encountered in natural environments. These observations contrast with those of Verworn21 who needed to mechanically prod Diffugia urceolata to ingest synthetic glass fragments. Figures 3 and 4 give insight into the theca construction mechanism and multiple skills of the Difflugia. These data are from experiments performed with 6 µm diameter silica particles. The Difflugia corrals, “scoops up,” and ingests via endocytosis, groups of particles. Presumably a composite of particles and bio-cement is formed within the Difflugia and divided from the cell as a seed of the theca. We are conjecturing, as we have no data on stages of construction prior to that shown in Fig. 4. The Difflugia leaves the seed, gathers more particles (Fig. 3) and returns to deposit more composite on/in the seed. The Difflugia molds the incipient theca by creating a bowl shape and forcing additional composite into the concave face. The walls of the theca are thus formed by biaxial expansion and must be plastic and self-adhesive to accommodate the


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addition of spherical particles. We surmise that the Difflugia continues this masonry until the interior of the theca is an appropriate volume and the walls withstand an appropriate applied pressure. We estimate that the theca shown in Fig. 4 is about 50% complete and we note that the pseudostome (opening) is approximately the same diameter as the final diameter of the thecae at this stage of construction. Later stages of wall construction must involve a constriction or narrowing of the opening. We have no insight into the interesting question of whether the Difflugia accomplishes this from the concave (inside) or convex (outside) side or both. All of the experiments reported were carried out with identical masses of 1, 3 and 6 µm diameter silica particles and 4 µm diameter polystyrene particles, thus the number (and concentration) of particles was not kept constant and varied significantly (by ~2 orders of magnitude) in these four cultures. The 6 µm diameter experiments afforded the best optical micrographs and these data could be interpreted to conclude that this diameter, or this concentration, or the combination of these two variable magnitudes is more optimal culture conditions for Difflugia globulosa than the other three in which both variables were changed. This is a rather weak conclusion and we emphasize that neither concentration nor diameter was changed independently. We also note that the 6 µm particles were significantly easier to focus on (in the microscope) than the smaller particles. We do believe, however, that the conditions of the other experiments could be optimized to prepare more easily observable thecae, and that, no doubt, more ideal conditions could be identified that used particle mixtures. Regardless of whether or not the diameter and/or the concentration used for the 6 µm particle cultures were fortuitous, these conditions were productive and were maintained and reproduced. Figure 1h-j as well as all of the micrographs in Fig. 2 suggest that very reproducible and robust


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theca are prepared from 6 µm diameter particles. These appear to be ideal building blocks that are assembled in a nearly close-packed fashion and are held together by a relatively low mass of bio-cement that the Difflugia provides. We cannot comment on whether or not the Difflugia prefer the synthetic particles over their natural building material or whether they discard particles that don’t fit well. We may have flooded the system and overwhelmed the natural choices. The “fresh” surfaces of the synthetic particles may be more obvious or accessible to the Difflugia than biofilm-coated diatom frustules. The optical images suggest that the 6 µm synthetic particles are the only solid construction material in the theca walls, but the presence of diatom fragments and other debris are clear from the SEM images (Figs. 2c and 2d). The optical micrographs (recorded from aqueous suspensions) also suggest that the “wet strength” of the composite wall is significant and that the particles are embedded in the bio-cement to give rather smooth walls. The bio-cement must adsorb to the silica surfaces from water and provide sufficient adhesive strength in the aqueous environment. We mention above, concerning the polystyrene particle control experiment, that the surface chemistry of the particles is not important to the increase in theca construction rate. But this interface is most certainly important to the wet strength of the composite walls. The images in Fig. 1h-j and Fig. 4 also show that silica particles are present and evenly distributed throughout the theca walls, right up to the edge of the circular pseudostomes. This is not obvious from the SEM images in Fig. 2 which show the general absence of particles on the exterior theca walls near the pseudostomes. We suspect that this inconsistency is due to fracture of the theca during the drying process of SEM sample preparation. The aqueous bio-cement shrinks significantly during dehydration and retracts almost completely from the particles leaving them attached by only a small area. The greater positive curvature near the pseudostomes causes particle release during drying. The SEM data show the


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presence of particles on the interior surfaces of the theca (most clear in Fig. 2c) and this is not obvious from the optical images. The micrographs indicate that the walls are comprised of an average of about 2 layers of particles.

SUMMARY AND IMPACT To our knowledge these are the first reported observations of the behavior of a single celled organism using primarily artificial particles as construction materials. This has obvious implications to taxonomy, since the species of Difflugia have been differentiated by the materials that they use to build their shell and such materials can affect the shape of the shell.15 The environmental implications are also obvious, because something man-made (Stöber particles, polystyrene spheres and likely microparticle pollutants) can clearly alter the growth and structure of a common microorganism. Materials scientists will be impressed with the biomicrofabrication skills of this Amoebozoan and will consider other compositions and other shaped particles to challenge their assembly/construction skills. The build-up of polystyrene microspheres to larger structures by Difflugia hints to potential application in bioremediation of plastic micropollutants.


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ASSOCIATED CONTENT Supporting Information. Figures S1 - S6. AUTHOR INFORMATION Corresponding Authors *Email: [email protected] *Email: [email protected]

ORCID Pei Bian: 0000-0003-0920-0028 Thomas J. McCarthy: 0000-0003-0414-010X Notes: The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the NSF-sponsored Materials Research Science and Engineering Center (DMR 0820506) for financial support and Northern Kentucky University for use of their optical microscopy and culture facilities.

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(3) Arrieira, R. L.; Schwind, L. T. F.; Joko, C. Y.; Alves, G. M.; Velho, L. F. M.; LansakToha, F. A. Relationships between Environmental Conditions and the Morphological Variability of Planktonic Testate Amoeba in Four Neotropical Floodplains. Eur. J. Protistology 2016, 56, 180-190. (4) Kosakyan, A.; Gomaa, F.; Lara, E.; Lahr, J. G. Current and Future Perspectives on the Systematics, Taxonomy and Nomenclature of Testate Amoebae. Eur. J. Protistology 2016, 55, 105-117. (5) Mazei, Y.; Warren, A. A Survey of the Testate Amoeba Genus Difflugia Leclerc, 1815 Based on Specimens in the E. Penard and C. G. Ogden Collections of the Natural History Museum, London. Part 1: Species with Shells that are Pointed Aborally and/or have Aboral Protuberances. Protistology 2012, 7, 121-171. (6) Mazei, Y.; Warren, A. A Survey of the Testate Amoeba Genus Difflugia Leclerc, 1815 Based on Specimens in the E. Penard and C. G. Ogden Collections of the Natural History Museum, London. Part 2: Species with Shells that are Pyriform or Elongate. Protistology 2014, 8, 133-171. (7) Mazei, Y.; Warren, A. A Survey of the Testate Amoeba Genus Difflugia Leclerc, 1815 Based on Specimens in the E. Penard and C. G. Ogden Collections of the Natural History Museum, London. Part 3: Species with Shells that are Spherical or Ovoid. Protistology 2015, 9, 3-49. (8) Geisen, S. et al., Soil Protistology Rebooted: 30 Fundamental Questions to Start with. Soil Biol. Biochem. 2017, 11, 94-103.


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(9) Reczuga, M. K.; Swindles, G. T.; Grewling, L.; Lamentowicz, M. Arcella peruviana sp. nov. (Amoebozoa: Arcellinida, Arcellidae), a New Species from a Tropical Peatland in Amazonia. Eur. J. Protistology 2015, 51, 437-449. (10) Boenigk, J.; Ereshefsky, M.; Hoef-Emden, K.; Mallet, J.; Bass, D. Concepts in Protistology: Definitions and Boundaries. Eur. J. Protistology 2012, 48, 96-102. (11) Oliverio, A. M.; Lahr, D. J. G.; Nguyen, T.; Katz, L. A. Cryptic Diversity within Morphospecies of Testate Amoebae (Amoebozoa: Arcellinida) in New England Bogs and Fens. Protist 2014, 165, 196-207. (12) Roland, T. P.; Amesbury, M. J.; Wilkinson, D. M.; Charman, D. J.; Convey, P.; Hodgson, D. A.; Royles, J.; Clauss, S.; Völcker, E. Taxonomic Implications of Morphological Complexity within the Testate Amoeba Genus Corythion from the Antarctic Peninsula. Protist 2017, 168, 565-585. (13) F. J. Gomaa, F. J.; Yang, J.; Mitchell, E. A. D.; Zhang, W.-J.; Yu, Z.; Todorov, M.; Lara, E. Morphological and Molecular Diversification of Asian Endemic Difflugia tuberspinifera (Amoebozoa, Arcellinida): A Case of Fast Morphological Evolution in Protists? Protist 2015, 166, 122-130. (14) Dumack, K.; Siemensma, F.; Bonkowski, M. Rediscovery of the Testate Amoeba Genus Penardeugenia (Thaumatomonadida, Imbricatea). Protist 2018, 169, 29-42. (15) Yu, Z.; Zhang, W.; Liu, L.; Yang, J. Evidence for Two Different Morphotypes of Difflugia tuberspinifera from China. Eur. J. Protistology 2014, 50, 205-211. (16) Armynot du Châtelet, E.; Noiriel, C.; Delaine, M. Three-Dimensional Morphological and Mineralogical Characterization of Testate Amebae. Microsc. Microanal. 2013, 19, 15111522.


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(17) Lahr, D. J. G.; Grant, J. R.; Katz, A. A. Multigene Phylogenetic Reconstruction of the Tubulinea (Amoebozoa) Corroborates Four of the Six Major Lineages, while Additionally Revealing that Shell composition does not Predict Phylogeny in the Arcellinida. Protist 2013, 164, 323-339. (18) Fiałkiewicz-Kozieł, B.; Smieja-Król, B.; Ostrovnaya, T. M.; Frontasyeva, M.; Siemińska, A.; Lamentowicz, M. Peatland Microbial Communities as Indicators of the Extreme Atmospheric Dust Deposition. Water Air Soil Pollut. 2015, 226, 97. (19) Delaine, M.; Armynot du Châtelet, E.; Recourt, P.; Potdevin, J. L.; Mitchell, E. A.; Bernard, N. Cinderella’s Helping Pigeons of the Microbial World: The Potential of Testate Amoebae for Identifying Cryptotephra. Eur. J. Protistol. 2016, 55, 152-164. (20) Delaine, M.; Bernard, N.; Gilbert, D.; Armynot du Châtelet, E. Origin and Diversity of Testate Amoebae Shell Composition: Example of Bullinularia indica Living in Sphagnum capillifolium. Eur. J. Protistology 2017, 59, 14-25. (21) Verworn, M. Biological Studies of Protista. Ann. Mag. Nat. Hist. 1888, 2, 155-169. DOI: 10.1080/00222938809460896. (22) Stӧber, W.; Fink, A. Controlled Growth of Monodisperse Silica Spheres in the Micron Size Range. J Coll. Int. Sci. 26, 62-69 (1968). (23) Zhang, J. H.; Chen, Z.; Wang, Z. L.; Zhang, W. Y.; Ming, N. B. Preparation of monodisperse polystyrene spheres in aqueous alcohol system. Materials Letters 57, 44664470 (2003). (24) Leidy, J. Fresh-Water Rhizopods of North America. Government Printing Office, Washington (1879). DOI: 10.5962/bhl.title.39676.


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Amoebae Assemble Synthetic Spherical Particles to form Reproducible Constructs Pei Bian,*,† Joseph Strano, ‡ Peiwen Zheng,† Miriam Steinitz-Kannan,‡ Stephen J. Clarson, § Ramamurthi Kannan,‡ Thomas J. McCarthy*,†

amoebae Stöber Particles 50 µm


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Amoebae Assemble Synthetic Spherical Particles to form

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