Pest insect control is an essential component for crop protection and public health. Neonicotinoids are a relatively new class of insecticides developed in the last four decades. Thiamethoxam, a member of the neonicotinoid class, has shown outstanding potency for crop protection against a variety of piercing-sucking pests. However, its use in industrial-volume packing and transportation is complicated by crystallization dynamics. In this work, a helium ion microscope (HIM) with a Protochips liquid cell was utilized to crystallize and image thiamethoxam in situ. The results of the study illustrate the growth and morphology of the thiamethoxam crystals at different He+ exposure doses, which is markedly different from what has been typically observed. Energy-dispersive x-ray spectroscopy results confirm the presence of the thiamethoxam on the liquid cell membrane. This imaging study illustrates the HIM ability to image and induce the crystallization in soft materials in liquid environments, and attempts to shed light onto the key processes involved in liquid imaging.

Pest insect control, an essential component of crop protection and public health, has evolved over a recorded history of three millennia.1,2 Starting with simple elements like sulfur, with a first recorded use in 1000 BC, the practice continues to evolve with more complex synthetically designed insecticides. However, with each new chemical class, insects develop resistance, thus limiting pesticide effectiveness. As the pesticides’ formulations become more intricate, additional unforeseen problems arise in delivery and distribution of the active component.

Neonicotinoids are a major new set of insecticide compounds developed in the last four decades.3 Thiamethoxam (TMX), a neonicotinoid, has outstanding potency and systemic action for crop protection against piercing-sucking pests, and are highly effective for flea control on cats and dogs. These compounds have generally low toxicity to mammals (acute and chronic), birds, and fish. TMX is an organic solid, with relatively high water solubility, 4 g/l at 25 °C, but is prone to problems with crystal growth and colloidal stability.4 Common industrial uses of TMX are foliar sprays of dilute aqueous solutions and seed coatings, along with binders, fillers, pigments, and other active ingredients. Among the practical challenges for TMX delivery are physical stability of the final concentrated products. These are typically crystal suspensions and are optimized to allow efficient seed application. However, achieving acceptable handling properties of the coated seeds during industrial-volume packing and transportation through elevators, conveyers, and planters remains a challenge.5,6

Maintaining the colloidal stability of TMX is essential for prolonged shelf life and effective performance. Typically, TMX is dispersed using a combination of conventional surfactants with molecular weights of up to around 1 kDa and polymeric dispersants with molecular weights of several tens of kilodaltons. Environmental considerations and the difficulty in optimizing these formulations motivate alternative strategies for controlling the interfacial properties of these organic solids. A key issue, slowing the optimization of these products, is limited knowledge of the crystallization dynamics of neonicotinoids from solution and how these processes can be altered by additives and agents.

Recent work demonstrated the control of supersaturation for the growth of TMX with high morphological uniformity through filamentous M13 bacteriophage with a high binding affinity to TMX crystals. This study acted as a “proof of concept” investigation intended to establish the feasibility of using a combinatorial phage display library7 for the preparation of organic crystals with morphological uniformity. However, the underlying mechanisms of TMX crystal formation remain unclear.

In this work, we utilize a helium ion microscope (HIM),8,9 in conjunction with a Protochips liquid cell,9 to crystallize and image TMX in situ. Due to its charge compensation capabilities, the imaging of insulating and biological samples is a natural application for the HIM.10–12 Furthermore, the HIM can provide sharp, well resolved images from electrically insulating biological samples without a conductive coating.13,14

Our HIM, and scanning electron microscopy (SEM) results, illustrate the growth and morphology of the TMX crystals at different He+ doses, which is markedly different from what has been typically observed (Fig. 1). Energy-dispersive x-ray spectroscopy (EDS) results confirm the presence of the elemental components of TMX on the liquid cell membrane. While the precise details of radiolysis behind the formation of TMX and its nonstandard morphology remain uncertain, this study illustrates the HIM’s ability to image and induce the crystallization process of these soft materials in liquid environments, and sheds light onto the processes involved.

Fig. 1

. HIM, with the use of e flood gun, displaying TMX crystals on a glass surface. (a) HIM image of many large crystallites that form upon drop casting an aqueous supersaturated solution of TMX and drying. (b) HIM image of two particular TMX crystals. (c) HIM image with a smaller field of view showing detail on the surface of TMX crystallites.

Fig. 1

. HIM, with the use of e flood gun, displaying TMX crystals on a glass surface. (a) HIM image of many large crystallites that form upon drop casting an aqueous supersaturated solution of TMX and drying. (b) HIM image of two particular TMX crystals. (c) HIM image with a smaller field of view showing detail on the surface of TMX crystallites.

Close modal

3-(2-chloro-1,3-thiazol-5ylmethyl)-5-methyl-1,3,5-oxadiazinan-4-ylidene(nitro)amine, commonly called TMX, was provided by Syngenta, Greensboro, NC. A 54 mM aqueous supersaturated solution of TMX was prepared by the following. TMX was added to UV-treated, millipore water and stirred for 10 min. The pH of the mixed solution was adjusted to 5.5 by adding sulfuric acid as needed and subsequently heated at 50 °C for 12 h. The solution was equilibrated back to room temperature for an additional 12 h and then filtered twice under vacuum using a Whatman #5 filter (2.5 μm cutoff) and a Buchner funnel.

In situ HIM imaging of the supersaturated TMX solution was performed in a static liquid cell (Protochips, Morrisville, NC). The liquid cell consists of two Si chips with ion-translucent Si3N4 membranes that are 50 nm in thickness. The details of the liquid cell are shown in Fig. S1 in the supplementary material.21 To clean the liquid cell, each chip was soaked in acetone for 1 min, followed by soaking in methanol for 1 min, and dried under streaming clean dry nitrogen. Afterwards, the Si chips were plasma cleaned in vacuum for 30 s. Immediately, 0.5 ml of the 54 mM aqueous supersaturated TMX solution was deposited onto one of the Si3N4 membranes, and the liquid cell was assembled. The liquid cell contained a ∼50 nm thick layer of the TMX solution in atmospheric conditions.15 

The assembled liquid cell was loaded into the vacuum chamber (2.5 × 107 Torr) of the HIM and imaged with a 10 μm aperture, 25 keV accelerator, and 1 pA beam current. The formation of bubbles and TMX growth were induced with the He+ beam and observed after an estimated dose of 6.25 × 1014 ions/cm2.

Figure 2 illustrates HIM images (top row) taken from the top of the Si3N4 liquid cell membrane during the TMX crystal formation phase. The dark dot in the center of Fig. 2(a) is the HIM scanned site where an estimated 6.25 × 1014 ions/cm2 have been delivered; which induces bubble formation in the liquid cell and catalyzes the crystallization process.16–18 Figures 2(b)–2(d) are additional areas where TMX growth has been induced at ion doses of 2.62 × 1015, 5.25 × 1015, 6.56 × 1015 ions/cm2, respectively. Figures 2(e)–2(h) are SEM images of the underside Si3N4 surface (opposite from where the He+ beam radiation occurred) in the disassembled liquid cell membrane, illustrating the growth features from an initial state [Fig. 2(e)], intermediate state [Figs. 2(f) and 2(g)], to the advanced state [Fig. 2(h)]. Although these are not the same features as in Figs. 2(a)–2(d), Figs. 2(e)–2(h) show the state of growth forming in Figs. 2(a)–2(d) at similar doses. The cell was disassembled and dried prior to collecting SEM data (Fig. S2 in the supplementary material21). Note the difference in the relative size of the structures. These have been formed at doses from ∼1 × 1014 ions/cm2 for the initial growth state to ∼1 × 1016 ions/cm2 for the advanced growth states. From smallest to largest crystallites, each central seed is surrounded by concentric circular arrays of smaller droplets. The authors believe that this is due to the bubble front motion as the He+ beam drives the hydrolytic process, discussed further below.

Fig. 2.

Microscopy images displaying initial to advanced growth of TMX on the Si3N4 membrane that was initiated by the He+ beam. (a) HIM image illustrating initial growth in situ with an estimated 6.25 × 1014 ions/cm2. (b) HIM image illustrating intermediate growth in situ with an estimated 2.62 × 1015 ions/cm2. (c) HIM image illustrating more developed intermediate growth in situ with an estimated 5.25 × 1015 ions/cm2. (d) HIM image illustrating advanced growth in situ with an estimated 6.56 × 1015 ions/cm2. (e) SEM image of the underside of the Si3N4 membrane illustrating TMX in an initial growth state formed from ∼1 × 1014 ions/cm2. (f) SEM image of the underside of the Si3N4 membrane showing TMX in an intermediate growth state formed from ∼5 × 1014 ions/cm2. (g) SEM image of the underside of the Si3N4 membrane showing TMX in a more developed intermediate growth state formed from ∼1 × 1015 ions/cm2. (h) SEM image of the underside of the Si3N4 membrane showing TMX in an advanced growth state formed from ∼1 × 1016 ions/cm2.

Fig. 2.

Microscopy images displaying initial to advanced growth of TMX on the Si3N4 membrane that was initiated by the He+ beam. (a) HIM image illustrating initial growth in situ with an estimated 6.25 × 1014 ions/cm2. (b) HIM image illustrating intermediate growth in situ with an estimated 2.62 × 1015 ions/cm2. (c) HIM image illustrating more developed intermediate growth in situ with an estimated 5.25 × 1015 ions/cm2. (d) HIM image illustrating advanced growth in situ with an estimated 6.56 × 1015 ions/cm2. (e) SEM image of the underside of the Si3N4 membrane illustrating TMX in an initial growth state formed from ∼1 × 1014 ions/cm2. (f) SEM image of the underside of the Si3N4 membrane showing TMX in an intermediate growth state formed from ∼5 × 1014 ions/cm2. (g) SEM image of the underside of the Si3N4 membrane showing TMX in a more developed intermediate growth state formed from ∼1 × 1015 ions/cm2. (h) SEM image of the underside of the Si3N4 membrane showing TMX in an advanced growth state formed from ∼1 × 1016 ions/cm2.

Close modal

Similar behavior has been recently revealed in electron and ion beam induced Pt nanoparticles.19,20 First principles density functional tight-binding molecular dynamics simulations for particle growth and interaction of He+ ions with water show that for the He+ in the 1–30 kV beam energy, the energy is primarily transferred to the nuclear motion of water, which results in vibrational excitation.20 Furthermore, these simulations show that increasing the He+ beam energy enhances the level of electronic excitation, which increases the rate of ionization and generates hydrated electrons, while simultaneously decreasing the amount of energy transferred to vibrational degrees of freedom. This effect acts as a local heating, and we propose that this drives a chemical activation step, where an intermediate species is formed that subsequently drives the crystallization.

Figure 3 is a more detailed image of the bubble as imaged from the top of the Protochips cell in a HIM, the formed TMX crystal, as well as its morphology illustrated by atomic force microscopy (AFM). An additional detailed HIM image of the bubble and TMX crystal in an advanced stage of growth is shown in Fig. S4 in the supplementary material.21 Figure 3(a) is a HIM image at 6.56 × 1015 ions/cm2, 10 μm aperture, 25 keV accelerator, and 1 pA beam current through the liquid cell Si3N4 membrane. A series of images showing growth of this feature is shown in Fig. S3 in the supplementary material.21 The dark region is the bubble forming on the other side of the membrane under the beam, the lighter contrast inside the dark region, highlighted by a green square, is the TMX crystal growing inside the bubble. Figure 3(b) is a contact mode AFM image of the same crystallite after the liquid cell has been disassembled. Note the characteristic smaller crystals decorating the large particle, as seen in Fig. 2 SEM images. Figure 3(c) is an AFM cross section taken from the blue line in Fig. 3(b). The formed structures have large aspect ratios: the width is ∼3.5 μm while the height is an order of magnitude lower that ∼300 nm. The formed TMX crystals grow in width and height from an initial to advanced state. This is evidenced by series of AFM images and corresponding cross sections of TMX in different states of growth shown in Fig. S5 in the supplementary material.21 TMX crystals can grow larger enough with the He+ beam to be visible with optical microscopy (Fig. S6 in the supplementary material21).

Fig. 3.

Additional details of the grown structures. (a) HIM image, through the Si3N4 membrane, displaying He+ beam induced growth from a supersaturated solution of TMX surrounded by a gas bubble. (b) The underside of the Si3N4 membrane was investigated with AFM and shows a growth feature of similar size as in (a). (c) The cross section indicates that these features grow in three dimensions.

Fig. 3.

Additional details of the grown structures. (a) HIM image, through the Si3N4 membrane, displaying He+ beam induced growth from a supersaturated solution of TMX surrounded by a gas bubble. (b) The underside of the Si3N4 membrane was investigated with AFM and shows a growth feature of similar size as in (a). (c) The cross section indicates that these features grow in three dimensions.

Close modal

To verify whether the formed structures were indeed TMX, we conducted EDS measurements to check the presence of the expected elemental components, as shown in Fig. 4. Figure 4(a) is an SEM image of the Si3N4 membrane after the cell has been disassembled. Figures 4(b)–4(d) are the nitrogen, chlorine, and the sulfur channels, respectively. Presence of these key elements in the image is a good indicator that the observed structures are TMX rich.

Fig. 4.

Energy-dispersive x-ray spectroscopy results for the grown deposits. (a) SEM image of the underside of the Si3N4 membrane, tilted by 30°, of a particular He+ induced growth feature. EDS confirms the chemical identity of these growths as TMX by mapping the main chemical and (b) displays an EDS chemical image of N in the grown deposits, (c) displays an EDS chemical image of Cl in the grown deposits, and (d) displays an EDS chemical image of S in the grown deposits.

Fig. 4.

Energy-dispersive x-ray spectroscopy results for the grown deposits. (a) SEM image of the underside of the Si3N4 membrane, tilted by 30°, of a particular He+ induced growth feature. EDS confirms the chemical identity of these growths as TMX by mapping the main chemical and (b) displays an EDS chemical image of N in the grown deposits, (c) displays an EDS chemical image of Cl in the grown deposits, and (d) displays an EDS chemical image of S in the grown deposits.

Close modal

We postulate that the mechanism of in situ TMX growth, initiated with the He+ beam, is driven by radiolysis (Fig. 5). We have observed TMX forming inside of, and concurrent with, the growth of bubbles, a by-product of radiolysis [Figs. 2(a)–2(d) and 3(a); Figs. S3 and S4 in the supplementary material21]. We suspect that an initial gas bubble forms inside the liquid cell as the incident He+ beam interacts with Si3N4 membrane creating secondary electrons, and also decomposes the solvent within the liquid cell creating a plethora of solvated by-products as well as a local TMX concentration gradient. This seeds and stabilizes TMX seed deposits on the face of the liquid cell membrane. With continued He+ beam exposure, the bubble grows and TMX precipitates at the gas-liquid interface of the bubble (the location of highest TMX concentration), while also depositing on the membrane. As the bubble front moves outward, additional TMX precipitation forms at the front to form concentric circles around the central TMX seed. A more detailed schematic of this postulated mechanism is presented in Fig. S7 in the supplementary material.21 

Fig. 5.

Postulated model to describe the He+ beam-initiated growth of TMX. (a) Scheme illustrating a radiolysis driven mechanism that is likely to be behind the formation of TMX. (b) AFM image of the underside of the Si3N4 membrane showing two grown TMX crystals and (c) corresponding cross section that lends support for the postulated model.

Fig. 5.

Postulated model to describe the He+ beam-initiated growth of TMX. (a) Scheme illustrating a radiolysis driven mechanism that is likely to be behind the formation of TMX. (b) AFM image of the underside of the Si3N4 membrane showing two grown TMX crystals and (c) corresponding cross section that lends support for the postulated model.

Close modal

In conclusion, we utilize a HIM in conjunction with a Protochips liquid cell to crystallize and image TMX in situ. Our HIM and SEM results illustrate growth and morphology of the TMX crystals at different He+ exposure doses, which are markedly different from what has been previously observed. EDS results confirm the presence of the key TMX elemental components on the liquid cell membrane. We postulate that the radiolysis driven mechanism behind the formation of TMX involves bubble formation inside the liquid cell, where the gas-liquid interface of the bubble aids in the crystallization process. This imaging study illustrates the HIM’s ability to image and induce the crystallization phenomena of soft materials in liquid environments, and sheds light onto the underlying processes involved.

This paper has been authored by UT-Battelle, LLC, under Contract No. DE-AC0500OR22725 with the U.S. Department of Energy. The United States Government retains, and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for the United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). The HIM imaging, image analytics, and simulations portion of this research were conducted at the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility. A.P. and J.F. were supported by Syngenta Crop Protection.

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See supplementary material at https://doi.org/10.1116/1.5040849 for additional HIM, SEM, and AFM data.

Supplementary Material