Analyzing the Interior of 3D Polymer Nanostructures by SEM Imaging of Ultrathin Sections

State-of-the-art three-dimensional (3D) laser nanoprinting allows the manufacturing of complex 3D polymer architectures that contain 50-100 nm minimum feature sizes (see, e.g., [1] and [2]). The detailed structural characterization of such polymer structures is important for further improving the printing processes, yet it poses a major challenge to any form of nano-characterization, especially when it comes to the structures’ interior.<br/><br/>We have developed ways to accomplish this task even on objects with outer dimensions of only a few tens of micrometers by adapting techniques used for electron-microscopy analysis of cells and tissues. By first “staining” the structures with suitable heavy metals (e.g., Ru, Os, U) to introduce contrast and then embedding them into an epoxide resin, we can produce ultrathin (70-200nm) cross sections which can be imaged in an SEM using secondary or back-scattered electrons. Because the sections are placed on Si wafers and due to the heavy-metal impregnation, the structures become electrically conductive without the need for metal sputter coating.<br/><br/>A broad variety of different 3D structure types can be investigated with the methods developed in our group: Assuming periodicity for the example of woodpile photonic crystals, the 3D structure can be reconstructed from individual sections [1]. For irregular structures, e.g., written with pore-forming photoresists [2], the 3D interior can be obtained by imaging sequential sections of the embedded material, aligning them, and reconstructing the volume in that way. This approach is called array tomography [3]. It has been introduced by neuroscientists to analyze the connectome of the brain. Meanwhile, it is widely used in biology. Depending on the type of photoresist or starting material used for printing, preparation of the fabricated objects has to be adopted. Dry structures produced, e.g., from acrylate-based photoresists may be embedded directly into resin after staining with OsO₄in acetone. Structures printed from a polyacrylamide hydrogel (PAM) need to be dehydrated gently [4] before infiltrating them with epoxide resin. We found that tannic acid is able to bind free water in PAM and pNIPAM hydrogels. It also acts as a mordant, mediating binding of heavy-metal salts in the bulk of the material and thus allowing high contrast imaging [5].<br/><br/>Introduction of heavy metals can also be achieved after sectioning – by so-called post-staining – to, e.g., differentially stain individual components in block-copolymers.<br/>The combination provides with a versatile toolbox to visualize the interior of complex polymer nanostructures at nanometer resolution.<br/><br/>Acknowledgements: The authors thank Vincent Hahn [1], Frederik Mayer [2], and Martin Wegener [1,2] (all KIT, Karlsruhe, Germany) for polymer samples and Christine Arndt [4], Tobias Spratte [5], and Christine Selhuber-Unkel [4,5] (all Heidelberg University, Germany) for hydrogel samples. Research funded by DFG (German Research Foundation) via the Excellence Cluster “3D Matter Made to Order” (EXC-2082/1-390761711) and by the Federal Ministry for Education and Research under Grant No. 13N14476.<br/><br/>[1] Hahn V et al. (2021) Two-step absorption instead of two-photon absorption in 3D nanoprinting. <i>Nature Photonics </i>accepted/in print<br/>[2] Mayer F et al. (2020) 3D Two-Photon Microprinting of Nanoporous Architectures. <i>Advanced Materials</i> 32, 32, 2002044. doi:10.1002/adma.202002044<br/>[3] Wacker I & Schröder RR (2013) Array tomography. <i>J Microscopy</i>252, 93-99.https://doi.org/10.1111/jmi.12087<br/>[4] Arndt C et al. (2021) Microengineered Hollow Graphene Tube Systems Generate Conductive Hydrogels with Extremely Low Filler Concentration.Nano Letters 21, 8, 3690-3697. doi:10.1021/acs.nanolett.0c04375.<br/>[5] Spratte T et al. (2021) Thermoresponsive Hydrogels with Improved Actuation Function by Interconnected Microchannels. <i>Adv. Intell. Syst. </i>in print, DOI: 10.1002/aisy.202100081