Kaberova Zhansaya, Karpushkin Evgeny, Nevoralová Martina, Vetrík Miroslav, Šlouf Miroslav, Dušková-Smrčková Miroslava
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského náměstí 2, 162 06 Praha 6, Prague, Czech Republic.
Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia.
Polymers (Basel). 2020 Mar 5;12(3):578. doi: 10.3390/polym12030578.
The exact knowledge of hydrogel microstructure, mainly its pore topology, is a key issue in hydrogel engineering. For visualization of the swollen hydrogels, the cryogenic or high vacuum scanning electron microscopies (cryo-SEM or HVSEM) are frequently used while the possibility of artifact-biased images is frequently underestimated. The major cause of artifacts is the formation of ice crystals upon freezing of the hydrated gel. Some porous hydrogels can be visualized with SEM without the danger of artifacts because the growing crystals are accommodated within already existing primary pores of the gel. In some non-porous hydrogels the secondary pores will also not be formed due to rigid network structure of gels that counteracts the crystal nucleation and growth. We have tested the limits of true reproduction of the hydrogel morphology imposed by the swelling degree and mechanical strength of gels by investigating a series of methacrylate hydrogels made by crosslinking polymerization of glycerol monomethacrylate and 2-hydroxyethyl methacrylate including their interpenetrating networks. The hydrogel morphology was studied using cryo-SEM, HVSEM, environmental scanning electron microscopy (ESEM), laser scanning confocal microscopy (LSCM) and classical wide-field light microscopy (LM). The cryo-SEM and HVSEM yielded artifact-free micrographs for limited range of non-porous hydrogels and for macroporous gels. A true non-porous structure was observed free of artifacts only for hydrogels exhibiting relatively low swelling and high elastic modulus above 0.5 MPa, whereas for highly swollen and/or mechanically weak hydrogels the cryo-SEM/HVSEM experiments resulted in secondary porosity. In this contribution we present several cases of severe artifact formation in PHEMA and PGMA hydrogels during their visualization by cryo-SEM and HVSEM. We also put forward empirical correlation between hydrogel morphological and mechanical parameters and the occurrence and intensity of artifacts.
水凝胶微观结构，主要是其孔隙拓扑结构的确切知识，是水凝胶工程中的关键问题。对于溶胀水凝胶的可视化，低温或高真空扫描电子显微镜（低温扫描电子显微镜或高真空扫描电子显微镜）经常被使用，而伪像偏差图像的可能性常常被低估。伪像的主要原因是水合凝胶冷冻时冰晶的形成。一些多孔水凝胶可以用扫描电子显微镜观察而不存在伪像风险，因为生长的晶体可以容纳在凝胶已有的初级孔隙中。在一些无孔水凝胶中，由于凝胶的刚性网络结构会抵消晶体的成核和生长，也不会形成次级孔隙。我们通过研究一系列由甘油单甲基丙烯酸酯和甲基丙烯酸2-羟乙酯交联聚合制备的甲基丙烯酸酯水凝胶，包括它们的互穿网络，测试了凝胶溶胀度和机械强度对水凝胶形态真实再现的限制。使用低温扫描电子显微镜、高真空扫描电子显微镜、环境扫描电子显微镜、激光扫描共聚焦显微镜和经典宽视野光学显微镜研究了水凝胶的形态。低温扫描电子显微镜和高真空扫描电子显微镜在有限范围的无孔水凝胶和大孔凝胶中产生了无伪像的显微照片。仅对于溶胀相对较低且弹性模量高于0.5 MPa的水凝胶观察到了无伪像的真正无孔结构，而对于高度溶胀和/或机械性能较弱的水凝胶，低温扫描电子显微镜/高真空扫描电子显微镜实验导致了次级孔隙的形成。在本论文中，我们展示了聚甲基丙烯酸羟乙酯和聚甲基丙烯酸缩水甘油酯水凝胶在低温扫描电子显微镜和高真空扫描电子显微镜观察过程中严重伪像形成的几个案例。我们还提出了水凝胶形态和力学参数与伪像的出现及强度之间的经验相关性。
