Development of SEM method for analysis of organ-containing objects using inverse opals

The purpose of this study is to test the possibility of using inorganic macroporous structures of inverse opal in sample preparation for scanning electron microscopy of biological objects. As an absorbent substrate we used silica inverse opals prepared by a sol-gel method to study the biological objects. The process of manufacturing the inverse opal involves a complex multi-stage technological process. First, we synthesized submicron spherical particles from polymethylmethacrylate by the method of emulsifier-free emulsion polymerization of methylmethacrylate in an aqueous medium in the presence of a diazoinitiator. This method can be used to obtain an ensemble of particles with high monodispersity, the average size of which can vary in the range from 100 to 500 nm. Then, by self-assembly technique, we deposited the beads of polymethylmethacrylate into ordered matrices (templates), mainly with a face-centered cubic lattice. The resulting mesoporous structures, called artificial opals or colloidal crystals, had lateral dimensions of about 10 × 10 × 2 mm. Then we heat-treated the opals to 120 °C to harden the template before being impregnated with the precursor. Further, we impregnated the opals with silica sol with a particle size distribution from 1 to 5 nm, obtained by hydrolysis of tetraethoxysilane in the presence of hydrochloric acid, and then, after curing and drying the impregnating composition in air at room temperature, we multi-stage fired them up to 550 °C at normal pressure in the air atmosphere to remove all organic components. As a result, the macroporous metamaterial (the so-called inverse opals) with an open system of pores up to 400 nm in size, occupying about 80 % of the volume, were obtained. We studied lactic acid bacteria of cucumber brine and human red blood cells with TM4000 Plus, SU3500 and S-5500 scanning electron microscopes. Auxiliary substance for the sample preparation was ionic liquid VetexQ EM (Interlab LLC). We showed that it is possible to use the inverse opal as an absorbent substrate for sample preparation and rapid analysis in scanning electron microscopy without pre-drying, chemical treatment, or temperature exposure. To improve imaging in the electron microscope, we used sputter coater to cover the inverse opal surface with a thin film of platinum. The use of ionic liquid in combination with the absorbent porous medium allows preserving an original shape of the biological structures. Using the human red blood cells and lactic acid bacteria, we showed that it is possible to carry out of the morphological analysis of the cells using various scanning electron microscopes. We found that on the basis of the inverse opal, there is a fundamental possibility of creating the absorbent substrate suitable for repeated use in the study of the biological objects. At the same time, trace remnants of previous samples remaining after annealing the plate do not introduce significant distortions when conducting new series of observations. In this study, we obtained high-quality electronic micrographs of the biological objects with high resolution and contrast. At the same time, due to the use of the inverse opals as the absorbent substrate, time and financial costs for research are reduced.