Enhanced electron microscopy imaging for a detailed structural study of alginate hydrogel containing the encapsulated...

Hydrogels are widely applicable in medicine, biotechnology, etc. A specific example is bacterial alginate produced by plant growth-promoting rhizobacterium Azotobacter vinelandii, which encapsulates the cells within its hydrogel network, offering promising applications in agriculture. To better understand the properties and behaviour of hydrogel, it is important to study its architecture. However, due to high-water content and fine structure of hydrogel samples, their preparation for electron microscopy is challenging. In this study, we developed an optimised protocol for preparing complex samples of A. vinelandii cells encapsulated in alginate hydrogel for imaging by low-voltage scanning transmission electron microscopy. Our approach addresses structural instability and artefact formation typically encountered during sample dehydration and staining. We demonstrated that careful timing of CaCl₂ addition, after initial fixation, is essential. Equally important is the careful selection of its concentration to maintain hydrogel integrity while preserving cellular morphology. The inclusion of lead citrate staining step enhanced the contrast within hydrogel matrix, allowing for improved visualisation of fine details. This refined protocol enables high-resolution imaging of alginate-based hydrogels containing embedded cells while preserving the key structural features. It is compatible with various transmission electron microscopy techniques and may be adapted for use with other soft biomaterials.