Atomic force microscopy (AFM) is a powerful tool for nanoscale surface characterization, and the choice of substrate plays a crucial role in experimental success. Mica (muscovite) is widely used as one of the most preferred AFM substrates, particularly in the study of biomolecules and nanomaterials. Freshly cleaved mica offers several advantages, including atomic smoothness, chemical inertness, and ease of surface preparation. The smooth surface of mica, which requires no complex polishing, provides a perfectly plain base for high-resolution imaging of individual molecules, such as DNA, proteins, and lipid bilayers. This property allows for accurate measurement of the topography and mechanical properties of samples without artifacts introduced by substrate roughness.

Biomacromolecules are typically stored in a salt buffer, which requires the substrate to be rinsed to remove these salts after sample deposition. Therefore, there are several methods avaiable for modifying the mica surface to bind the target molecules. Here, we present three types of surface modifications of freshly cleaved mica for various AFM experiments that we use in our practice.

1) Mica surface modification with divalent metal ions. This modification compensates for the negative surface charge of mica, which is important when studying negatively charged biomolecules like DNA and RNA.

2) Binding of amino groups of APTES molecules to the mica surface. This is a universal method that allows for the active capture of various biomacromolecules from solution onto the surface.

3) Mica surface modification with active antibodies. With the A-protein, antibodies can be oriented with a specific epitope facing outward. This allows for the specific binding of viruses, bacteria, and their fragments.

The work was performed within the framework of the Program for Basic Research in the Russian Federation for a long-term period (2021-2030) (№122030100168-2).