**2. Sample preparation**

Sample preparation art is in fact a simple procedure of critical-path steps, where every single step makes a large difference. The sample preparation in AFM is easier as compared to the other electron microscope techniques [8]. Further, AFM provides advantage of operating in almost any environment conditions, such as aqueous solutions, in air, vacuum, or other gases. Typically, AFM is operated at three different modes namely contact mode, noncontact mode, and tapping mode. Contact is a static mode, and tapping and noncontact are dynamic modes, as the cantilever oscillates in tapping and noncontact modes. This is achieved by adding an extra piezoelectric element that oscillates up and down between 5 and 400 kHz to the cantilever holder. The contact mode is the mode where the tip of the cantilever scans the sample in close contact with the surface. This mode is used usually for surface force measurements. In noncontact mode, the tip flies about 5–15 nm above the sample surface. Whereas in tapping mode the tip of probe touches the sample, and moves completely away from the sample in each oscillation cycle. The tip usually taps the sample during each oscillation in tapping mode, hence it is often the most stable mode used in air. In noncontact mode the cantilever stays close to the sample all the times and possess much smaller oscillation amplitude. The contact mode imaging is heavily influenced by frictional and adhesive forces which may damage samples and distort image data. The non-contact imaging mostly provides low resolution and can get hindered by the contaminant thus producing interfere with oscillation. On the other side the tapping mode imaging overcome the disadvantages of the other two modes. It eradicates the frictional forces by spasmodically contacting the surface and oscillating with appropriate amplitude to avoid the tip from being trapped by adhesive meniscus forces from the contaminant layer.

In general, for particle analysis in AFM the smaller the size of the particles the flatter/smoother the substrate should be that is the size of the particles should be greater than the topographical features of the substrate. Commonly used substrates are glass cover slips, highly ordered pyrolytic graphite, silicon oxide wafers, mica and atomically flat gold. For biological samples like imaging DNA12 and proteins, atomically flat substrates are used while for fine-size features examination like bio-cells, colloids, quantum dots and carbon nanotubes, glass, mica and silicon substrate are used. If a sample comes in the form of a bulk material such as wood or epoxy-resin, metal discs are used as a substrate. The adhesive used in this case is typically carbon tape or thermal wax [3].

In case of biological samples, in order to observe biological structures in their native state, they are supposed to be attached to a smooth solid substrate to resist the lateral forces exerted by the scanning tip, in that reverence, mica, glass and silicon oxide have proved to be excellent substrates for the examination. Muscovite mica, is a non-conducting layered mineral composed of multiple 1 nm thick layers [9]. It can be cleaved simply with the help of adhesive tape to yield clean, atomically flat surfaces which are negatively charged. Mica is most normally used substrate for imaging double-stranded DNA, DNA-protein complexes, protein arrays, densely packed proteins, supported lipid films and animal cells. Also, the mica surface can be modified with silanes which helps in both to promote adsorption or to allow covalent binding of the

biomolecules [10]. Glass represents another suitable substrate for imaging biological samples. For imaging cells and other large structures glass cover slips are flat enough for imaging adsorbed molecules while in some cases, silicon oxide wafer scan also be used instead of glass. Though they are more expensive and difficult to handle, they offer much smoother surface than glass. Hydrophobic substrates, highly orientated pyrolytic graphite, which is atomically flat over large areas [11] are also preferred for biological sample preparation. Hydrophobic surface can be obtained by coating the mica surface with carbon for immobilizing DNA [12, 13].

For imaging document, adhesives and fibers sample under AFM, the frequently used substrate is microscope slide. Usually the samples are cut as per the required area to be imagined under the AFM and then double-sided adhesives are applied to affixed the sample at its fixed position as when the AFM tip is scanning it does not get deviated from the position. For soil sample analysis usually the grains are pressed into pressure-sensitive adhesive putty to provide suitable support during the scanning process thus this allows for retrieval of the grains afterwards or realignment if necessary during analysis [14–16].

The hair samples are priory washed before been examined under microscope using solvent namely sodium dodecyl sulfate solution or doubled distilled water. The substrate like metal discs or glass slide can be used as they are stable and shown eligible drift or creep. Adhesives such as conductive sticky carbon pads or doublesided tape are used to fix the sample at its position. If conductive sticky carbon pads are used as an adhesive then the hair sample are lowered onto the pads and pressed into place using tweezers, so that the fibers did not roll on the pad and hence pick up any contamination from the adhesive [14, 17, 18]. Researchers have also used epoxy as an adhesives at the sample ends to ensure that no interference with the top surface occurs and the adhesion between the hair sample and AFM disc keeps the middle of the sample fixed to the disc during AFM measurements [19].
