# Nanomechanical Characterization of Bacterial Polyhydroxyalkanoates Using Atomic Force Microscopy
## 1 Introduction  
Mechanical characterization of materials is fundamental for gaining information about their microstructure and, hence, their possible applicability. Hardness, elasticity, plasticity, strength, and fracture are commonly measured mechanical characteristics. Well-established techniques such as tensile testing, hardness testing, or dynamic mechanical analysis are employed for this purpose [1]. However, since these measurements require macroscopic samples, they cannot be compatible in cases of poor availability of materials. In addition, they mainly provide macroscopic information about the tested samples, and heterogeneities or features at the nano-scale cannot be detected. A solution to these problems is represented by the use of atomic force microscopy (AFM). The AFM, introduced in 1986 and belonging to the scanning probe microscope (SPM) family [2], consists of a micro-machined cantilever probe, a sharp tip mounted to a piezoelectric actuator and a position-sensitive photodiode detector for receiving the reflected laser beam to provide cantilever deflection feedback [3]. AFM spreading over the years is caused by the possibility to analyze the sample, not only obtaining high-resolution topographic images, but also performing a characterization of the nanomechanical properties [4]. In particular, it can provide valuable information on local properties such as elasticity, viscoelasticity, hardness, and adhesion [5, 6, 7, 8, 9, 10]. To obtain such information, the so-called force-distance curve must be first obtained through AFM in quasi-static nanoindentation. Force-distance curves can be then fitted with mathematical methods which contain relevant system properties as parameters [3]. This peculiarity caused a strong diffusion of AFM in biology and biomedicine fields, where the understanding of the behavior of complex biological systems is fundamental. In the last decades, AFM was used to evaluate the local elasticity of small, soft, and inhomogeneous samples, such as bacteria, cells but also bone tissues, and a large variety of biomaterials [11, 12, 13, 14]. In addition, recent works include the properties investigation of many classes of materials related to different fields of application. By way of example, it is possibleto mention geological materials [15], metallic alloys [16], ceramics [17], polymers [18], nanocomposites [19] and functional nanostructures [20]. Since AFM allows to determine both the microstructure and the local mechanical properties of soft matter, it has been revealed as a particularly interesting tool for probing polymers, whose morphology presents relevant features at the nanoscale [21]. As mentioned above, in the last decade, several pioneering studies using AFM nanomechanical mapping on polymers have been published [22]. Among the large variety of studied polymers, it is possible to cite homopolymers such as polyvinyl acetate, polystyrene or polymethylmethacrylate [23; 24], blends like polystyrene/poly(n-butyl methacrylate) or polyolefin elastomer/polyamide [25; 26], and even polymer-based composites materials [27; 28]. In particular, the Elastic modulus is the most diffusely measured parameter, having been quantitatively deduced for several polymeric bulk samples and thin films with elastic modulus varying in a wide range, from a few kPa, e.g., hydrated hyaluronic acid hydrogels, to several GPa, e.g., polymethylmethacrylate, polyethylene naphthalene, or oxidized polyimides [29; 30]. It should be mentioned, the use of AFM for the microstructure mechanical investigation of biopolymers. Indeed, the knowledge of the relationships between structure, properties, and performance is essential for prospective safe applications of biodegradable and/or bio-based polymers [31]. Both natural and synthetic biopolymers have been reported in more recent years. Cellulose fibers, polycaprolactone, polyvinyl acetate, polylactic acid (PLA) and cellulose-reinforced starch-gelatin polymer composite are a few of the investigated materials [32; 33; 34; 35].