## 3 Results and Discussion  
### SERS-Active Substrates Surface Analysis  
It is well-known, that one of the key parameters of the silver SERS substrate, which dramatically affects the quality and Raman signal amplification, is the character of thin film surface morphology. That is why in the past, the general idea of SERS substrates engineering was to deposit and investigate the different kinds of disrupted (or island-like) metal films with a various type of rough surface. The nanostructure and morphology of PVD metal thin films could be controlled by the substrate crystalline structure and temperature, deposition rate, material purity, vacuum conditions, etc. Precisely controlling these deposition recipe parameters makes it possible to obtain self-assembled metal thin films with reproducible and controllable sophisticated surface morphology. For example, silver thin films can be deposited with pore-like semi-regular structures, where the \"pore\" sizes and the surface roughness can be adjusted in a controlled manner. For substrate temperature in the range of 550-650 K, and deposition rate of approximately 1.0 nm/s, one can get films with large plateaus and deep holes. Smaller substrate temperature in the range of 450-550 K and higher deposition rates lead to smaller plateaus and shallow grooves. Each type of structures like this has its own surface morphology, which is characterized by a number of semi-regular structures, characteristic size, depth and shape. By optimizing the combination of these surface morphology singularities, we determine the best \"pattern\" providing the highest Raman signal amplification. To provide this analysis we first performed the high-resolution AFM surface scans of all the seven types of SERS-active substrates (see Materials and Methods).  
Samples of type I (Figure 1a) and type II (Figure 1b) have smooth surfaces with relief height of about several nanometers. At high resolution it can be seen (Figure 1c), that a smooth sample (type I) has micro-relief on the surface with typical feature size of about 30 nm. There are around 130 pieces in the 1 \(\times\) 1 \(\upmu\)m\({}^{2}\) area (the average quantity). Silver films on glass have more corrugated surface of two different types, depending on the deposition conditions. For example, the structures type III (Figure 1d) are fine-grained films with a characteristic surface grain size of about 50 nm. There are significant defects of 100 \(\div\) 120 nm on the type IV samples (Figure 1e) on the background of a similar fine-grained structure. A typical surface morphology of silver films formed on mica under described evaporation conditions (the samples type V and VI) is shown in Figure 1f,g. Such structures are self-assembled silver film with nanoscale single-crystalline surface features (or pore-like semi-regular nanostructures). The microstructure of these films can be controlled by vacuum conditions, substrate temperature and deposition rate. By optimizing these parameters it is possible to manage the number of pores and their size, or even completely get rid of them.  
The last sample (type VII) consist of 100 nm thick silver film grown on a thick (3000 nm) aluminum film on a glass substrate. The silver film has a uniform nanostructured surface with the structural elements size in the range of 130-180 nm (Figure 1h). In the next step all the samples were experimentally analyzed in term of Raman signal amplification of myoglobin probe deposited on the surface using air-drying method from the low concentration solution (10 \(\upmu\)g/mL).