## 4 Results  
Figure 1 shows the difference between the individual silver layer thicknesses. The layer thicknesses were carefully selected to form four different types of AgNPs. As mentioned above, the difference between the average diameters of the nanoparticles created during heat treatment is to be clearly defined as a function of the layer thickness.  
The deposition of silver layers was followed by several analyzing steps of the sample surface to control the size of the growing silver particles. After short, one minute heat treatment, the silver-deposited titanium samples were investigated by SEM to obtain a visual image of the beaded surface. Surface morphology was analyzed by applying National Instruments Visual Assistance image analysis program. We could obtain the number, the size (diameter), and even the distribution function of the silver nanoparticles changes in an area of about 9 \(\upmu\)m\({}^{2}\).  
The parameters of AgNPs preparation were varied by: (i) changing the thickness of the silver layer during the sputtering process (see Figure 1); (ii) changing the temperature during the heat treatment.  
In the case of fully beaded surfaces, there is a monotonic relationship between the diameter of the silver nanoparticles and the temperature of the heat treatment. The larger the amount of material deposited on the surface is, the larger were the nanoparticles that were obtained. It is also clear that, at thinner film thicknesses, the diameter of the nanoparticles is independent of the heat treatment temperature. By this method, silver nanoparticles of any average diameter can be produced between 40 and 400 nm (Figure 2). The cell growth investigations were performed at 600 \({}^{\circ}\)C heat-treated samples because, in this case, the nanoparticles were well separated, even after 1 min of heat treatment. The surface modification (Stages I, II, and III) of the samples was repeated on 24 samples. As a result, the difference in diameter between the AgNPs was visible depending on the thickness of the layers.  
The SEM image in Figure 3 shows the surface of a sample having an initial layer thickness of 15 nm before 600 \({}^{\circ}\)C 1 min heat treatment. The red spots in Figure 3B correspond to the Ag-L peak  
Figure 1: Depth profile of the four samples with different silver thicknesses measured by secondary neutral mass spectrometry (SNMS) method. The thickness of the layers was determined by the position of the half interface width obtained from the position of the distances between 90 to 10% decay of measured intensity.  
Figure 2: Scanning electron microscopy images taken of silver nanoparticles as a function of temperature and thickness of the deposited layer (see the details in the text).  
and the green to the Ti-K peak, which are superimposed on the SEM image. The EDS mapping image (Figure 3B) is illustrated and colored into Figure 3C according to the peaks defined by EDS.  
Figure 4 shows how the size distribution of silver nanoparticles on the TiO surface changed as a function of the thickness of the silver layer previously sputtered on the surface. There is a linear relationship between the average diameter of the AgNPs and the thickness of the sputtered silver layer (see Figure 4B).  
Figure 4: (**A**) Gaussian distribution of the average diameter of AgNPs; (**B**) linear relationship between average nanoparticle diameter and silver layer at T = 600 \({}^{\circ}\)C.  
Figure 3: Scanning electron microscopy image (**A**) with the corresponding composition mapping image (**B**) and spectra (**C**). The typical characteristic lines of the elements are marked in the spectra.