Figure 1.  Schematic illustration of time-dependent evolution of concentrations of precursor reactant (red), nanoparticle precursor (green), and size of nanoparticles (blue) involved in a typical synthesis of collagen nanoparticles. The major steps are (I) nucleation, (II) early growth, and (III) late growth stages. The background sketches the evolution of NPs from reactive. The lower part highlights the appropriate in situ X-ray techniques for different evolution periods ( S. Wu et al., 2019 ) .

When a synthesis reaction begins, the process of converting the reactant(s) into the NP precursor, which is required for the generation of solid nuclei, will occur. As the process proceeds, more NP precursor is built up until it reaches a supersaturation point , at which case it spontaneously condenses to form solid nuclei in the liquid solution (ie, nucleation stage I in Figure 1). In a liquid solution of supersaturated NP precursor, a solid nucleus is regarded as the smallest stable solid particle local. To ascertain chemical environment of target elements, in situ X-ray absorption fine structure (XAFS) can be used to examine the transformation of reactant into NP precursor (pre-nucleation). The emergence of solid nuclei is better characterized by the size-sensitive in situ SAXS (ie, condensation of NP precursor, or nucleation). The additional reaction promotes NP growth, or the enlargement of nuclei to NPs. Due to the high surface energy of microscopic NPs, Ostwald ripening and NP coalescence may take place at the early development stage II (Figure 1) together with growth. In order to follow the growth kinetics, in situ SAXS is the most suitable method. The development of NPs and intra-particle reconstruction take control at the late development stage III (Figure 1) of the process. The associated growth kinetics and reconstruction evolution can be observed using both wide-angle X-ray scattering (WAXS) and in situ synchrotron SAXS. In order to follow the growth kinetics, in situ SAXS is the most suitable method. The development of NPs and intra-particle reconstruction take control at the late development stage III (Figure 1) of the process.The associated growth kinetics and reconstruction evolution can be observed using both wide-angle X-ray scattering (WAXS) and in situ synchrotron SAXS. In order to follow the growth kinetics, in situ SAXS is the most suitable method. The development of NPs and intra-particle reconstruction take control at the late development stage III (Figure 1) of the process. The associated growth kinetics and reconstruction evolution can The associated growth kinetics and reconstruction evolution can be observed using both wide-angle X-ray scattering (WAXS) and in situ synchrotron SAXS. Growth kinetics and reconstruction evolution can be observed using both wide-angle X-ray scattering (WAXS) and in situ synchrotron SAXS.