Figure 5: Real-time imaging of the nucleation and growth of 2D glucose isomerase crystals with molecular resolution.

The mica surface was initially exposed to a high [NaCl] solution (condition of middle panel of Fig. 3) to wet the surface without inducing nucleation. The AFM liquid cell was subsequently flushed with 0.01 mg ml⁻¹, 10 mM Hepes pH 7.0, 10 mM NaCl and 50 mM MgCl₂ to trigger crystal formation (t=0, scan direction for all images upwards). Crystalline clusters rapidly emerge with sizes ranging from 4 to ~40 molecules. Cross-correlating successive images reveal that smaller clusters (III and IV) have a predisposition to shrink or dissolve completely, whereas larger clusters (I and II) tend to amass monomers, notwithstanding temporal fluctuations. This is in accordance with one of the core concepts of classical nucleation, that is, the existence of a critical size that subdivides clusters in groups of either sub- or supercritical. More importantly, it suggests the presence of a local maximum in the cluster size dependence (the relevant order parameter) of the free energy. The presence of such an activation barrier demonstrates that the formation of the 2D crystalline phase occurs through nucleation and not by means of spinodal decomposition. Shortcuts across the nucleation barrier do occur by means of coalescence of independently formed subcritical clusters (black arrows), a pathway outside the scope of CNT.