## 3 Results  
Surface Pressure Area/Isocycles of nMGS and dMGS Films and Their Morphology as Visualized by Brewster Angle Microscopy  
It can be seen (Figure 1) that both nMGS and dMGS formed surface films capable of increasing the surface pressure upon area compression. As previously reported, the effect is proportional to the amount of meibum deposited at the air-water interface, both for healthy and for diseased MGS (Figure 1, upper row). Figure 1: Surface pressure/area (solid lines) and surface pressure/reciprocal compressibility (dotted lines) isotherms of nMGS (**a**) and dMGS (**b**) at 14 \(\upmu\)g (blue lines) and 28 \(\upmu\)g meibum (red lines) deposited at the trough surface, as well as (**c**) dependence of the maximum surface pressure on the total amount of meibum and the health status of MGS samples. The data of panel (**c**) represent mean \(\pm\) standard deviation of samples from five healthy individuals and four MGD patients, respectively. Although there is a tendency observed that at the same amount of deposited meibum, healthy samples can reach higher maximum \(\pi\) upon completion of film contraction (Figure 1, bottom row), there is great variability between the specimens and due to the small sample size no statistically significant conclusion can be reached. The films' in-plane reciprocal compressibility (or rigidity) modulus, or \(\mathrm{C_{s}}^{-1}\) (Figure 1, dotted lines) was calculated as follows (Equation (1)):  
\[\mathrm{C_{s}}^{-1}=\mathrm{A_{\pi}}\;(-\mathrm{d}\pi/\mathrm{d}\mathrm{A}), \tag{1}\]  
where \(\mathrm{A_{\pi}}\) is the area at the indicated \(\pi\). The inflexions in the \(\pi/\mathrm{C_{s}}^{-1}\) curves denote surface pressures at which notable modifications in the layers texture occur at compression. It can be seen that both nMGS and dMGS had \(\mathrm{C_{s}}^{-1}<50\;\mathrm{mN/m}\), which corresponds to the liquid expanded state of the film at the meibum/water interface [14]. Figure 2 summarizes the dependences of the transient elasticity modulus on time which were analyzed via the Kohlrausch-Williams-Watts (KWW) equation (Equation (2)):  
\[\Delta\pi=\mathrm{A_{m}}.\;\mathrm{exp}\;(-(\mathrm{t}/\tau)^{\beta})+\Delta \pi_{\mathrm{EQ}}, \tag{2}\]  
Typically the \"stretched\" exponent \(\beta\leq 1\) lowers relative to unity because of augmentation of (i) the film domains interaction and/or (ii) the film heterogeneity (i.e., increased amount of interacting arrays) [15]. Here \(\mathrm{A_{m}}\) represents the relaxation amplitude and \(\Delta\pi_{\mathrm{EQ}}\) is the stationary plateau value. It can be seen (Figure 2) that while \(\beta\) values varied greatly among both \"healthy\" and \"diseased\" Meibomian films, the plateau values of \(\Delta\pi\) achieved at the end of the relaxation transients were \(\geq\)0.8 mN/m for nMGS, while the transients of dMGS layers cannot stabilize to a plateau value and \(\Delta\pi\) increment dropped to zero. The latter feature is known to be an indication of mechanically unstable \"brittle\" layers [16; 17; 18; 19; 20]. This was indeed confirmed when experiments were performed to test the ability of Meibomian layers to sustain their surface area at 10 mN/m surface pressure. This value was chosen as it provides a reasonable estimate for the TFLL film pressure in terms of difference between the surface tension of delipidated (i.e., the aqueous tear surface tension) and intact human tears [21]. It can be seen (Figure 3) that, while nMGS films maintained constant surface pressure and surface area values, dMGS layers were indeed mechanically unstable and brittle and displayed constant contraction of film area, probably due to the redistribution of lipid molecules from the aqueous interface to the nonpolar lipid stratum of the layers [2]. In turn, such behavior is expected to result in facilitated instability of the tear film at interblink periods [2; 22]. Such distinction in the performance of nMGS and  
Figure 2: Stress relaxation transients of nMGS (**a**) and dMGS (**b**) films.