## 4 Discussion  
As demonstrated via Brewster Angle Microscopy here and in other studies, because of the extreme hydrophobicity of its lipids, MGS does not exist as a monomolecular film at the air-water interface, even at surface pressures as low as 0.5-10 mN/m. This is also reaffirmed by the inspection of the apparent area per molecule at which the MGS surface pressure/area isotherms take place, i.e., from a lift-off area of \(\sim\)40-50 A\({}^{2}\)/molecule to up to 10 A\({}^{2}\)/molecule, which also clearly proves the multilayer structure of the Meibomian films [5; 6]. Instead, \"healthy\" MGS forms rough heterogeneous layers that become more homogeneous with increases in \(\pi\) and maintain a liquid extended-like reciprocal compressibility modulus [5; 6; 24]. The behavior at the air-water interface of mixtures of triglycerides and cholesterol esters which compositionally resemble meibum is similar, as they also form films of multilayer thickness that display non-collapsibility and reversibility [24; 25]. An inherent characteristic of such thick multilayers enriched with NPL is that the PL molecules (situated at the oil/aqueous interface) evade the formation of tightly packed and collapsed domains upon area contraction and instead PL relocate to the upper NPL stratum of the films. During the subsequent area expansion (i.e., a stage similar to the impact of eye opening at blink), this upper stratum of the layer acts as a reservoir, allowing polar lipids to quickly reappear at the interface, thus maintaining the stability of the films upon continuous blink-like area changes. The so-called duplex film structure, which consists of a continuous liquid-like oily suspension placed on top of a polar lipid monolayer [5; 6], renders the MGS layers' capability to evade collapse and to resist material loss for many consecutive blinks in the course of the day. Due to these distinctive characteristics, it is shown that in vivo at the ocular surface, the TFLL is able to maintain stable composition and structure for hours [26]. This latter effect decreases the need for the Meibomian glands to supply novel lipid material in the TFLL and ensures a TFLL turnover rate of 0.93 \(\pm\) 0.36%/min (i.e., a notably lower rate, compared to 10.3 \(\pm\) 3.7%/min AT turnover). These inherent features of \"healthy\" MGS layers constitute the capacity of the TFLL to sustain its structure between blinks and to contribute to the strength of the tear film. It was recently found that MGS films' elasticity plays important role in ensuring the structural recovery of the TFLL (revealed by image cross-correlation assay of lipid layer dynamics in vivo) in successive blinks, which in turn aligns with the general stability of the TF in healthy and in dry eyes [5; 6; 24; 27; 28]. Indeed, the ability of TFLL in vivo to spread quickly and uniformly while maintaining its texture (i.e., a display of elasticity) upon blinks has been strongly correlated with TF overall stability in numerous clinical studies [2; 27; 28]. In particular, it was found that the lipid layer spread took much longer in MGD patients (3.54 s in MGD vs. 0.36 s in healthy individuals) [22] and the spatial heterogeneity of TFLL thickness distribution was notably higher (lipid map uniformity = 125 nm\({}^{2}\)), compared to healthy eyes (lipid map uniformity = 14 nm\({}^{2}\)) [29]. The clinical studies and in vitro laboratory results that found that MGS and tear specimens from dry-eye patients had diminished surfactant potency and were unable to establish a continuous duplex film in the course of compression, contrary to samples from healthy eyes, also show excellent agreement [2]. Similar trends are also revealed in the current study, where the inability of MGD samples to form continuous layers (as revealed by BAM) is accompanied by inferior performance of MGD samples in stress relaxation transients and in formation of brittle layers that fail to maintain constant surface area at 10 mN/m.