How best to measure tear film formation: an opinion

Healthy eyes are covered by a tear film that is stable and renewed with each blink. A funcAonal tear film has a surface lipid layer blankeAng a complex aqueous layer that contains a cocktail of proteins and mucins. About 5 -50% of people suffer from a condiAon known as dry eye where the tear film is faulty (Stapleton et al., 2017). Clinically, the gold standard diagnosAc test is the tear film break-up Ame which determines how quickly the tear film breaks down aPer a blink. There have been vast amounts of research into how and why the tear film is unstable in paAents with dry eye disease (King-Smith et al., 2018). Some invesAgators, on the other hand, have examined how quickly the tear film forms upon blinking, typically referred to as tear film build-up Ame (Nemeth et al., 2002). Such studies aim to provide insight into the physiology of the tear film and why healthy tear films are stable and resilient through different environmental condiAons. Knowing when the tear film has fully formed is clinically relevant for biometry and reAnal imaging because this is when the opAcal quality of the eye is opAmal (Montés-Micó et al., 2010).Following a blink, the formaAon of the tear film occurs in two steps (Holly, 1973). First with raising of the upper lid, the cornea is covered iniAally by aqueous and mucin and then there is a slower upward moAon of the lipid layer which thickens the tear film, redistribuAng the mucoaqueous layer to the upper part of the ocular surface (Brown and Dervichian, 1969). This final step of tear film formaAon can be imaged.Measuring tear film formaAon in vivo has been inconsistent (Table ) but this is expected because of the many consideraAons which influence experimental design.Views differ about when a measurement should start and finish, and what indicates that the tear film has fully formed. Experiments are also constrained by what can be measured.Invisibility and thinness of the tear film have meant that surrogate parameters have been used and not the tear film itself.The most used surrogate methods for measuring tear film formaAon have been tracking lipid layer spread and movement of reflecAve parAcles embedded in the lipid layer using interferometry or slit lamp biomicroscopy. This has allowed determinaAon of when tear film spread is complete because the colourful fringes or parAcles stop moving (lipid layer stabilisaAon). Despite differences in experimental details, it takes about 1 s for the lipid layer/tear film to stabilise following a blink. An outlier study found a lipid spread Ame of only 0.36 ± 0.22 seconds (Goto and Tseng, 2003). Few have examined aqueous layer dynamics (Benedeco et al., 1984;Zhu et al., 2006). Other studies measured ocular surface parameters such as Surface Regularity Index, Surface Asymmetry Index, or Tear Film Surface Quality to evaluate tear film formaAon. They determined when these parameters reached their lowest value, i.e. the Ame when the tear film was opAmal. These studies consistently report longer Ames: 2.5 -7 s. A Ame of 7 s to reach an opAmal tear film is unexpected since the average interblink interval is around 6 s in duraAon (Johnston et al., 2013). One publicaAon (King-Smith et al., 2009) reports that the tear film never stabilized which might be a result of subtle eye and head movements (Szczesna-Iskander and Robert Iskander, 2025). Overall, techniques have included tear film fluorophotometry, slit-lamp biomicroscopy, high speed videokeratoscopy, interferometry, and dynamic wavefront sensing (Table ).Velocity studies of tear film formaAon commonly use either lipid layer features or parAcles. King-Smith et al. (King-Smith et al., 2009) idenAfied an iniAal fast and then a slow phase of tear film build-up, whereas Szczesna-Iskander (Szczesna-Iskander, 2018) introduced a mulA-phase model of tear film surface kineAcs with an iniAal fast and then a slow tear film buildup followed by a stable phase. Importantly, because lipid layer spread slows rapidly aPer a blink, to compare so-called "iniAal velociAes" between studies (as recommended by (Owens and Phillips, 2002)), a common Ame origin (t0) is required, i.e., the starAng point of a measurement. This is not possible when "aPer a blink" is a frequent descriptor of t0 (Table ) as the exact end point of a blink is difficult to define because of frequent low amplitude twitching of the upper eyelid once it reaches the almost fully open posiAon (Kwon et al., 2013).The posiAon of the parAcle being measured also requires consideraAon because more superior parAcles (more distant from the lower lid) tend to have higher velociAes than more inferior parAcles during and aPer the upstroke of a blink (Berger and Corrsin, 1974;King-Smith et al., 2009). This means that any instantaneous velocity measurement will depend on the Ame aPer a blink when they are recorded and the posiAon on the ocular surface.Moreover, calculaAng an average velocity using the starAng and end point of a parAcle would be invalid because it does not reflect the substanAal changes in velocity over Ame. It appears that most publicaAons have chosen to track parAcles near the center of the cornea presumably because they are more visible and in focus (Owens and Phillips, 2002;Varikooty et al., 2012).Blinking condiAons are another important variable. Normally, there is no awareness of natural blinking. However, once being made aware of blinking or instructed to blink, blinking becomes varied in terms of frequency and force (Palakuru et al., 2007;McMonnies, 2020;Szczesna-Iskander and Llorens Quintana, 2020). Natural blinking condiAons (complete, natural and spontaneous) have been used in more recent studies, but in many studies, blinking has been under instrucAon or acAvely suppressed (Table ). It is surprising that in some publicaAons, blinking condiAons have not been described. This suggests that this was not considered as a variable.There is no gold standard when it comes to the study tear film formation. Each publication exhibits unique experimental design not allowing direct comparisons. Therefore, common baseline factors are needed to enable comparison between studies. In our view, natural blinking is preferred and if instructed blinking or suppressed blinking conditions are to be used, then it needs to be explicitly stated. We also recommend the turning point of a blink, i.e., the first frame of the beginning of eye opening, to represent t0. In terms of measuring velocity, instantaneous velocities relative to t0 should be recorded. In this regard, having a higher framerate makes identification of the turning point more precise (Navascues-Cornago et al., 2026). When particles are studied, it appears that most publications have chosen to track particles near the center of the cornea (more visible and in focus). This means there has likely been a bias towards higher velocities. We recommend using particles that are convenient to measure and consistently visible. However, in all cases, the particle position on the eye should be noted relative to central cornea.Progress towards understanding the physiology of tear film formation is only possible with a common research foundation, which in turn can guide future studies in developing standardized and meaningful guidelines for tear film measurements.Author Timon Ax has received travel funding from Beyond 700 Pty Ltd. Author Thomas Millar is employed by Beyond 700 Pty Ltd. The other authors report no conflicts of interest.This table only refers to in vivo studies of tear film formaAon in humans. It reports on a selecAon of studies which were deemed most relevant and included, with few excepAons, normal subjects. Data from dry eye paAents or otherwise non-normal condiAons (like contact lens wear) have been highlighted in gray shading. The table does not cover mathemaAcal models, animal experiments or in vitro experiments.