Volume 6, Issue 6 , Pages 707-713, December 2010
Nanoscale phase dynamics of the normal tear film
Article Outline
Abstract
The tear film is a dynamic multilayered structure. The interactions and the interfacial dynamics between the layers that occur during a blink cycle must be such that they allow for maintenance of a stable tear film. Attempts to understand these dynamics have been limited by the techniques and biomarkers used. Quantum dots (qdots) offer a new potential to monitor the dynamics of the tear film layers in vivo without the drawbacks of previously used methodologies. Indium phosphide–gallium qdots were used to differentially assess the dynamics of the lipid and aqueous layers of the tear film in real time. In the aqueous, qdots dispersed to form a stable local region that was swept away into the upper and lower menisci during a blink. They did not redisperse onto the ocular surface but were progressively removed from the menisci through the puncta. Some of these qdots adhered to the mucin layer on the ocular surface in a meshlike pattern and remained there for five to six blinks before they were removed. The organic qdots dispersed quickly but patchily over the whole outer surface of the tear film. They also strongly marked both eyelid margins and slowly dispersed onto the skin and eyelashes and not through the puncta. Some were trapped in the menisci as blobs that rolled along the meniscus. These data support the view of a distinct three-layered tear film: an inner mucin layer attached to the epithelial cells, a fluid aqueous layer, and an outer viscoelastic lipid layer.
From the Clinical Editor
Indium galium posphide quantum qdots were used to differentially assess the tear film in real time. These data support the view of a distinct three layered tear film: an inner mucin layer attached to the epithelial cells; a fluid aqueous layer; and an outer viscoelastic lipid layer.
Graphical Abstract
The dynamics of the lipid, aqueous and mucin layers of the tear film were observed in real time with fluorescent indium gallium phosphate quantum dots. About 0.5μL of lipophilic and hydrophilic qdots were instilled on the ocular surface and their movements were monitored using a slit lamp biomicroscope.
Key words: Tear dynamics, Quantum dots, Tear interaction, Nano-ophthalmology, Biological fluid, Tear film
The tear fluid represents a typical complex multilayered thin fluid with intrinsic dynamicity.1, 2, 3 It is a 7-μm-thick dynamic three-layered biological fluid on the ocular surface4 (Figure 1) that is made up of mucins attached to the ocular surface, overlaid by proteins, salts, and water comprising over 90% of the tears, and the approximately 100-nm-thick outer lipids. In terms of the dynamicity of the tear fluid, a complexity arises from various interactive interfaces; a liquid-solid interface between the tear film and the epithelial cells of the ocular surface, a liquid-liquid interface between the lipid and aqueous layers, and a gas-liquid interface between the lipids and the air.5 Furthermore, frequent blinking, once every 4–5 seconds, puts the tear film in a constant flux motion and also requires it to repeatedly reorganize itself on the ocular surface after each blink within a few milliseconds to adequately perform its physiological and protective functions.
Figure 1.
The three layers of the tear film. The outermost hydrophobic lipid layer is approximately 100 nm thick; the middle aqueous layer comprises over 90% of the tear film; and the inner hydrophilic mucin layer is embedded at the corneal epithelium.
In complex fluids such as the tear film, interfacial characteristics of fluids are entirely different from their behavior as a pure gas or liquid,6 making it almost impossible to evaluate the phase dynamics in vitro. Taking advantage of the near-perfect optical properties of the tear film and the cornea, noninvasive optical observational techniques such as interferometry7 and optical coherence tomography8 have been tailored to evaluate different aspects of tear dynamics. Interferometry is used to observe the surface spread and, in some instances, thickness of the lipid layer. By its very nature, the information is restricted to the surface of the tear film and does not give any information about the removal of the lipids from the ocular surface. High-resolution optical coherence tomography has also been used to investigate tear dynamics and in particular for monitoring the thickness of the aqueous layer on the ocular surface and at the meniscus between blinks. Although aspects about tear flow have been inferred, there is little information about the spread of the tear film across the ocular surface and interactions between the different layers. Because of these limitations of optical observational techniques, computational modeling has been applied to the tear film9, 10; however, these models are based on theoretical assumptions and are difficult to verify. If the interfacial flow at phase boundaries could be observed directly in real time in vivo, it would enable a greater understanding of the overall tear fluid dynamics and their implications, particularly at lipid-aqueous and aqueous-mucin/epithelium interfaces.
Observations of the dynamics of the layers of the tear film at a molecular level can be made with fluorescent organic dyes and radioactive tracers. The most extensively used biomarker to evaluate the stability of the tear film for dry eye diagnosis is fluorescein,11 but it loses fluorescence rapidly, has a scatter effect making it difficult to assess tear dynamics, and also stains exposed corneal and conjunctival epithelium. Other commonly used organic biomarkers on the ocular surface for diagnostic purposes are rose Bengal and lissamine green, but these stain ocular surface tissues and not the tear film per se.12 In addition, the volume of these organic dyes (2–5 μL) required to observe the tear film is also a concern, in that addition of such relatively high amounts to the nonstimulated tears, which have a total volume of as low as 4 μL, can artificially alter the structure, dynamics, and interfacial interactions of the preexisting layers. In laboratory-based studies, a radioactive tracer, technetium 99, has been used for tracking the drainage of aqueous layer through the lacrimal duct,13 and the lipids on meibomian gland orifices have been observed with Sudan III.14, 15 These markers do not disrupt the tear film layers, but they do not give any information on the dynamics of the layers on the ocular surface and the interlayer interactions.
The relatively recent availability of organic and aqueous fluorescent quantum dots16, 17 (qdots) provides a new possibility to view movement of particles of the tear fluid at a molecular level in real time and evaluate interfacial interactions. Qdots are nanocomposites of a nanocrystal-semiconductor core enclosed within an outer shell that adsorb light strongly in the ultraviolet and deep blue regions and emit at higher wavelengths according to their diameter with up to 95% efficiency for qdots with a heavy-metal core. Moreover, qdots have a fluorescence life approximately 10 times longer than the organic dyes because they do not have a quenching effect and their outer coating can be modified to make them either hydrophilic or lipophilic. Because the qdots can be used in very small quantities to observe the tear layers at a molecular level over a longer period of time, they mitigate all of the drawbacks of existing biomarkers and currently used optical techniques for the assessment of tear film.
In this study we monitored the intrinsic dynamics of the aqueous and lipid layers of the normal tear film with hydrophilic and lipophilic qdots in real time to evaluate the functioning and interactions between the layers. Understanding these fundamentals of tear dynamics is essential for developing treatment modalities for ocular surface pathologies such as dry eye and chronic blepharitis and also for developing effective drug delivery mechanisms to intraocular tissues. The increasing possibilities of tissue-targeted drug delivery for ocular tissues using nanoparticles have also made it essential to understand the dynamics of the tear film at a molecular level.
Methods
This study was approved by the University of Western Sydney and the University of New South Wales human research ethics committees. After obtaining written informed consent, seven subjects with no positive symptoms of dry eye on the McMonnies dry eye questionnaire18 were recruited for the study.
Normality of the ocular surface was determined by direct observation using a slit-lamp biomicroscope (Carl Zeiss, Jena, Germany), and the normal stability of the tear film was established with the tearscope (Keeler Diagnostics, Windsor, United Kingdom), which is a commonly used noninvasive observational grid reflectometry system for the ocular surface.
Application and observation of the qdots
Transferpettor (Brand G.m.b.H. & Co., Wertheim, Germany), a positive displacement pipette, was used to apply 0.5 μL of 8 μM INGAP qdots solution (Evident Technologies Inc., Troy, New York) above the horizontal meridian of the lateral and medial bulbar conjunctiva. InGaP qdots are bioconjugated lipid-coated qdots with a hydrodynamic diameter of 20–25 nm and comprising a core of indium phosphide and gallium, nanocrystals of III-V semiconductors, and a zinc sulfide shell. These qdots are inert and stable at standard temperatures and can be surface-modified to make them either water- or lipid-soluble, which made them attractive as biomarkers for the tear film on the ocular surface.
Eyes were kept open without blinking for as long as possible, and a video of the movement of the fluorescent qdots was recorded at 25 frames per second using a charge-coupled device color video camera (Sony, Tokyo, Japan) attached to a slit-lamp biomicroscope at 8× magnification. The eye was illuminated using the cobalt blue filter of the slit lamp.
Hydrophobic qdots were applied to the lower lid margin and recorded as described above.
Evaluation of qdots movements
Adobe Premiere (Adobe Inc., San Jose, California), a video editing software, was used to remove the excessive blue coloration of the cobalt blue filters from the videos. This was not near the emission wavelengths of the qdots (∼600 nm), and therefore their appearance was unaffected by the editing. These videos were analyzed frame by frame at a later time to track the movement of qdots on the ocular surface and the menisci.
Results
When the aqueous qdots were pipetted into the aqueous of the tear film, they dispersed immediately from the concentrated center to surrounding areas of low concentration in all directions uniformly. Following the rapid dispersion, the qdots remained stable on the ocular surface (Figure 2, A–C), and reflective dust particles in the outer lipid layer could be seen above the aqueous qdots in the aqueous layer. Upon a blink (Figure 2, D) the qdots flowed off the surface (Figure 2, E and F) and strongly stained both the upper and lower aqueous lakes (Figure 2, G). The qdots were not drawn back onto the ocular surface by blinking but instead were removed through the puncta into the lacrimal ducts (Figure 2, H). As a consequence the fluorescence in the aqueous lakes gradually decreased until there were no qdots remaining after five to six blinks. This was unexpected, because it implies that the aqueous on the ocular surface is being replaced with each blink by new secretion from the lacrimal gland and that there is minimal mixing from the pool of tears in the meniscus.
Figure 2.
Aqueous qdots applied to the ocular surface. (A) qdots being applied to the surface; (B) 4 seconds after application the qdots have spread from the local application point; (C) 17 seconds after application just before blink. (D) Blink showing surface of eyelid; (E) 0.02 seconds after the blink, note that few qdots remain on the surface (arrows). (F) After the second blink qdots have all but disappeared from the surface; (G) the qdots accumulated in the aqueous lake (arrow) between the lower lid and the ocular surface. LM, lower lid margin. (H) The eyelid has been everted to reveal the lower punctum, which shows the lumen filled with qdots (arrow). The aqueous lake between the lower eyelid and the ocular surface can also be seen to be strongly stained. Scale = 2 mm.
Some of the aqueous qdots attached to the mucins of the ocular surface in a meshlike pattern. Interestingly, they did not flow with the qdots in the aqueous layer after blinks but remained embedded in the mucous layer. It could be clearly seen that they were adhered to the mucins and not to the ocular surface tissues. They remained attached to the mucins on the ocular surface for five to six blinks, after which they appeared to have flowed into the lacrimal lakes.
Lipophilic qdots were applied onto the eyelid margin between the meibomian gland orifices and the eyelashes. They appeared to disperse rapidly across the whole tear film surface as a series of small circular areas and did not stain the eyelid margin (Figure 3, A), until a blink occurred. After the blink, qdots were visible a short time later assimilated into the outer layer of the tear film and moved in a similar manner to the trapped debris in this layer (Figure 3, B). At the same time the staining extended the length of the upper and lower lid margins and into the inner canthus. It did not spill over onto the skin or onto the ocular surface side of the eyelid margin; in other words, there was a distinct boundary for this hydrophobic region on the eyelid margin. Histologically the eyelid margin that corresponds to the hydrophobic region is covered by the thinnest transitional stratum corneum in the body, indicating a distinct difference in the epithelia of this region.19 The staining of the eyelid margins remained for at least 2 hours but the fluorescent regions on the surface of tear film disappeared. Qdots were seen to make slow upward movements immediately after the opening phase of each blink but the staining remained stationary during inter-blink intervals. In a few instances it seemed that some of these qdots had accumulated over the meibomian gland orifices and upon a hard blink could be seen jetting into the tear film (presumably being carried by meibomian lipids being excreted from the meibomian glands), where they dispersed rapidly (Figure 3, C). Some of the qdots were also seen a short time after application as small fluorescent balls rolling to and fro within the meniscus (Figure 3, D). It appeared that these had formed some sort of large droplets or micelles perhaps with proteins or mucins in the aqueous.
Figure 3.
Lipophilic qdots after placing them on the lower lid margin. (A) Just after application the qdots spread as rafts all over the ocular surface (arrows), and there was no staining of the eyelid margin. (B) After a blink the qdots could be seen dispersed as diffuse areas (arrows) in the outer layer of the tear film. (C) qdots jetting from a meibomian gland orifice. (D) Blobs of qdots within the aqueous lake beneath the superficial lipid layer. The lower lid margin (LM) is strongly stained after the first blink. (E) No fluorescence is seen inside the punctum (arrow). (F) The eyelashes are stained with the lipophilic qdots (long white arrow), and there is some staining of the periocular skin (short white arrow); the ocular side of the eyelid is unstained (black arrow). (G) The lipophilic barrier, a distinct stained line on the eyelid margin (white arrow), seen after a few lipophilic qdots were placed at the base of an eyelash; the meibomian gland orifices (black arrows) can be seen on the ocular side of this line. OS, ocular surface. Scale = 2 mm.
The lipophilic qdots did not drain from the ocular surface through the puncta (Figure 3, E) but appeared on eyelashes (Figure 3, F), and after some time a small amount was seen on the skin at the eyelid margin. Some lipophilic qdots were also observed in the region of the inner canthus. It is therefore most likely that the lipids are removed from the eye via the eyelashes and the inner canthus where they are periodically wiped away as we rub our eyes. A small amount of lipid escaping by flowing beyond the eyelid margin onto the skin of the eyelid could not be excluded, but if this were the case it was probably a minor route. Similarly, it is possible that the lipid is trapped in the aqueous and forms micelles or becomes bound to proteins that could be removed via the lacrimal duct, but more probable that it ends up as the hardened condensation with mucins deposited at the inner canthus.14 On placing a small amount of qdots at the base of the eyelids, they rapidly dispersed to form a distinct line along the eyelid margin just exterior to (skin side of) the meibomian gland orifices (Figure 3, G).
Discussion
The tear film has always interested scientists across multidisciplinary fields, and this is to our knowledge the first instance when the in vivo dynamics of its layers have been observed at the nanoscale in real time. It is indeed remarkable that a microns-thick dynamic fluid protects the exposed ocular tissues and contributes to the functioning of one of the most important sensory processes in a living being. The complexity of structuralization and dynamics of the tear fluid is compounded by its constituents, all coming from different sources; the outer lipid layer consists of various polar and nonpolar lipids; the middle aqueous layer contains antibodies, electrolytes, and metabolites; and the inner mucins are attached to the corneal epithelium. In recent years the potential molecular interactions between different components of the tear film, particularly of the lipids with proteins and mucins,20, 21, 22 have led researchers to believe that the tear film is a multiphasic density-gradient fluid and not distinctly three-layered as proposed in 1946 by Wolff.23 However, our study shows that the dynamics of lipid and aqueous layers are completely independent of one another, thus supporting the traditional view of distinct layers of the tear film. If the tear film were a density-gradient fluid, minimal difference in the patterns of flow would have been seen for the qdots: the flow of the layers would have been in the same direction, although the speed of movement in the different layers may have been different.
Another interesting finding was that the aqueous qdots did not return to the ocular surface from the upper and lower lid menisci after a blink and were drained progressively from the eye. This could have been due to the high capillary forces in the menisci24 which supersede the gravitational effect and other forces on the tear film, thereby restricting the outward flow of aqueous. The outer lipid layer, however, spread back onto the surface following blinks with identical patterns, indicating that the lipid components are well-integrated and also that the layer is viscoelastic. Recent models of the outer layer of the tear film includes proteins from the aqueous layer,22 suggesting that there has to be some interaction between the two layers. Although the lipid and aqueous layers have disparate flow patterns, the aqueous layer is remarkably still during interblink periods, and this would allow adsorption of proteins and other molecules into the lipid layer. The large interfacial surface area and the high concentrations of proteins in the aqueous would facilitate this adsorption.
The hydrophobic line we observed between the lid margin and the periocular skin differs from another line on the ocular side of the meibomian gland orifices known as Marx's line or the lid wiper,25 and to our knowledge, this is the first observation of such a boundary and its hydrophobic characteristics. Marx's line is not known to have any barrier function for the tears, but the line stained with the hydrophobic qdots appears to represent a boundary of the epithelium that acts as a barrier for the tears spilling out of the eye, and skin lipids and other skin contaminants from entering the eye. Such a boundary is consistent with the very low contamination (4%) of the ocular surface and tears by cosmetics applied to the periocular skin and eyelashes.19
Through this study we have demonstrated a novel bottom-up approach in the understanding of dynamics of the layers of the tear film in real time using qdots. Current techniques used in evaluating thin fluid film interfaces are based on optical and electromagnetic properties or the surface tension of the tear components. Independent optical systems are suitable only if the thickness of the fluid is at least half the wavelength of light and there is a difference in refractive index of the fluids, and the interacting surfaces allow sufficient light waves to pass through without scattering and aberrations. Moreover, the Fresnel effect also has to be overcome.26 Electromagnetic techniques cannot be used in fluids without electric or magnetic properties, and further, this technique requires one of the layers to be solid, which reduces its applicability for the tear film considerably. Surface tension is not a direct measurement of fluid-fluid dynamics. More importantly, the above tests do not encompass all the rheological properties of the different components of the tears that could influence the interfacial interactions such as viscoelasticity, newtonian properties, shear stress, and oscillatory flow, all of which have an influence on tear film dynamics.27 With qdots all of these factors are accounted for. Furthermore, because the qdots are stable,28 their addition does not affect the intrinsic dynamics of the multilayered tear film components and their interactions within themselves or with the corneal epithelium.
It is known from previously published studies that tear dynamics can differ between normal individuals and those with dry eye,29 although quantifying these differences has been difficult due to unavailability of a biomarker to evaluate the tear layers in real time without disrupting the tear film. We have demonstrated through this study that qdots can be used to monitor the dynamics of different layers of the tear film highly effectively in real time. Furthermore, because the qdots have considerably longer fluoresence life than organic dyes, qdots can be used to monitor flow for longer periods. This opens a huge potential of clinical applicability of qdots in the diagnosis of tear film–related disorders such as dry eye and its subtypes and in assessing the suitability of the ocular surface for contact lens wear. The slit-lamp biomicroscope, which is a standard clinical instrument used to view the anterior eye, has a light source of wavelength 400–450 nm, and therefore, no modifications would be required on the current clinical setup for a successful clinical uptake of qdots. It is now necessary to categorize the dynamics of the layers of the tear film clinically to develop paradigms for dry eye diagnosis and assessment of patient suitability for contact lens wear.
The dynamics of the tear film layers can have important implications for devising effective drug delivery mechanisms to ocular tissues. It is thought that about 80% of the volume of an eye drop instilled on the ocular surface is removed through the lacrimal duct and becomes available for systemic absorption at the nasal cavity,30 thus lessening the benefits of topical administration. Because topical medications must be made amphiphilic to pass through the cornea, it seems that they dissolve in the relatively highly abundant aqueous layer and are removed with the aqueous to the nasal cavity through the puncta. If molecules could be retained on the ocular surface for longer periods, there would be a higher level of adsorption through the corneal and conjunctival epithelium. Based on our study, mucoadhesive polymers31 would be the most effective drug delivery system, because they would attach to the mucin layer and remain on the ocular surface for a longer period than other molecules.
Acknowledgments
The authors thank Professor Mark Willcox for his valuable advice and the Brien Holden Vision Institute for access to their clinical facilities.
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This research was supported by a seed grant from the University of Western Sydney and the Australian government linkage project scheme. T.J.M. has received financial support from Alcon Inc. for an unrelated study on the in vitro assessment of the physiochemical properties of human meibum.
PII: S1549-9634(10)00163-2
doi:10.1016/j.nano.2010.06.002
© 2010 Elsevier Inc. All rights reserved.
Volume 6, Issue 6 , Pages 707-713, December 2010
