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Thematic Review Series Thematic Review Series: Seeing 2020: Lipids and Lipid-Soluble Molecules in the Eye| Volume 62, 100039, January 01, 2021

Lipid conformational order and the etiology of cataract and dry eye

Open AccessPublished:February 05, 2021DOI:https://doi.org/10.1194/jlr.TR120000874

      Abstract

      Lens and tear film lipids are as unique as the systems they reside in. The major lipid of the human lens is dihydrosphingomylein, found in quantity only in the lens. The lens contains a cholesterol to phospholipid molar ratio as high as 10:1, more than anywhere else in the body. Lens lipids contribute to maintaining lens clarity, and alterations in lens lipid composition due to age are likely to contribute to cataract. Lens lipid composition reflects adaptations to the unique characteristics of the lens: no turnover of lens lipids or proteins; the lowest amount of oxygen of any tissue; and contains almost no intracellular organelles. The tear film lipid layer (TFLL) is also unique. The TFLL is a thin (100 nm) layer of lipid on the surface of tears covering the cornea that contributes to tear film stability. The major lipids of the TFLL are wax esters and cholesterol esters that are not found in the lens. The hydrocarbon chains associated with the esters are longer than those found anywhere else in the body (as long as 32 carbons), and many are branched. Changes in the composition and structure of the 30,000 different moieties of TFLL contribute to the instability of tears. The focus of the current review is how spectroscopy has been used to elucidate the relationships between lipid composition, conformational order and function, and the etiology of cataract and dry eye.

      Supplementary key words

      Abbreviations:

      BR (blink rate), CE (cholesteryl ester), MHSCT (meibum from donors with dry eye due to hematopoietic stem cell transplantation), MMGD (meibum from donors with dry eye due to Meibomian gland dysfunction), Mn (meibum from donors without dry eye), TBUT (tear breakup time), TFLL (tear film lipid layer), WE (wax ester)
      The focus of the current review is how spectroscopy has been used to elucidate the relationships between lipid composition, conformational order and function, and the etiology of cataract and dry eye (keratoconjunctivitis sicca). Cataracts are a major cause of progressive irreversible blindness, particularly in underdeveloped countries, afflicting over 20 million people (
      • Pascolini D.
      • Mariotti S.P.
      Global estimates of visual impairment.
      ); but, unlike most other blinding eye diseases that are progressive, age-related, and irreversible, there is an easy corrective treatment for cataracts: cataract extraction and insertion of a synthetic intraocular lens. Dry eye is the major reason worldwide for seeking medical help and affects 5–50% of people worldwide, especially Asians (
      • Stapleton F.
      • Alves M.
      • Bunya V.Y.
      • Jalbert I.
      • Lekhanont K.
      • Malet F.
      • Na K.
      • Schaumberg D.
      • Uchino M.
      • Vehof J.
      • et al.
      TFOS DEWS II epidemiology report.
      ). Since the last review of lens lipids and the etiology of cataracts in this journal a decade ago (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ), insightful spectroscopic studies have been published suggesting a need for this review. Although numerous reviews related to the composition of tear lipids and the etiology of dry eye have been published recently (
      • Green-Church K.B.
      • Butovich I.
      • Willcox M.
      • Borchman D.
      • Paulsen F.
      • Barabino S.
      • Glasgow B.
      The International Workshop on Meibomian Gland Disfunction: report of the Subcommittee on Tear Film Lipids and Lipid-Protein Interactions in Health and Disease.
      ,
      • Pucker A.D.
      • Nichols J.J.
      Analysis of meibum and tear lipids.
      ,
      • Butovich I.A.
      • Millar T.J.
      • Ham B.M.
      Understanding and analyzing meibomian lipids–a review.
      ,
      • Knop E.
      • Knop N.
      • Millar T.
      • Obata H.
      • Sullivan D.A.
      The International Workshop on Meibomian Gland Dysfunction: report of the Subcommittee on Anatomy, Physiology, and Pathophysiology of the Meibomian Gland.
      ,
      • Murube J.
      The origin of tears. III. The lipid component in the XIX and XX centuries.
      ,
      • Georgiev G.A.
      • Eftimov P.
      • Yokoi N.
      Structure-function relationship of tear film lipid layer: a contemporary perspective.
      ,
      • Foulks G.N.
      • Borchman D.
      Meibomian gland dysfunction: the past, the present, the future.
      ,
      • Foulks G.N.
      The correlation between the tear film lipid layer and dry eye disease.
      ,
      • Butovich I.A.
      Meibomian glands, meibum, and meibogenesis.
      ,
      • Borchman D.
      From bench to bedside: infrared spectroscopy and the diagnosis and treatment of dry eye and cataracts.
      ,
      • Millar T.J.
      • Schuett B.S.
      The real reason for having a meibomian lipid layer covering the outer surface of the tear film - A review.
      ,
      • Knop E.
      • Knop N.
      • Schirra F.
      Meibomian glands. Part II: physiology, characteristics, distribution and function of meibomian oil.
      ,
      • Pucker A.D.
      • Haworth K.M.
      The presence and significance of polar meibum and tear lipids.
      ), this is the first review article related to publications that use spectroscopic techniques such as infrared, NMR, fluorescence, and Brewster’s angle spectroscopies to elucidate relationships between tear lipid hydrocarbon chain conformational order and function related to the etiology of dry eye. Human lens lipid composition is unique, as the major phospholipid is dihydrosphingomyelin, found at high concentration (47%) only in human lenses, and it has an unusually high cholesterol to phospholipid molar ratio of 2:9 (Table 1). Tear lipids are also unique, as the predominant lipids are wax esters (WEs) and cholesteryl esters (CEs) (80%), with branched and very long hydrocarbon chains (Table 1). Whereas phospholipids make up most of the lens lipids, they only make up about 6% of the lipids found in tears. The relationships of these unusual lipids with structure and function form the basis of this review.
      Table 1Major lipids of the human lens and tear film
      Lipid SpeciesMajor Human Lens Lipids (
      • Deeley J.M.
      • Mitchell T.W.
      • Wei X.
      • Korth J.
      • Nealon J.R.
      • Blanksby S.J.
      • Truscott R.J.
      Human lens lipids differ markedly from those of commonly used experimental animals.
      )
      Major Human Tear Film Lipids (
      • Lam S.M.
      • Tong L.
      • Duan X.
      • Petznick A.
      • Wenk M.R.
      • Shui G.
      Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles.
      )
      Dihydrosphingomyelin
      Values are molar percent of phospholipids.
      470
      Sphingomyelin
      Values are molar percent of phospholipids.
      1918.3
      Phosphatidylcholine
      Values are molar percent of phospholipids.
      1130.2
      Phosphatidylethanolamine (1-O-alkyl ether)
      Values are molar percent of phospholipids.
      150
      Phosphatidylethanolamine
      Values are molar percent of phospholipids.
      08.5
      Phosphatidylserine
      Values are molar percent of phospholipids.
      81.3
      Phosphatidylinositol
      Values are molar percent of phospholipids.
      18.8
      Phosphatidic acid
      Values are molar percent of phospholipids.
      00.9
      Lyso phosphatidylethanolamine
      Values are molar percent of phospholipids.
      015.8
      Lyso phosphatidylcholine
      Values are molar percent of phospholipids.
      06.0
      Lyso phosphatidylserine
      Values are molar percent of phospholipids.
      05.6
      Ceramide
      Values are molar percent of phospholipids.
      03.2
      Cholesterol
      Values are molar percent of all lipids.
      200% (equatorial), 900% (nucleus) (
      • Borchman D.
      • Delamere N.A.
      • McCulley L.A.
      • Paterson C.A.
      Studies on the distribution of cholesterol, phospholipids and protein in the human and bovine lens.
      ,
      • Li L.K.
      • Spector A.
      Age-dependent changes in the distribution and concentration of human lens cholesterol and phospholipids.
      )
      6
      Phospholipids
      Values are molar percent of all lipids.
      1008.2
      CEs
      Values are molar percent of all lipids.
      044.8
      WEs
      Values are molar percent of all lipids.
      035.2
      Triacylglycerides
      Values are molar percent of all lipids.
      02.8
      Diacylglycerides
      Values are molar percent of all lipids.
      00.3
      O-acyl-ω-hydroxy-fatty acid
      Values are molar percent of all lipids.
      02.5
      a Values are molar percent of phospholipids.
      b Values are molar percent of all lipids.

      Function and unique characteristics of the lens

      The purpose of the lens is to focus light onto the back of the eye (retina) where the light is transduced into an electric signal and is then interpreted by the brain as a visual image. Zonules attached to the equatorial region of the lens capsule surrounding the lens are attached to ciliary muscles that control zonular tension and change the shape and focus of the lens (Fig. 1). In order to be and remain clear, the lens is unique: it has no blood supply and thus, less oxygen than other organs (
      • Barbazetto I.A.
      • Liang J.
      • Chang S.
      • Zheng L.
      • Spector A.
      • Dillon J.P.
      Oxygen tension in the rabbit lens and vitreous before and after vitrectomy.
      ); all of the cells are arranged in a crystalline hexagonal array; the space between the cells is smaller than the wavelength of light to avoid scattering; there are almost no intracellular organelles; and all of the biomolecules, such as the crystalline proteins, are arranged in a symmetrical crystalline array (
      • Trokel S.
      The physical basis for transparency of the crystalline lens.
      ,
      • Taylor V.L.
      • al-Ghoul K.J.
      • Lane C.W.
      • Davis V.A.
      • Kuszak J.R.
      • Costello M.J.
      Morphology of the normal human lens.
      ). It is remarkable and yet unexplained how the lens adapted all of the features above, the absence of any one of which would cause the lens to become opaque and useless. The lens contains a thin monolayer of epithelial cells on the posterior surface that contains organelles (Fig. 1). The epithelial cells differentiate and elongate at the equator into fiber cells that are centimeters long, which, over time, migrate toward the center of the lens (
      • Brown N.P.
      • Bron A.J.
      ). As there is no turnover of lipids (
      • Hughes J.R.
      • Levchenko V.A.
      • Blanksby S.J.
      • Mitchell T.W.
      • Williams A.
      • Truscott R.J.
      No turnover in lens lipids for the entire human lifespan.
      ) and proteins (
      • de Vries A.C.
      • Vermeer M.A.
      • Hendriks A.L.
      • Bloemendal H.
      • Cohen L.H.
      Biosynthetic capacity of the human lens upon aging.
      ) due to the lack of intracellular organelles (
      • Bassnett S.
      The fate of the Golgi apparatus and the endoplasmic reticulum during lens fiber cell differentiation.
      ,
      • Bassnett S.
      Fiber cell denucleation in the primate lens.
      ), with time, the lens increases in size and weight and many lens lipids and proteins are as old as the individual.
      Figure thumbnail gr1
      Fig. 1Top: Cross-section through a human eye. Bottom: Schematic of the human lens. Used with permission from LifeMap Sciences, Inc. (https://discovery.lifemapsc.com).

      Function and unique characteristics of the tear film lipid layer

      The tear film lipid layer (TFLL) is a thin (100 nm) lipid layer on the surface of tears (
      • King-Smith P.E.
      • Hinel E.A.
      • Nichols J.J.
      Application of a novel interferometric method to investigate the relation between lipid layer thickness and tear film thinning.
      ,
      • King-Smith P.E.
      • Fink B.A.
      • Fogt N.
      • Nichols K.K.
      • Hill R.M.
      • Wilson G.S.
      The thickness of the human precorneal tear film: evidence from reflection spectra.
      ) covering the cornea that is 80 times thinner than the aqueous tear layer below (Fig. 2). The major source of the TFLL is the Meibomian gland that contributes about 80% of the TFLL (Fig. 3) (
      • Mudgil P.
      • Borchman D.
      • Gerlach D.
      • Yappert M.C.
      Sebum/meibum surface film interactions and phase transitional differences.
      ,
      • Ashraf Z.
      • Pasha U.
      • Greenstone V.
      • Akbar J.
      • Apenbrinck E.
      • Foulks G.N.
      • Borchman D.
      Quantification of human sebum on skin and human meibum on the eye lid margin using sebum tape, spectroscopy and chemical analysis.
      ,
      • Borchman D.
      • Yappert M.C.
      • Milliner S.
      • Bhola R.
      Confirmation of squalene in human eye lid lipid by heteronuclear single quantum correlation spectroscopy.
      ,
      • Ivanova S.
      • Borchman D.
      • Yappert M.C.
      • Tonchev V.
      • Yokoi N.
      • Georgiev G.
      Surface properties of squalene/meibum films and NMR confirmation of squalene in tears.
      ,
      • Glasgow B.J.
      • Abduragimov A.R.
      Interaction of ceramides and tear lipocalin.
      ). Meibomian glands (also known as “tarsal glands”) are named after Heinrich Meibom, a German physician who first described them; hence, the lipid secreted from the Meibomian gland is called “meibom” (
      • Murube J.
      The origin of tears. III. The lipid component in the XIX and XX centuries.
      ). It is speculated that some of the TFLL comes from the sebaceous glands in the eyelid (
      • Mudgil P.
      • Borchman D.
      • Gerlach D.
      • Yappert M.C.
      Sebum/meibum surface film interactions and phase transitional differences.
      ) and lipid bound to lipocalin (
      • Glasgow B.J.
      • Abduragimov A.R.
      Interaction of ceramides and tear lipocalin.
      ) originating from the lacrimal gland. The Meibomian gland is a sebaceous gland consisting of acini cells that constantly fill the gland with lipids. Upon blinking, which involves the contraction of the orbicularis and Riolan’s muscles, a small amount of lipid is squeezed out of the Meibomian glands onto the tear film surface. Thus, unlike the lens discussed above in which there is no turnover of lipid, fresh lipid is layered onto the tear film surface as often as one blinks. Upon blinking, the TFLL is drawn upward and the tear film spreads, driven by the Marangoni effect (
      • Knop E.
      • Knop N.
      • Schirra F.
      Meibomian glands. Part II: physiology, characteristics, distribution and function of meibomian oil.
      ,
      • Yokoi N.
      • Bron A.J.
      • Georgiev G.A.
      The precorneal tear film as a fluid shell: the effect of blinking and saccades on tear film distribution and dynamics.
      ,
      • Berger R.E.S.
      • Corrsin S.
      A surface tension gradient mechanism for driving the pre-corneal tear film after a blink.
      ,
      • Bron A.J.
      • Tiffany J.M.
      • Gouveia S.M.
      • Yokoi N.
      • Voon L.W.
      Functional aspects of the tear film lipid layer.
      ). Shortly after blinking, which occurs about every 10 s, tears break up and the process starts over again with another blink. Thus, the major function of the TFLL is to aid in the spreading of tears. Other functions of the TFLL are to dam, lubricate, and stabilize the tear film to allow for proper refraction, to degrade mucinic clots, to provide an antibacterial effect, and to suppress exposure to UV rays (
      • Murube J.
      The origin of tears. III. The lipid component in the XIX and XX centuries.
      ).
      Figure thumbnail gr2
      Fig. 2Schematic of the TFLL on the surface of the cornea adapted from slideshare.net (https://www.pharmaccutical-journal.com).
      Figure thumbnail gr3
      Fig. 3Cross-section through the eyelid. Sebum (red) is shown mixing with meibum (yellow), which forms a continuous TFLL film over the ocular surface and eyelid. From (
      • Ashraf Z.
      • Pasha U.
      • Greenstone V.
      • Akbar J.
      • Apenbrinck E.
      • Foulks G.N.
      • Borchman D.
      Quantification of human sebum on skin and human meibum on the eye lid margin using sebum tape, spectroscopy and chemical analysis.
      ).

      Structure/conformation of lens membranes

      In passing through the human lens, light traverses through thousands of cellular membranes that scatter most of the light passing through the lens (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ,
      • Borchman D.
      From bench to bedside: infrared spectroscopy and the diagnosis and treatment of dry eye and cataracts.
      ,
      • Bettelheim F.A.A.
      • Ali A.
      Light scattering of normal human lens III. Relationship between forward and back scatter of whole excised lenses.
      ,
      • Tang D.
      • Borchman D.
      • Schwarz A.K.
      • Yappert M.C.
      • Vrensen G.F.J.M.
      • van Marle J.
      • DuPré D.B.
      Light scattering of human lens vesicles in vitro.
      ). The amount of light scattered by lens membranes is related to lipid structural order or stiffness with age, cataract, and species (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ). Lens membranes are important to the clarity of the lens, as membrane proteins such as aquaporin, plasma membrane Ca2+-ATPase, and Na,K-ATPase reside in the lens membranes of the epithelium and equatorial fibers and are necessary for maintaining the homeostasis of lens water, calcium, sodium, and potassium [references in (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ,
      • Borchman D.
      From bench to bedside: infrared spectroscopy and the diagnosis and treatment of dry eye and cataracts.
      )]. Typical membranes are similar to the Singer fluid-mosaic model in which proteins float in a sea of fluid phospholipids with lateral mobility within the bilayer (Fig. 4, left) (
      • Singer S.J.
      • Nicolson G.L.
      The fluid mosaic model of the structure of cell membranes.
      ). Lens membranes are atypical, as membranes of adult human lenses are some of the most saturated and ordered membranes in the human body, and their high level of cholesterol leads to the formation of patches of pure cholesterol bilayers (Fig. 4, right) (
      • Lam S.M.
      • Tong L.
      • Duan X.
      • Petznick A.
      • Wenk M.R.
      • Shui G.
      Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles.
      ,
      • Ashraf Z.
      • Pasha U.
      • Greenstone V.
      • Akbar J.
      • Apenbrinck E.
      • Foulks G.N.
      • Borchman D.
      Quantification of human sebum on skin and human meibum on the eye lid margin using sebum tape, spectroscopy and chemical analysis.
      ). Furthermore, most of the lipids are associated with crystalline and membrane proteins, thus limiting their mobility (Fig. 4, right) (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ,
      • Borchman D.
      From bench to bedside: infrared spectroscopy and the diagnosis and treatment of dry eye and cataracts.
      ).
      Figure thumbnail gr4
      Fig. 4Left: A typical membrane. Right: Human lens membrane. Typical membranes contain fluid lipids with relatively few cholesterol molecules (red cylinders). Human lens membranes are unique. Most of the lipid is associated with proteins such as α-crystallin (α-crystallin assembly shown as gray balls, one large ball and one small ball for each α-crystallin) and aquaporin, which limits their mobility. Human lens membranes are some of the most saturated ordered (stiff) membranes in the human body. The major lipid of the human lens is dihydrosphingomyelin (green shaded balls). Found in quantity only in the human lens. From (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ).
      Hydrocarbon chain conformation may be used to measure hydrocarbon structural order, a static measure of lipid fluidity. Conformation is the spatial arrangement of atoms in a molecule that can come about through the rotation of the atoms about a chemical bond. When lipids are completely ordered, the hydrocarbons are arranged in an all-trans conformation of rotamers (Fig. 5, top). This allows the lipid hydrocarbon chains to pack tightly together maximizing van der Waal’s interactions between chains. When the hydrocarbon chains are disordered, the number of gauche rotamers increases, the lipids pack more loosely, and van der Waal’s interactions are minimal (Fig. 5, bottom). Most membranes are disordered; however, lens membranes are exceptionally ordered and the degree of lipid order increases linearly with sphingolipid content (
      • Borchman D.
      • Yappert M.C.
      • Afzal M.
      Lens lipids and maximum lifespan.
      ) (Fig. 6) and increases in the human lens with age (
      • Huang L.
      • Rasi V.
      • Grami V.
      • Marrero Y.
      • Borchman D.
      • Tang D.
      • Yappert M.C.
      Human lens phospholipid changes with age and cataract.
      ) and cataract (
      • Borchman D.
      • Ozaki Y.
      • Lamba O.P.
      • Byrdwell W.C.
      • Yappert M.C.
      Age and regional structural characterization of clear human lens lipid membranes by infrared and near-infrared Raman spectroscopies.
      ,
      • Paterson C.A.
      • Zeng J.
      • Husseini Z.
      • Borchman D.
      • Delamere N.A.
      • Garland D.
      • Jiminez-Asensio J.
      Calcium ATPase activity and membrane structure in clear and cataractous human lenses.
      ,
      • Borchman D.
      • Lamba O.P.
      • Yappert M.C.
      Structural characterization of lipid membranes from clear and cataractous human lenses.
      ). The relationships between lens membrane order and lens clarity are discussed later in this article.
      Figure thumbnail gr5
      Fig. 5Schematic of phospholipids and the conformation of hydrocarbon chains that define lipid order. The more trans rotamers, the tighter the packing, the greater the van der Waal’s interactions between lipids, and the greater the lipid order (stiffness). The opposite is true for gauche rotamers. From (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ).
      Figure thumbnail gr6
      Fig. 6The relationship between lens sphingolipid content and hydrocarbon chain order. Hydrocarbon chain order reflects the structural stiffness of the hydrocarbon chain region of lipids in membranes. Clear human lens cortex and nucleus (closed square); cataractous human lenses (closed triangle). This figure was adapted from Figure 5 in (
      • Huang L.
      • Rasi V.
      • Grami V.
      • Marrero Y.
      • Borchman D.
      • Tang D.
      • Yappert M.C.
      Human lens phospholipid changes with age and cataract.
      ). All the data except those related to cataractous lens lipid are from Borchman, Yappert, and Afzal (
      • Borchman D.
      • Yappert M.C.
      • Afzal M.
      Lens lipids and maximum lifespan.
      ). Cataractous lipid order information is extracted from Paterson et al. (
      • Paterson C.A.
      • Zeng J.
      • Husseini Z.
      • Borchman D.
      • Delamere N.A.
      • Garland D.
      • Jiminez-Asensio J.
      Calcium ATPase activity and membrane structure in clear and cataractous human lenses.
      ).

      Structure/conformation of the TFLL

      Like lens lipids discussed above, tear film lipid structural order has been measured by quantifying hydrocarbon chain conformation and structure using infrared (
      • Mudgil P.
      • Borchman D.
      • Gerlach D.
      • Yappert M.C.
      Sebum/meibum surface film interactions and phase transitional differences.
      ,
      • Ramasubramanian A.
      • Blackburn T.
      • Sledge S.M.
      • Yeo H.
      • Yappert M.C.
      • Gully Z.N.
      • Singh S.
      • Mehta S.
      • Mehta A.
      • Borchman D.
      Structural differences in meibum from donors after hematopoietic stem cell transplantations.
      ,
      • Sledge S.
      • Henry C.
      • Borchman D.
      • Yappert M.C.
      • Bhola R.
      • Ramasubramanian A.
      • Blackburn R.
      • Austin J.
      • Massey K.
      • Sayied S.
      • et al.
      Human meibum age, lipid-lipid interactions and lipid saturation in meibum from infants.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Ho D.V.
      Temperature-induced conformational changes in human tearlipids hydrocarbon chains.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Tang D.
      • Ho D.V.
      Spectroscopic evaluation of human tear lipids.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      Confirmation of changes in human meibum lipid infrared spectra with age using principal component analysis.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Kakar S.
      • Podoll N.
      • Rychwalski P.
      • Schwietz E.
      Physical changes in human meibum with age as measured by infrared spectroscopy.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      Changes in human meibum lipid with meibomian gland dysfunction using principal component analysis.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Bell J.
      • Wells E.
      • Neravetla S.
      • Greenstone V.
      Human meibum lipid conformation and thermodynamic changes with meibomian-gland dysfunction.
      ,
      • Hunter M.
      • Bhola R.
      • Yappert M.C.
      • Borchman D.
      • Gerlach D.
      Pilot study of the influence of eyeliner cosmetics on the molecular structure of human meibum.
      ,
      • Foulks G.N.
      • Borchman D.
      • Yappert M.C.
      • Sung-Hye K.
      • McKay J.W.
      Topical azithromycin therapy of meibomian gland dysfunction: clinical response and lipid alterations.
      ,
      • Foulks G.N.
      • Borchman D.
      • Yappert M.C.
      • Kakar S.
      Topical azithromycin and oral doxycycline therapy of meibomian gland dysfunction: a comparative clinical and spectroscopic pilot study.
      ,
      • Mudgil P.
      • Borchman D.
      • Yappert M.C.
      • Duran D.
      • Cox G.W.
      • Smith R.J.
      • Bhola R.
      • Dennis G.R.
      • Whitehall J.S.
      Lipid order, saturation and surface property relationships: A study of human meibum saturation.
      ,
      • Sledge S.M.
      • Borchman D.
      • Oliver A.
      • Michael H.
      • Dennis E.K.
      • Gerlach D.
      • Bhola R.
      • Stephen E.
      Evaporation and hydrocarbon chain conformation of surface lipid films.
      ,
      • Borchman D.
      • Ramakrishnan V.
      • Henry C.
      Differences in meibum and tear lipid composition and conformation.
      ,
      • Ramasubramanian A.
      • Borchman D.
      Structural differences in meibum from teenage donors with and without dry eye induced by allogeneic hematological stem cell transplantations.
      ,
      • Georgiev G.A.
      • Borchman D.
      • Eftimov P.
      • Yokoi N.
      Lipid saturation and the rheology of human tear lipids.
      ,
      • Borchman D.
      The optimum temperature for the heat therapy for meibomian gland dysfunction.
      ,
      • Mudgil P.
      • Borchman D.
      • Ramasubramanian A.
      Insights into tear film stability from babies and young adults; a study of human meibum lipid conformation and rheology.
      ), Raman (
      • Oshima Y.
      • Sato H.
      • Zaghloul A.
      • Foulks G.N.
      • Yappert M.C.
      • Borchman D.
      Characterization of human meibum lipid using Raman spectroscopy.
      ), Brewster’s angle (
      • Ivanova S.
      • Borchman D.
      • Yappert M.C.
      • Tonchev V.
      • Yokoi N.
      • Georgiev G.
      Surface properties of squalene/meibum films and NMR confirmation of squalene in tears.
      ,
      • Georgiev G.A.
      • Borchman D.
      • Eftimov P.
      • Yokoi N.
      Lipid saturation and the rheology of human tear lipids.
      ,
      • Nencheva Y.
      • Ramasubramanian A.
      • Eftimov P.
      • Yokoi N.
      • Borchman D.
      • Georgiev G.
      Effects of lipid saturation on the surface properties of human meibum films.
      ), and fluorescence anisotropy (
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Tang D.
      • Ho D.V.
      Spectroscopic evaluation of human tear lipids.
      ) spectroscopies. Meibum lipid hydrocarbons align to maximize van der Waal’s interactions between chains. Since the seminal model proposed for the packing of the TFLL in 1997 (
      • McCulley J.P.
      • Shine W.
      A compositional based model for the tear film lipid layer.
      ), a revised model has been proposed based on X-ray crystallographic studies of pure WEs and CEs (Fig. 7) (
      • Borchman D.
      • Ramasubramanian A.
      • Foulks G.N.
      Human meibum cholesteryl and wax ester variability with age, gender and Meibomian gland dysfunction.
      ). For a 100 nm-thick TFLL, the structure of the bulk lipids above the phospholipid monolayer consisting of esters would stack 16 times with a repeating motif. Note that the hydrocarbon chains of the WEs and CEs are not randomly oriented, as they are in an oil phase as many schematic pictures in literature show them to be. Meibum exists in a liquid crystalline phase at lower temperatures. The term “liquid crystalline phase” is used because meibum is never a solid with 100% trans, as the meibum hydrocarbon chains contain about 72% trans rotamers allowing them to pack tightly together (Fig. 5, top) (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ). Thus, the term liquid crystalline phase is used rather than “solid phase”. At higher temperatures, meibum is in the gel phase and the conformation of the meibum lipid hydrocarbon chains is about 18% trans rotamers and 82% gauche rotamers (Fig. 5, bottom). Thus, meibum is not a liquid (0% trans) but rather in the “gel phase”. It is unknown how mixtures of WE and CE pack together, but based on X-ray crystallographic studies of pure WE and CE, it is reasonable that the minimal energy structure of the lipids is maintained in a mixture of the two (
      • Borchman D.
      • Ramasubramanian A.
      • Foulks G.N.
      Human meibum cholesteryl and wax ester variability with age, gender and Meibomian gland dysfunction.
      ). Thus, the lipids are shown to align to maximize hydrocarbon chain interactions (Fig. 7). Below the phase transition temperature, meibum lipids pack in an orthorhombic geometry, whereas above the phase transition temperature, the meibum lipids pack in a monoclinic geometry (
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Bell J.
      • Wells E.
      • Neravetla S.
      • Greenstone V.
      Human meibum lipid conformation and thermodynamic changes with meibomian-gland dysfunction.
      ). The arrangement of molecules in Fig. 7 allows for the interdigitation of CE side chains and maximizes the adjacent packing of the steroid nuclei and CE carbonyl moieties, as they are for pure CE. Phospholipids are shown with their hydrophilic head group facing the tear aqueous layer. Phospholipids do not interact with CE (
      • Salmon A.
      • Hamilton J.A.
      Magic-angle spinning and solution C-13 nuclear magnetic resonance studies of medium- and long-chain cholesteryl esters in model bilayers.
      ,
      • Janiak M.J.
      • Small D.M.
      • Shipley G.G.
      Interactions of cholesterol esters with phospholipids – cholesteryl myristate and dimyristoyl lecithin.
      ,
      • Souza S.L.
      • Hallock K.J.
      • Funari S.S.
      • Vaz W.L.C.
      • Hamilton J.A.
      • Melo E.
      Study of the miscibility of cholesteryl oleate in a matrix of ceramide, cholesterol and fatty acid.
      ); so, they are likely to form a monolayer alone with other amphipathic molecules such as (O-acyl)-ω-hydroxy fatty acids. A limitation of the model is that proteins, especially mucin, are likely to associate with the TFLL, but are not included in the model (
      • Faheem S.
      • Kim S.
      • Nguyen J.
      • Neravetla S.
      • Ball M.
      • Foulks G.N.
      • Yappert M.C.
      • Borchman D.
      Wax-tear and meibum protein, wax-β-carotene interactions in vitro using infrared spectroscopy.
      ).
      Figure thumbnail gr7
      Fig. 7Schematic of WE and CE packing from X-ray crystallography. A: Molecular size of CE and WE with 22 carbon hydrocarbon chains. B:) Potential lamellar packing of WE. B (top): Shows rhombic packing of the hydrocarbon chains. B (right): The trans orientation for ordered hydrocarbons, gauche rotamer orientations for disordered hydrocarbon chains. C: Smectic phase packing of CE. D: Speculative packing of a WE, CE, and phospholipid mixture on an aqueous surface. From (
      • Borchman D.
      • Ramasubramanian A.
      • Foulks G.N.
      Human meibum cholesteryl and wax ester variability with age, gender and Meibomian gland dysfunction.
      ).

      Relationships between lens membrane lipid composition, structure, and function and the etiology of cataract

      Human lenses differ significantly from animal lenses in regard to compaction and oxidation with age, UV filters, protein and crystallin content, synthesis of ascorbate, and antioxidant enzymes, prompting an author to state: “Unfortunately, due to marked variability in the lenses of different species, there appears at present to be no ideal animal model system for studying human ARN cataract” (Ref. (
      • Truscott R.J.W.
      Age-related nuclear cataract—oxidation is the key.
      ); p. 709). Indeed, even the lens phospholipid content of numerous species, such as chickens, cows, elephants, guinea pigs, pigs, sheep, mice, and rats, varies greatly between species [see references in (
      • Huang L.
      • Rasi V.
      • Grami V.
      • Marrero Y.
      • Borchman D.
      • Tang D.
      • Yappert M.C.
      Human lens phospholipid changes with age and cataract.
      ,
      • Borchman D.
      • Stimmelmayr R.
      • George J.C.
      Whales, lifespan, phospholipids and cataracts.
      ,
      • Stimmelmayr R.
      • Borchman D.
      Lens lipidomes among Phocidae and Odobenidae.
      )]. Nevertheless, spectroscopic studies on rat, porcine, and bovine lenses have provided insights into the contribution of glucose (
      • Alghamdi A.H.S.
      • Mohamed H.
      • Sledge S.M.
      • Borchman D.
      Absorbance and light scattering of lenses organ cultured with glucose.
      ), glucocorticoids (
      • Bree M.
      • Borchman D.
      The optical properties of rat, porcine and human lenses in organ culture treated with dexamethasone.
      ), and cholesterol (
      • Borchman D.
      • Cenedella R.I.
      • Lamba O.P.
      Role of cholesterol in the structural order of lens lipids.
      ) on light scattering and membrane structure. Guinea pig hyperbaric oxygen (
      • Borchman D.
      • Giblin F.J.
      • Leverenz V.R.
      • Reddy V.N.
      • Lin L.R.
      • Yappert M.C.
      • Tang D.
      • Li L.
      Impact of aging and hyperbaric oxygen in vivo on guinea pig lens lipids and nuclear light scatter.
      ) and UV light (
      • Giblin F.J.
      • Leverenz V.R.
      • Padgaonkar V.A.
      • Unakar N.J.
      • Dang L.
      • Lin L.R.
      • Lou M.F.
      • Reddy V.N.
      • Borchman D.
      • Dillon J.P.
      UVA light in vivo reaches the nucleus of the guinea pig lens and produces deleterious, oxidative effects.
      ) as well as numerous other model systems have been reviewed and provided insights into the contribution of numerous factors related to human cataractogenesis that would be difficult to obtain from human lenses (
      • Graw J.
      Mouse models for microphthalmia, anophthalmia and cataracts.
      ,
      • Thiagarajan R.
      • Manikandan R.
      Antioxidants and cataract.
      ,
      • Rowan S.
      • Bejarano E.
      • Taylor A.
      Mechanistic targeting of advanced glycation end-products in age-related diseases.
      ,
      • Harkema L.
      • Youssef S.A.
      • de Bruin A.
      Pathology of mouse models of accelerated aging.
      ,
      • Tripathi B.J.
      • Tripathi R.C.
      • Borisuth N.S.
      • Dhaliwal R.
      • Dhaliwal D.
      Rodent models of congenital and hereditary cataract in man.
      ,
      • Graw J.
      Mouse models of cataract.
      ).
      Interspecies comparison of lens lipid composition and structure has provided insights into factors related to the etiology of cataracts. Short-lived species such as rats have a very low lens sphingomyelin content and a very high phosphatidylcholine content, whereas humans and whales that live much longer have a high lens sphingolipid content and a low lens phosphatidylcholine content. One explanation of why rats get cataracts at 2 years, dogs at 8 years, humans at 60 years, and whales do not get cataracts even after living 200 years comes from studies of lens lipid compositional and structural differences with age and species (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ,
      • Borchman D.
      From bench to bedside: infrared spectroscopy and the diagnosis and treatment of dry eye and cataracts.
      ,
      • Borchman D.
      • Yappert M.C.
      • Afzal M.
      Lens lipids and maximum lifespan.
      ,
      • Borchman D.
      • Stimmelmayr R.
      • George J.C.
      Whales, lifespan, phospholipids and cataracts.
      ,
      • Stimmelmayr R.
      • Borchman D.
      Lens lipidomes among Phocidae and Odobenidae.
      ). At the heart of the explanation is oxidation from reactive oxidative species (
      • Huang L.
      • Yappert M.C.
      • Jumblatt M.
      • Borchman D.
      Hyperoxia and thyroxine-treatment and the relationships between reactive oxygen species generation, mitochondrial membrane potential and cardiolipin in human lens epithelial cell cultures.
      ,
      • Huang L.
      • Tang D.
      • Yappert M.C.
      • Borchman D.
      Oxidation induced changes in human lens epithelial cells. 2. Mitochondria and the generation of reactive oxygen species.
      ), likely to originate from the mitochondria (
      • Muller F.L.
      • Lustgarten M.S.
      • Jang Y.
      • Richardson A.
      • Van Remmen H.
      Trends in oxidative aging theories.
      ,
      • Balaban R.S.
      • Nemoto S.
      • Finkel T.
      Mitochondria, oxidants, and aging.
      ,
      • McNulty R.
      • Wang H.
      • Mathias R.T.
      • Ortwerth B.J.
      • Truscott R.J.
      • Bassnett S.
      Regulation of tissue oxygen levels in the mammalian lens.
      ). Oxidative damage to lipids accumulates in the lens with age because there is no turnover of lipids (
      • Hughes J.R.
      • Levchenko V.A.
      • Blanksby S.J.
      • Mitchell T.W.
      • Williams A.
      • Truscott R.J.
      No turnover in lens lipids for the entire human lifespan.
      ) or proteins (
      • de Vries A.C.
      • Vermeer M.A.
      • Hendriks A.L.
      • Bloemendal H.
      • Cohen L.H.
      Biosynthetic capacity of the human lens upon aging.
      ) disrupting crystalline structure, resulting in an increase of light scattering (
      • Varma S.D.
      • Chand D.
      • Sharma Y.R.
      • Kuck Jr., J.F.
      • Richards D.
      Oxidative stress on lens and cataract formation: role of light and oxygen.
      ). Furthermore, products of lipid oxidation impede membrane function and alter relevant cellular processes such as growth, respiration, and ATPase and phosphate transport, as well as DNA, RNA, and protein synthesis (
      • Esterbauer H.
      • Schaur R.J.
      • Zollner H.
      Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.
      ). The association between lipid oxidation and lens opacity is very strong and has led many to state that lipid oxidation may be the initiating pathogen of human cataract (
      • Varma S.D.
      • Chand D.
      • Sharma Y.R.
      • Kuck Jr., J.F.
      • Richards D.
      Oxidative stress on lens and cataract formation: role of light and oxygen.
      ,
      • Esterbauer H.
      • Schaur R.J.
      • Zollner H.
      Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.
      ,
      • Bhuyan K.C.
      • Master R.W.
      • Coles R.S.
      • Bhuyan D.K.
      Molecular mechanisms of cataractogenesis: IV. Evidence of phospholipid-malondialdehyde adduct in human senile cataract.
      ,
      • Micelli-Ferrari T.
      • Vendemiale G.
      • Grattagliano I.
      • Boscia F.
      • Arnese L.
      • Altomare E.
      • Cardia L.
      Role of lipid peroxidation in the pathogenesis of myopic and senile cataract.
      ,
      • Simonelli F.
      • Nesti A.
      • Pensa M.
      • Romano L.
      • Savastano S.
      • Rinaldi E.
      • Auricchio G.
      Lipid peroxidation and human cataractogenesis in diabetes and severe myopia.
      ,
      • Tomba M.C.
      • Gandolfi S.A.
      • Maraini G.
      Search for an oxidative stress in human senile cataract. Hydrogen peroxide and ascorbic acid in the aqueous humour and malondialdehyde in the lens.
      ,
      • Babizhayev M.A.
      • Deyev A.I.
      • Linberg L.F.
      Lipid peroxidation as a possible cause of cataract.
      ,
      • Bhuyan K.C.
      • Bhuyan D.K.
      Molecular mechanism of cataractogenesis: III. Toxic metabolites of oxygen as initiators of lipid peroxidation and cataract.
      ,
      • Bhuyan K.C.
      • Bhuyan D.K.
      • Podos S.M.
      Lipid peroxidation in cataract of the human.
      ). Changes in lens lipid composition with age and cataract are due to the preferential oxidation of glycerophospholipids, as explained below (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ,
      • Borchman D.
      From bench to bedside: infrared spectroscopy and the diagnosis and treatment of dry eye and cataracts.
      ,
      • Huang L.
      • Rasi V.
      • Grami V.
      • Marrero Y.
      • Borchman D.
      • Tang D.
      • Yappert M.C.
      Human lens phospholipid changes with age and cataract.
      ,
      • Borchman D.
      • Stimmelmayr R.
      • George J.C.
      Whales, lifespan, phospholipids and cataracts.
      ,
      • Stimmelmayr R.
      • Borchman D.
      Lens lipidomes among Phocidae and Odobenidae.
      ).
      Lifespan, age, and cataract are related (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ,
      • Borchman D.
      From bench to bedside: infrared spectroscopy and the diagnosis and treatment of dry eye and cataracts.
      ,
      • Huang L.
      • Rasi V.
      • Grami V.
      • Marrero Y.
      • Borchman D.
      • Tang D.
      • Yappert M.C.
      Human lens phospholipid changes with age and cataract.
      ,
      • Borchman D.
      • Stimmelmayr R.
      • George J.C.
      Whales, lifespan, phospholipids and cataracts.
      ,
      • Stimmelmayr R.
      • Borchman D.
      Lens lipidomes among Phocidae and Odobenidae.
      ). The longer animals live, the longer they require clear lenses. Lens sphingolipid content and lifespan are correlated (Fig. 8). Does this correlation have a scientific basis or is it coincidental? Sphingolipids resist oxidation better than phospholipids (
      • Oborina E.M.
      • Yappert M.C.
      Effect of sphingomyelin versus dipalmitoylphosphatidylcholine on the extent of lipid oxidation.
      ) because they have fewer double bonds (
      • Deeley J.M.
      • Mitchell T.W.
      • Wei X.
      • Korth J.
      • Nealon J.R.
      • Blanksby S.J.
      • Truscott R.J.
      Human lens lipids differ markedly from those of commonly used experimental animals.
      ,
      • Byrdwell W.C.
      • Borchman D.
      Liquid chromatography/mass-spectrometric characterization of sphingomyelin and dihydrosphingomyelin of human lens membranes.
      ). Sphingolipids resist oxidative degradation so much so that they were the only biomolecules found in a mammoth buried in ice for 40,000 years (
      • Kreps E.M.
      • Chirkovskaia E.V.
      • Pomazanskaia L.F.
      • Avrova N.F.
      • Levitina M.V.
      Issledovanie lipidov mozga mamonta Elephas primigenius, pogibshego bolee 40 tysiach let tomu nazad. Russian.
      ). Thus, it has been suggested that humans have adapted so that their lens membranes have a high sphingolipid content that confers resistance to oxidation, allowing their lenses to stay clear for a longer time relative to those in many other species (
      • Borchman D.
      • Yappert M.C.
      • Afzal M.
      Lens lipids and maximum lifespan.
      ). Similarly, bowhead whales (Balaena mysticetus) that can live over 200 years (
      • Rozell N.
      Bowhead Whales May Be the World’s Oldest Mammals.
      ,
      • de Magalhães J.P.
      • Costa J.
      A database of vertebrate longevity record and their relation to other life-history traits.
      ,
      • George J.
      • Bada J.L.
      • Zeh J.
      • Scott L.
      • Brown S.E.
      • O’Hara T.
      • Suydam R.
      Age and growth estimates of bowhead whales (Balaena mysticetus) via aspartic acid racemization.
      ,
      • George J.
      • Follmann E.
      • Zeh J.
      • Sousa M.
      • Tarpley R.J.
      • Suydam R.
      • Horstmann L.
      A new way to estimate the age of bowhead whales (Balaena mysticetus) using ovarian corpora counts.
      ,
      • Zeh J.
      • Craig G.J.
      • Oliver B.
      • Zauscher M.
      Age and growth estimates of bowhead whales (Balaena mysticetus) harvested in 1998-2000 and the relationship between racemization rate and body temperature.
      ,
      • Philo L.M.
      • Shotts Jr., E.B.
      • George J.C.
      Morbidity and mortality.
      ) do not get cataracts (
      • Philo L.M.
      • Shotts Jr., E.B.
      • George J.C.
      Morbidity and mortality.
      ), and, like humans, they have likely adapted to have the highest amount of sphingolipids in their lenses compared with other species (Fig. 8). Rats have a relatively lower level of lens sphingolipids and get cataracts relatively early, at about 2 years of age. “The strong correlation between sphingolipid and lifespan may form a basis for future studies which are needed since correlations do not necessitate cause. One could hope that if human lenses could be made to have a lipid composition similar to whales, like the bowhead whale, humans would not develop cataracts for over 100 years” (Ref. 74; p. 2289).
      Figure thumbnail gr8
      Fig. 8Lifespan versus lipid phase transition parameters from (
      • Borchman D.
      • Stimmelmayr R.
      • George J.C.
      Whales, lifespan, phospholipids and cataracts.
      ) (black stars). Data from (
      • Huang L.
      • Rasi V.
      • Grami V.
      • Marrero Y.
      • Borchman D.
      • Tang D.
      • Yappert M.C.
      Human lens phospholipid changes with age and cataract.
      ,
      • Borchman D.
      • Stimmelmayr R.
      • George J.C.
      Whales, lifespan, phospholipids and cataracts.
      ) (circles).
      Diet and exposure to UV radiation are unlikely to contribute to the significant differences observed in the correlation between lifespan and phospholipid composition (Fig. 8), as rats receive less UV radiation over their lifespan and lens lipid composition does not change with extreme diets (
      • Nealon J.R.
      • Blanksby S.J.
      • Abbott S.K.A.
      • Hulbert J.
      • Mitchell T.W.
      • Truscott R.J.
      Phospholipid composition of the rat lens is independent of diet.
      ).
      In addition to the relationship between lens membrane sphingolipid content and lifespan (Fig. 8), the sphingolipid content of many animals is directly related to lipid hydrocarbon order (Fig. 9). Because of higher lipid order, membranes may be less susceptible to oxidative damage because oxygen is five times more soluble in lipid membranes than it is in the aqueous around the membrane (
      • Power G.G.
      • Stegall H.
      Solubility of gases in human red blood cell ghosts.
      ,
      • Kimmich R.
      • Peters A.
      Solvation of oxygen in lecthin bilayers.
      ,
      • Peters A.
      • Kimmich R.
      The heterogeneous solubility of oxygen in aqueous lecithin dispersions and its relation to chain mobility.
      ,
      • Kimmich R.
      • Peters A.
      • Spohn K.H.
      Solubility of oxygen in lecithin bilayers and other hydrocarbon lamellae as a probe for free volume and transport properties.
      ,
      • Subczynski W.K.
      • Hyde J.S.
      Concentration of oxygen in lipid bilayers using a spin-label method.
      ,
      • Vanderkooi J.M.
      • Wright W.W.
      • Erecinska M.
      Oxygen gradients in mitochondria examined with delayed luminescence from excited-state triplet probes.
      ). In addition, oxygen is five to ten times more soluble in fluid membranes (
      • Smotkin E.S.
      • Moy F.T.
      • Plachy W.Z.
      Dioxygen solubility in aqueous phosphatidylcholine dispersions.
      ), such as membranes low in sphingolipids, than it is in the aqueous. It would be of value to test to determine whether organ-cultured rat lenses are more susceptible to oxidation and cataract compared with whale lenses.
      Figure thumbnail gr9
      Fig. 9Correlation between lens sphingolipid content and hydrocarbon chain order (stiffness) from (
      • Stimmelmayr R.
      • Borchman D.
      Lens lipidomes among Phocidae and Odobenidae.
      ). Black stars, pinniped lipid [data from (
      • Huang L.
      • Rasi V.
      • Grami V.
      • Marrero Y.
      • Borchman D.
      • Tang D.
      • Yappert M.C.
      Human lens phospholipid changes with age and cataract.
      ,
      • Borchman D.
      • Stimmelmayr R.
      • George J.C.
      Whales, lifespan, phospholipids and cataracts.
      ,
      • Borchman D.
      • Tang D.
      • Yapper M.C.
      Lipid composition, membrane structure relationships in lens and muscle sarcoplasmic reticulum membranes.
      ,
      • Broekhuyse R.M.
      • Kuhlmann E.D.
      • Jap P.H.K.
      Lens membranes. IX. Some characteristics of fiber membranes in relation to ageing and cataract formation.
      )]; filled squares, human lens nuclear lipid; open squares, human lens cortical lipids [from (
      • Pascolini D.
      • Mariotti S.P.
      Global estimates of visual impairment.
      ,
      • Micelli-Ferrari T.
      • Vendemiale G.
      • Grattagliano I.
      • Boscia F.
      • Arnese L.
      • Altomare E.
      • Cardia L.
      Role of lipid peroxidation in the pathogenesis of myopic and senile cataract.
      ,
      • Simonelli F.
      • Nesti A.
      • Pensa M.
      • Romano L.
      • Savastano S.
      • Rinaldi E.
      • Auricchio G.
      Lipid peroxidation and human cataractogenesis in diabetes and severe myopia.
      )]; open circles, lenses from various species [from (
      • Stimmelmayr R.
      • Borchman D.
      Lens lipidomes among Phocidae and Odobenidae.
      )].
      In vitro studies demonstrated that ordered lipids scatter more light than disordered lipids (
      • Tang D.
      • Borchman D.
      • Schwarz A.K.
      • Yappert M.C.
      • Vrensen G.F.J.M.
      • van Marle J.
      • DuPré D.B.
      Light scattering of human lens vesicles in vitro.
      ). With cataract, light scattering increases by 20%, and it is speculated that the increase is due to the increase in the lipid order of lens membranes (
      • Tang D.
      • Borchman D.
      • Schwarz A.K.
      • Yappert M.C.
      • Vrensen G.F.J.M.
      • van Marle J.
      • DuPré D.B.
      Light scattering of human lens vesicles in vitro.
      ). It is plausible that the increase in lipid-lipid interactions may contribute to myopia by causing greater compaction and overall stiffness of the lens. In addition, lipid order influences the activity of three lens proteins, the plasma membrane and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase activity, and aquaporin function, structure, quaternary assembly, and stability as reviewed in this journal (
      • Borchman D.
      • Yappert M.C.
      Lipids and the ocular lens.
      ).

      Factors other than hydrocarbon chain structure that relate to membrane function

      The levels of human lens sphingolipid and cholesterol are correlated (Fig. 10); however, cholesterol probably plays a minor role in determining lens membrane structure (
      • Borchman D.
      • Cenedella R.I.
      • Lamba O.P.
      Role of cholesterol in the structural order of lens lipids.
      ). It is likely to play a role in raft formation (
      • Rujoi M.
      • Jin J.
      • Borchman D.
      • Tang D.
      • Yappert M.C.
      Isolation and lipid characterization of cholesterol-enriched fractions in cortical and nuclear human lens fibers.
      ) and antagonize the binding of α-crystallin to lens membranes (
      • Tang D.
      • Borchman D.
      • Cenedella R.J.
      • Yappert M.C.
      Influence of cholesterol on the interaction of α-cystallin with phospholipid.
      ). It has been proposed that α-crystallin binding to lens membranes may serve as a “crystallization seed” for the binding of other proteins to the membrane, resulting in protein aggregation and light scattering (
      • Tang D.
      • Borchman D.
      • Cenedella R.J.
      • Yappert M.C.
      Influence of cholesterol on the interaction of α-cystallin with phospholipid.
      ). Thus, in addition to the inhibition of cataracts due to sphingolipids, cholesterol could inhibit protein aggregation and cataract. The relationship between cholesterol and cataract development time may also be important because cholesterol causes lens membranes to be less permeable to oxygen that may serve to keep oxygen in the outer regions of the lens long enough for the mitochondria to degrade it (
      • Subczynski W.K.
      • Hyde J.S.
      Concentration of oxygen in lipid bilayers using a spin-label method.
      ,
      • Plesnar E.
      • Szczelina R.
      • Subczynski W.K.
      • Pasenkiewicz-Gierula M.
      Is the cholesterol bilayer domain a barrier to oxygen transport into the eye lens?.
      ).
      Figure thumbnail gr10
      Fig. 10Relationship between the molar amounts of lens sphingolipid and cholesterol. Pinnipeds from (
      • Stimmelmayr R.
      • Borchman D.
      Lens lipidomes among Phocidae and Odobenidae.
      ) and bowhead whale (100% SL) from (
      • Borchman D.
      • Stimmelmayr R.
      • George J.C.
      Whales, lifespan, phospholipids and cataracts.
      ) (black stars). Calf lens cortex and nucleus and 2- to 6-year-old cow from (
      • Rozell N.
      Bowhead Whales May Be the World’s Oldest Mammals.
      ) and 1-year-old cow from (
      • Krause A.C.
      The chemistry of the lens: VI. Lipids.
      ) (open squares). Cow, sheep, human, rat, mouse, pig, and chicken from (
      • Deeley J.M.
      • Mitchell T.W.
      • Wei X.
      • Korth J.
      • Nealon J.R.
      • Blanksby S.J.
      • Truscott R.J.
      Human lens lipids differ markedly from those of commonly used experimental animals.
      ) (open circles). human lens from references (
      • Berger R.E.S.
      • Corrsin S.
      A surface tension gradient mechanism for driving the pre-corneal tear film after a blink.
      ,
      • George J.
      • Bada J.L.
      • Zeh J.
      • Scott L.
      • Brown S.E.
      • O’Hara T.
      • Suydam R.
      Age and growth estimates of bowhead whales (Balaena mysticetus) via aspartic acid racemization.
      ) (open triangles). Mice (10 and 45 days old) from (
      • Andrews J.S.
      • Leonard-Martin T.
      • Kador P.F.
      Membrane lipid biosynthesis in the Philly mouse lens. I. The major phospholipid classes.
      ) (open inverted triangles). Figure from (
      • Stimmelmayr R.
      • Borchman D.
      Lens lipidomes among Phocidae and Odobenidae.
      ).
      The human (
      • Byrdwell W.C.
      • Borchman D.
      Liquid chromatography/mass-spectrometric characterization of sphingomyelin and dihydrosphingomyelin of human lens membranes.
      ,
      • Kreps E.M.
      • Chirkovskaia E.V.
      • Pomazanskaia L.F.
      • Avrova N.F.
      • Levitina M.V.
      Issledovanie lipidov mozga mamonta Elephas primigenius, pogibshego bolee 40 tysiach let tomu nazad. Russian.
      ) and whale (
      • Borchman D.
      • Stimmelmayr R.
      • George J.C.
      Whales, lifespan, phospholipids and cataracts.
      ) lenses contain a higher amount of dihydrosphingolipid than any other organ that may inhibit lens growth by slowing the multiplication and elongation of lens cells (
      • Yappert M.C.
      • Rujoi M.
      • Borchman D.
      • Vorobyov I.
      • Estrada R.
      Glycero- versus sphingo-phospholipids: correlations with human and non-human mammalian lens growth.
      ). As there is no turnover of lipids (
      • Hughes J.R.
      • Levchenko V.A.
      • Blanksby S.J.
      • Mitchell T.W.
      • Williams A.
      • Truscott R.J.
      No turnover in lens lipids for the entire human lifespan.
      ) and proteins (
      • de Vries A.C.
      • Vermeer M.A.
      • Hendriks A.L.
      • Bloemendal H.
      • Cohen L.H.
      Biosynthetic capacity of the human lens upon aging.
      ) in the lens, slow lens growth is essential in longer-lived species so that the lens does not become too large.
      In summary, the long-lived species, such as humans and the bowhead whale, exhibit lens lipid adaptations that confer resistance to oxidation, thereby allowing the lens to stay clear for a relatively longer time than is the case in many other species.

      Relationships between tfll structure and function and the etiology of dry eye

      Age-related relationships between tear film stability and TFLL structure

      The measurement of tear breakup time (TBUT) and blink rates (BRs), both measures of tear film stability, and TFLL structural order (see STRUCTURE/CONFORMATION OF THE TFLL above) varies greatly from person to person, complicating the analysis of correlations between these parameters. None-the-less, generalities and trends can be surmised when large cohorts are examined.

      Tear stability with age

      TBUT decreases dramatically by 2.3% per year between 0.5 and 20 years of age (Fig. 11A). Between 21 and 50 years of age, TBUT decreases less dramatically by 1.03% per year, and then, above 50 years of age decreases by only 0.9% per year (Fig. 11A). Therefore, the major decline in tear film stability occurs between birth and 20 years of age.
      Figure thumbnail gr11
      Fig. 11Tear breakup and BR are a measure of tear stability. Human meibum structural order was assessed by quantifying hydrocarbon chain order measured in vitro using infrared spectroscopy and (Cs−1)max, reciprocal compressibility modulus, measured using Langmuir trough technology. A: Changes in TBUT with age. The first bar is from (
      • Isenberg S.J.
      • Del Signore M.
      • Guillon J.-P.
      • Chen A.
      • Wei J.
      The lipid layer and stability of the preocular tear film in newborns and infants.
      ); the last three bars are from (
      • Patel S.
      • Farrell J.C.
      Age-related changes in precorneal tear film stability.
      ). B: Changes in BR with age. The first bar is from (
      • Sledge S.
      • Henry C.
      • Borchman D.
      • Yappert M.C.
      • Bhola R.
      • Ramasubramanian A.
      • Blackburn R.
      • Austin J.
      • Massey K.
      • Sayied S.
      • et al.
      Human meibum age, lipid-lipid interactions and lipid saturation in meibum from infants.
      ,
      • Lawrenson J.G.
      • Birhah R.
      • Murphy P.J.
      Tear-film lipid layer morphology and corneal sensation in the development of blinking in neonates and infants.
      ); the middle two bars are from (
      • Sledge S.
      • Henry C.
      • Borchman D.
      • Yappert M.C.
      • Bhola R.
      • Ramasubramanian A.
      • Blackburn R.
      • Austin J.
      • Massey K.
      • Sayied S.
      • et al.
      Human meibum age, lipid-lipid interactions and lipid saturation in meibum from infants.
      ); the last bar is from (
      • Nosch D.S.
      • Pult H.
      • Albon J.
      • Purslow C.
      • Murphy P.J.
      Relationship between corneal sensation, blinking, and tear film quality.
      ). C: Relationship between tear film breakup time and BR from a cohort of 28 year olds (
      • Nosch D.S.
      • Pult H.
      • Albon J.
      • Purslow C.
      • Murphy P.J.
      Relationship between corneal sensation, blinking, and tear film quality.
      ). D: The first and third bars are from (
      • Mudgil P.
      • Borchman D.
      • Ramasubramanian A.
      Insights into tear film stability from babies and young adults; a study of human meibum lipid conformation and rheology.
      ); the second and last bars are from (
      • Ramasubramanian A.
      • Blackburn T.
      • Sledge S.M.
      • Yeo H.
      • Yappert M.C.
      • Gully Z.N.
      • Singh S.
      • Mehta S.
      • Mehta A.
      • Borchman D.
      Structural differences in meibum from donors after hematopoietic stem cell transplantations.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Tang D.
      • Ho D.V.
      Spectroscopic evaluation of human tear lipids.
      ,
      • Ramasubramanian A.
      • Borchman D.
      Structural differences in meibum from teenage donors with and without dry eye induced by allogeneic hematological stem cell transplantations.
      ). E: A larger reciprocal compressibility indicates a stiffer more elastic lipid layer. Filled circles and squares are from (
      • Mudgil P.
      • Ramasubramanian A.
      • Borchman D.
      Meibum lipid hydrocarbon chain branching and rheology after hematopoietic stem cell transplantation.
      ); filled triangles are from (
      • McCulley J.P.
      • Shine W.
      A compositional based model for the tear film lipid layer.
      ). Data are the average ± the standard error of the mean. The number of subjects are in parentheses.
      BRs may also be used as an indirect measure of tear film stability, as BR and TBUT were inversely correlated for a cohort with an average age of 28 years (Fig. 11C) (
      • Kimmich R.
      • Peters A.
      Solvation of oxygen in lecthin bilayers.
      ). Many factors contribute to BRs, such as dopaminergic activity and psychological and physiological conditions (
      • Nosch D.S.
      • Pult H.
      • Albon J.
      • Purslow C.
      • Murphy P.J.
      Relationship between corneal sensation, blinking, and tear film quality.
      ), but, in general, BRs reflect the level of tear film stability. Infants less than one-half of a year old blink as little as once every 2 min. The BR rises sharply between birth and 20 years of age, and then rises more gradually between 20 and 80 years of age (Fig. 11B). Therefore, like TBUT, the major increase in BR (decrease in stability) occurs between birth and 20 years of age. The slight decrease in TBUT above 50 years of age is not evident in the BR.

      Tear stability and structure

      The major change in tear film stability occurs between birth and 20 years of age (Fig. 11A, B). This age-related stability change correlates with a change in the stiffness or order of the TFLL estimated from the inverse of the in-plane elasticity modulus (Cs−1), also called the reciprocal compressibility modulus (Fig. 11E) (
      • Mudgil P.
      • Ramasubramanian A.
      • Borchman D.
      Meibum lipid hydrocarbon chain branching and rheology after hematopoietic stem cell transplantation.
      ). (Cs−1)max is measured using Langmuir trough technology. The (Cs−1)max for human meibum was measured in vitro using Langmuir trough technology and found to increase with age up to 50 years (Fig. 11E), indicating that with age, the TFLL becomes more elastic and stiff as tear film stability decreases (Fig. 11A, B) (
      • Nosch D.S.
      • Pult H.
      • Albon J.
      • Purslow C.
      • Murphy P.J.
      Relationship between corneal sensation, blinking, and tear film quality.
      ).
      Like (Cs−1)max measurements, meibum structural order measured using infrared spectroscopy also shows that meibum hydrocarbon chains become more ordered (stiff) between birth and 25 years of age (Fig. 11D) (
      • Borchman D.
      • Lamba O.P.
      • Yappert M.C.
      Structural characterization of lipid membranes from clear and cataractous human lenses.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Tang D.
      • Ho D.V.
      Spectroscopic evaluation of human tear lipids.
      ,
      • Ramasubramanian A.
      • Borchman D.
      Structural differences in meibum from teenage donors with and without dry eye induced by allogeneic hematological stem cell transplantations.
      ,
      • Mudgil P.
      • Borchman D.
      • Ramasubramanian A.
      Insights into tear film stability from babies and young adults; a study of human meibum lipid conformation and rheology.
      ). Individual meibum order measurements were similar to measurements from pooled meibum, in agreement with a previous study (
      • Borchman D.
      • Ramakrishnan V.
      • Henry C.
      Differences in meibum and tear lipid composition and conformation.
      ). Stiffer meibum lipid hydrocarbon chains with dry eye, discussed in the next section, are also associated with a decrease in tear film stability.
      One must note that changes in meibum hydrocarbon chain structural order measured using infrared spectroscopy are not always related to the elasticity or stiffness, (Cs−1)max, measured in rheological studies. For instance, meibum and tear lipid saturation causes an increase in both the (Cs−1)max of tear lipid (
      • Georgiev G.A.
      • Borchman D.
      • Eftimov P.
      • Yokoi N.
      Lipid saturation and the rheology of human tear lipids.
      ) and meibum (
      • Nencheva Y.
      • Ramasubramanian A.
      • Eftimov P.
      • Yokoi N.
      • Borchman D.
      • Georgiev G.
      Effects of lipid saturation on the surface properties of human meibum films.
      ) and the phase transition temperature of tear lipid (
      • Georgiev G.A.
      • Borchman D.
      • Eftimov P.
      • Yokoi N.
      Lipid saturation and the rheology of human tear lipids.
      ) and meibum (
      • Sledge S.
      • Henry C.
      • Borchman D.
      • Yappert M.C.
      • Bhola R.
      • Ramasubramanian A.
      • Blackburn R.
      • Austin J.
      • Massey K.
      • Sayied S.
      • et al.
      Human meibum age, lipid-lipid interactions and lipid saturation in meibum from infants.
      ,
      • Mudgil P.
      • Borchman D.
      • Yappert M.C.
      • Duran D.
      • Cox G.W.
      • Smith R.J.
      • Bhola R.
      • Dennis G.R.
      • Whitehall J.S.
      Lipid order, saturation and surface property relationships: A study of human meibum saturation.
      ). However, as the phase transition increases above 33.4°C, the lipid order at 33.4°C reaches a maximum value of about 80% ordered and does not change further with saturation. Thus, elasticity and meibum hydrocarbon chain order are not necessarily related when meibum structural order is high.
      Like stability measurements, meibum structural order varies greatly from person to person, and large sample sizes (greater than 10) are necessary to make meaningful correlations. For instance, it was pointed out that two of seven meibum samples, 3 and 4 years of age, had an order of 62% and 67%, two standard deviation units above samples of a similar age (
      • Mudgil P.
      • Borchman D.
      • Ramasubramanian A.
      Insights into tear film stability from babies and young adults; a study of human meibum lipid conformation and rheology.
      ). Thus, generalities made from a few samples should be made cautiously, and more measurements are needed to be certain of the structural order of meibum from babies and infants.
      Above 20 years of age, the slight 1% decrease per year in TBUT is not reflected in the BR that does not change above 20 years of age (Fig. 11A, B). Perhaps this is because the BR is associated with factors such as dopaminergic activity and psychological and physiological conditions, and TBUT is not (
      • Nosch D.S.
      • Pult H.
      • Albon J.
      • Purslow C.
      • Murphy P.J.
      Relationship between corneal sensation, blinking, and tear film quality.
      ). Paradoxically, the slight 0.9% decline with age in TBUT above 50 years of age is associated with a decrease in meibum hydrocarbon chain order, not with an increase in age, as observed between birth and 20 years of age and in dry eye, as discussed in the next section. Perhaps hydrocarbon structural order drives the large decrease in tear stability with age between birth and adulthood, and one may speculate that above 50 years of age, TFLL compositional and macromolecular structural changes drive the decrease in tear film stability with age that is associated with a decrease in hydrocarbon chain order. (Cs−1)max measurements on meibum from donors above 50 years old are needed to confirm and make further correlations between tear stability and TFLL elasticity and meibum structural order for individuals older than 50 years.
      In conclusion, the major decline in tear film stability between birth and 20 years of age measured by BRs and TBUT occurs concomitantly with an increase in elasticity and meibum hydrocarbon chain order measured by Langmuir trough technology and infrared spectroscopy, respectively. This correlation fits with the correlation between hydrocarbon chain order and dry eye discussed below. The relationships between elasticity and hydrocarbon structural order above 50 years of age is less clear. The functional consequences of a stiff TFLL and ordered meibum are discussed in the section below.

      Disease-related relationships between tear film stability and TFLL structure

      Tear film instability (Fig. 12A, B) is related to elevated hydrocarbon chain order (Fig. 12C) and elevated TFLL elasticity or stiffness (Fig. 12D) with dry eye. As with age (Fig. 11A), tear film stability measured by TBUT decreases with dry eye symptoms (Fig. 12B) (
      • Isenberg S.J.
      • Del Signore M.
      • Guillon J.-P.
      • Chen A.
      • Wei J.
      The lipid layer and stability of the preocular tear film in newborns and infants.
      ,
      • Patel S.
      • Farrell J.C.
      Age-related changes in precorneal tear film stability.
      ,
      • Vu C.H.V.
      • Kawashima M.
      • Yamada M.
      • Suwaki K.
      • Uchino M.
      • Shigeyasu C.
      • Hiratsuka Y.
      • Yokoi N.
      • Tsubota K.
      Dry Eye Cross-Sectional Study in Japan Study Group
      Influence of meibomian gland dysfunction and friction-related disease on the severity of dry eye.
      ). TBUT and dry eye have recently been reviewed (
      • Yokoi N.
      • Georgiev G.A.
      Tear-film-oriented diagnosis for dry eye.
      ). Similarly, as TBUT and BR are inversely related (Fig. 11C) (
      • Kimmich R.
      • Peters A.
      Solvation of oxygen in lecthin bilayers.
      ), tear film stability measured by BR also decreases with dry eye (Fig. 12A) (
      • Tsubota K.
      Tear dynamics and dry eye.
      ). Patients with stages 1 and 2 of Parkinson’s disease who were treated with dopamine agonist therapy and are susceptible to dry eye also have high BRs (Fig. 12A) (
      • Karson C.N.
      • LeWit P.A.
      • Caine D.B.
      • Wyatt R.J.
      Blink rates in Pakinsonism.
      ). As with aging (see the section above), hydrocarbon chain order is higher with dry eye due to Meibomian gland dysfunction (
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Bell J.
      • Wells E.
      • Neravetla S.
      • Greenstone V.
      Human meibum lipid conformation and thermodynamic changes with meibomian-gland dysfunction.
      ,
      • Borchman D.
      The optimum temperature for the heat therapy for meibomian gland dysfunction.
      ), dry eye in patients after hematopoietic stem cell transplantation (
      • Ramasubramanian A.
      • Blackburn T.
      • Sledge S.M.
      • Yeo H.
      • Yappert M.C.
      • Gully Z.N.
      • Singh S.
      • Mehta S.
      • Mehta A.
      • Borchman D.
      Structural differences in meibum from donors after hematopoietic stem cell transplantations.
      ,
      • Borchman D.
      The optimum temperature for the heat therapy for meibomian gland dysfunction.
      ), and dry eye associated with Parkinson’s disease (
      • Ramasubramanian A.
      • Borchman D.
      Structural differences in meibum from teenage donors with and without dry eye induced by allogeneic hematological stem cell transplantations.
      ) (Fig. 12C). The TFLL of meibum from patients with dry eye associated with hemopoietic stem cell transplantation was more elastic and stiff, having a higher (Cs−1)max (Fig. 12D) compared with normal age-matched controls, in agreement with hydrocarbon chain order measured using infrared spectroscopy (Fig. 12C). Thus, hydrocarbon chain order and meibum and TFLL stiffness were related to a decrease in tear film stability. Correlation does not necessitate cause, but the relationship between hydrocarbon chain order and tear film stability is intriguing. When tear film stability is restored with treatment, lipid order is also restored to normal levels (Fig. 12E) (
      • Foulks G.N.
      • Borchman D.
      • Yappert M.C.
      • Sung-Hye K.
      • McKay J.W.
      Topical azithromycin therapy of meibomian gland dysfunction: clinical response and lipid alterations.
      ), suggesting that the relationship between lipid order and tear film stability may be more than coincidental. It is reasonable to speculate that more ordered lipid could inhibit the flow of meibum from the Meibomian glands and contribute to the formation of a discontinuous patchy TFLL, which in turn results in deteriorated spreading and decreased surface elasticity (
      • Georgiev G.A.
      • Eftimov P.
      • Yokoi N.
      Structure-function relationship of tear film lipid layer: a contemporary perspective.
      ). Indeed, one of the earliest meibum surface film studies performed in 1969 demonstrated that more ordered meibum does not spread (
      • Brown S.I.
      • Dervichian D.G.
      The oils of the meibomian glands: physical and surface characteristics.
      ), and that ordered meibum has a high surface tension resulting in poor spreading (
      • Holly F.J.
      Formation and rupture of the tear film.
      ,
      • Butovich I.A.
      • Arciniega J.C.
      • Wojtowicz J.C.
      Meibomian lipid films and the impact of temperature.
      ). One may also speculate that more ordered lipid results in the attenuated capability to restore TFLL structure between blinks.
      Figure thumbnail gr12
      Fig. 12Structural functional relationships and dry eye. Tear breakup and BR are a measure of tear stability. Human meibum structural order was assessed by quantifying hydrocarbon chain order measured in vitro using infrared spectroscopy and (Cs−1)max, the reciprocal compressibility modulus, measured using Langmuir trough technology. A: Tear film stability measured by the BR. The first and last bars are from (
      • Tsubota K.
      Tear dynamics and dry eye.
      ); the middle bar is from (
      • Karson C.N.
      • LeWit P.A.
      • Caine D.B.
      • Wyatt R.J.
      Blink rates in Pakinsonism.
      ) and is for Parkinson’s patients at stages 1 and 2 receiving dopamine agonist therapy. B: Tear film stability measured by noninvasive TBUT (NTBUT). The first bar is from (
      • Isenberg S.J.
      • Del Signore M.
      • Guillon J.-P.
      • Chen A.
      • Wei J.
      The lipid layer and stability of the preocular tear film in newborns and infants.
      ); the second bar is from (
      • Patel S.
      • Farrell J.C.
      Age-related changes in precorneal tear film stability.
      ); the last two bars are from (
      • Vu C.H.V.
      • Kawashima M.
      • Yamada M.
      • Suwaki K.
      • Uchino M.
      • Shigeyasu C.
      • Hiratsuka Y.
      • Yokoi N.
      • Tsubota K.
      Dry Eye Cross-Sectional Study in Japan Study Group
      Influence of meibomian gland dysfunction and friction-related disease on the severity of dry eye.
      ). C: Hydrocarbon chain order, a measure of lipid stiffness using infrared spectroscopy. The first bar is from (
      • Ramasubramanian A.
      • Blackburn T.
      • Sledge S.M.
      • Yeo H.
      • Yappert M.C.
      • Gully Z.N.
      • Singh S.
      • Mehta S.
      • Mehta A.
      • Borchman D.
      Structural differences in meibum from donors after hematopoietic stem cell transplantations.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Tang D.
      • Ho D.V.
      Spectroscopic evaluation of human tear lipids.
      ,
      • Foulks G.N.
      • Borchman D.
      • Yappert M.C.
      • Sung-Hye K.
      • McKay J.W.
      Topical azithromycin therapy of meibomian gland dysfunction: clinical response and lipid alterations.
      ) for donors 68 ± 8 years old; the second bar is from (
      • Borchman D.
      The optimum temperature for the heat therapy for meibomian gland dysfunction.
      ,
      • Lawrenson J.G.
      • Birhah R.
      • Murphy P.J.
      Tear-film lipid layer morphology and corneal sensation in the development of blinking in neonates and infants.
      ) for donors 66 ± 6 years old; the third bar is from (
      • Singer S.J.
      • Nicolson G.L.
      The fluid mosaic model of the structure of cell membranes.
      ,
      • Mudgil P.
      • Borchman D.
      • Yappert M.C.
      • Duran D.
      • Cox G.W.
      • Smith R.J.
      • Bhola R.
      • Dennis G.R.
      • Whitehall J.S.
      Lipid order, saturation and surface property relationships: A study of human meibum saturation.
      ) for donors 54 ± 2 years old; the last bar is from (
      • Hunter M.
      • Bhola R.
      • Yappert M.C.
      • Borchman D.
      • Gerlach D.
      Pilot study of the influence of eyeliner cosmetics on the molecular structure of human meibum.
      ) for donors 66 ± 10 years old. D: Reciprocal compressibility modulus, a measure of TFLL elasticity or stiffness (
      • Mudgil P.
      • Ramasubramanian A.
      • Borchman D.
      Meibum lipid hydrocarbon chain branching and rheology after hematopoietic stem cell transplantation.
      ). E: A pilot study showing how when dry eye symptoms are ameliorated with treatment, lipid order is restored (
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      Confirmation of changes in human meibum lipid infrared spectra with age using principal component analysis.
      ). Data are the average ± the standard error of the mean. The number of subjects is in parentheses. MGD, Meibomian gland dysfunction; HSCT, dry eye associated with hematopoietic stem cell transplantation.
      A warm compress on the eyelid is one of the oldest successful therapies to treat dry eye (
      • Borchman D.
      The optimum temperature for the heat therapy for meibomian gland dysfunction.
      ). The phase transitional parameters of meibum lipids have been used to estimate the ideal temperature needed to fluidize meibum in the eyelid and yet be safe (
      • Borchman D.
      The optimum temperature for the heat therapy for meibomian gland dysfunction.
      ). For dry eye due to Meibomian gland dysfunction, heating the eye to 41.5°C safely fluidizes meibum by 90%. For dry eye due to hematopoietic stem cell transplantation, an unsafe temperature of 52°C is needed to fluidize meibum by 90%, suggesting that other therapies are needed (
      • Borchman D.
      The optimum temperature for the heat therapy for meibomian gland dysfunction.
      ).

      Animal model studies

      Most of the studies discussed in the current review involve the spectroscopy of human meibum. It is worth mentioning that, like the lens studies discussed above, insights into the etiology of dry eye can be gained from animal models that often cannot be obtained from humans. Animal models for dry eye have been reviewed (
      • Stern M.E.
      • Pflugfelder S.C.
      What we have learned from animal models of dry eye.
      ). Antioxidants have been tested in rabbits (
      • Bucolo C.
      • Fidilio A.
      • Platania C.B.M.
      • Geraci F.
      • Lazzara F.
      • Drago F.
      Antioxidant and osmoprotecting activity of taurine in dry eye models.
      ). Mucins (
      • Portal C.
      • Gouyer V.
      • Gottrand F.
      • Desseyn J.L.
      Ocular mucins in dry eye disease.
      ) and dendritic cells (
      • Maruoka S.
      • Inaba M.
      • Ogata N.
      Activation of dendritic cells in dry eye mouse model.
      ) have been studied, and a lacrimal gland excision model was developed in mice (
      • Shinomiya K.
      • Ueta M.
      • Kinoshita S.
      A new dry eye mouse model produced by exorbital and intraorbital lacrimal gland excision.
      ). TRP channels were explored (
      • Bereiter D.A.
      • Rahman M.
      • Thompson R.
      • Stephenson P.
      • Saito H.
      TRPV1 and TRPM8 channels and nocifensive behavior in a rat model for dry eye.
      ) and artificial tears have been tested in rats (
      • Ahn S.
      • Eom Y.
      • Kang B.
      • Park J.
      • Lee H.K.
      • Kim H.M.
      • Song J.S.
      Effects of menthol-containing artificial tears on tear stimulation and ocular surface integrity in normal and dry eye rat models.
      ). It has been suggested that a good animal model for human dry eye should have a similar meibum composition (
      • Butovich I.A.
      • Lu H.
      • McMahon A.
      • Eule J.C.
      Toward an animal model of the human tear film: biochemical comparison of the mouse, canine, rabbit, and human meibomian lipidomes.
      ), surface properties, and BRs (
      • Eftimov P.
      • Yokoi N.
      • Tonchev V.
      • Nencheva Y.
      • Georgiev G.A.
      Surface properties and exponential stress relaxations of mammalian meibum films.
      ,
      • Doughty M.J.
      Tear film stability and tear break up time (TBUT) in laboratory rabbits-a systematic review.
      ). Lipidomics (
      • Butovich I.A.
      • Lu H.
      • McMahon A.
      • Eule J.C.
      Toward an animal model of the human tear film: biochemical comparison of the mouse, canine, rabbit, and human meibomian lipidomes.
      ), Brewster angle spectroscopy, and surface rheology (
      • Eftimov P.
      • Yokoi N.
      • Tonchev V.
      • Nencheva Y.
      • Georgiev G.A.
      Surface properties and exponential stress relaxations of mammalian meibum films.
      ,
      • Doughty M.J.
      Tear film stability and tear break up time (TBUT) in laboratory rabbits-a systematic review.
      ) suggest that mice, cats, and canines meet that criterion. However, much can be learned from animal models with vastly different meibum compositions compared with humans. For instance, the tree shrew has extremely stable tears and blinks less than one time per minute (
      • Chen J.
      • Panthi S.
      Lipidomic analysis of meibomian gland secretions from the tree shrew: Identification of candidate tear lipids critical for reducing evaporation.
      ). Is the stability of the tree shrew’s tears due to the observed longer hydrocarbon chain lengths of the tree shrew compared with human meibum lipids (
      • Nicolaides N.
      • Santos E.C.
      The di- and tri-esters of the lipids of steer and human meibomian glands.
      )? Koalas have extremely stable tears and can go without blinking for over 10 min (
      • Leiske D.L.
      • Raju S.R.
      • Ketelson H.A.
      • Millar T.J.
      • Fuller G.G.
      The interfacial viscoelastic properties and structures of human and animal Meibomian lipids.
      ). Could the unusual surface properties and tear film thickness measured using Brewster angle spectroscopy (
      • Leiske D.L.
      • Raju S.R.
      • Ketelson H.A.
      • Millar T.J.
      • Fuller G.G.
      The interfacial viscoelastic properties and structures of human and animal Meibomian lipids.
      ) contribute to the stability of Koalas? Future spectroscopic studies could address the question: If humans had meibum with longer chain lengths, like the tree shrew, or the unique surface properties of koalas, could humans have more stable tears?

      Relationships between TFLL composition and structure

      Since the seminal studies of meibum composition in the 1970s (
      • Andrews J.S.
      Human tear film lipids: I. Composition of the principal non-polar component.
      ,
      • Tiffany J.M.
      Individual variations in human meibomian lipid composition.
      ) and ’80s (
      • Nicolaides N.
      • Santos E.C.
      The di- and tri-esters of the lipids of steer and human meibomian glands.
      ), many techniques have been applied to separate and quantify human tear lipid and meibum moieties such as: thin-layer chromatography, high-pressure liquid chromatography, gas chromatography, and many spectrometric techniques (
      • Lam S.M.
      • Tong L.
      • Duan X.
      • Petznick A.
      • Wenk M.R.
      • Shui G.
      Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles.
      ,
      • McCulley J.P.
      • Shine W.
      A compositional based model for the tear film lipid layer.
      ,
      • Andrews J.S.
      Human tear film lipids: I. Composition of the principal non-polar component.
      ,
      • Tiffany J.M.
      Individual variations in human meibomian lipid composition.
      ,
      • Nicolaides N.
      • Santos E.C.
      The di- and tri-esters of the lipids of steer and human meibomian glands.
      ,
      • Hancock S.E.
      • Ailuri R.
      • Marshall D.L.
      • Brown S.H.J.
      • Saville J.T.
      • Narreddula V.R.
      • Boase N.R.
      • Poad B.L.J.
      • Trevitt A.J.
      • Willcox M.D.P.
      • et al.
      Mass spectrometry-directed structure elucidation and total synthesis of ultra-long chain (O-acyl)-ω-hydroxy fatty acids.
      ,
      • Chen J.
      • Green K.B.
      • Nichols K.K.
      Characterization of wax esters by electrospray ionization tandem mass spectrometry: double bond effect and unusual product ions.
      ,
      • Butovich I.A.
      Fatty acid composition of cholesteryl esters of human meibomian gland secretions.
      ,
      • Butovich I.A.
      • Uchiyama E.
      • McCulley J.P.
      Lipids of human meibum: mass-spectrometric analysis and structural elucidation.
      ,
      • Lam S.M.
      • Tong L.
      • Reux B.
      • Lear M.J.
      • Wenk M.R.
      • Shui G.
      Rapid and sensitive profiling of tear wax ester species using high performance liquid chromatography coupled with tandem mass spectrometry.
      ,
      • Lam S.M.
      • Tong L.
      • Yong S.S.
      • Li B.
      • Chaurasia S.S.
      • Shui G.
      • Wenk M.R.
      Meibum lipid composition in Asians with dry eye disease.
      ,
      • Butovich I.A.
      • Uchiyama E.
      • Pascuale M.A.D.
      • McCulley J.P.
      Liquid chromatography–mass spectrometric analysis of lipids present in human meibomian gland secretions.
      ,
      • Butovich I.A.
      Cholesteryl esters as a depot for very long chain fatty acids in human meibum.
      ,
      • Butovich I.A.
      • Borowiak A.M.
      • Eule J.C.
      Comparative HPLC-MS analysis of canine and human meibomian lipidomes: many similarities, a few differences.
      ,
      • Butovich I.A.
      Tear film lipids.
      ,
      • Butovich I.A.
      • Arciniega J.C.
      • Lu H.
      • Molai M.
      Evaluation and quantitation of intact wax esters of human meibum by gas-liquid chromatography-ion trap mass spectrometry.
      ,
      • Hancock S.E.
      • Poad B.L.J.
      • Willcox M.D.P.
      • Blanksby S.J.
      • Mitchell T.W.
      Analytical separations for lipids in complex, nonpolar lipidomes using differential mobility spectrometry.
      ,
      • Butovich I.A.
      • Wojtowicz J.C.
      • Molai M.
      Human tear film and meibum. Very long chain wax esters and (O-acyl)-omegahydroxy fatty acids of meibum.
      ,
      • Nicolaides N.
      • Kaitaranta J.K.
      • Rawdah T.N.
      • Macy J.I.
      • Boswell III, F.M.
      • Smith R.
      Meibomian gland studies: comparison of steer and human lipids.
      ,
      • Nicolaides N.
      • Ruth E.C.
      Unusual fatty acids in the lipids of steer and human meibomian gland excreta.
      ,
      • Cory C.C.
      • Hinks W.
      • Burton J.L.
      • Shuster S.
      Meibomian gland secretion in the red eyes of rosacea.
      ,
      • Dougherty J.M.
      • Osgood J.K.
      • McCulley J.P.
      The role of wax and sterol ester fatty acids in chronic blepharitis.
      ,
      • Shine W.E.
      • McCulley J.P.
      The role of cholesterol in chronic blepharitis.
      ,
      • Shine W.E.
      • McCulley J.P.
      Meibomian gland triglyceride fatty acid differences in chronic blepharitis patients.
      ,
      • Shine W.E.
      • McCulley J.P.
      Polar lipids in human meibomian gland secretions.
      ,
      • Chen J.
      • Nichols K.K.
      • Wilson L.
      • Barnes S.
      • Nichols J.J.
      Untargeted lipidomic analysis of human tears: A new approach for quantification of O-acyl-omega hydroxy fatty acids.
      ,
      • Shine W.E.
      • McCulley J.P.
      Role of wax ester fatty alcohols in chronic blepharitis.
      ,
      • Harvey D.J.
      • Tiffany J.M.
      • Duerden J.M.
      • Pandher K.S.
      • Mengher L.S.
      Identification by combined gas chromatography-mass spectrometry of constituent long-chain fatty acids and alcohols from the meibomian glands of the rat and a comparison with human Meibomian lipids.
      ,
      • Joffre M.
      • Souchier C.
      • Grégoire S.
      • Viau S.
      • Bretillon L.
      • Acar N.
      • Bron A.M.
      • Creuzot-Garcher C.
      Differences in meibomian fatty acid composition in patients with meibomian-gland dysfunction and aqueous-deficient dry eye.
      ,
      • Nichols K.K.
      • Ham B.M.
      • Nichols J.J.
      • Ziegler C.
      • Green-Church K.B.
      Identification of fatty acids and fatty acid amides in human Meibomian gland secretions.
      ,
      • Saville J.T.
      • Zhao Z.
      • Willcox M.D.
      • Ariyavidana M.A.
      • Blanksby S.J.
      • Mitchell T.W.
      Identification of phospholipids in human meibum by nano-electrospray ionisation tandem mass spectrometry.
      ,
      • Chen J.
      • Green-Church K.B.
      • Nichols K.K.
      Shotgun lipidomic analysis of human meibomian gland secretions with electrospray ionization tandem mass spectrometry.
      ,
      • Butovich I.A.
      On the lipid composition of human meibum and tears: comparative analysis of nonpolar lipids.
      ,
      • Butovich I.A.
      Lipidomic analysis of human meibum using HPLCMSn.
      ,
      • Chen J.
      • Nichols K.K.
      Comprehensive shotgun lipidomics of human meibomian gland secretions using MS/MS(all) with successive switching between acquisition polarity modes.
      ,
      • Brown S.H.
      • Kunnen C.M.
      • Duchoslav E.
      • Dolla N.K.
      • Kelso M.J.
      • Papas E.B.
      • de la Jara P.L.
      • Willcox M.D.
      • Blanksby S.J.
      • Mitchell T.W.
      A comparison of patient matched meibum and tear lipidomes.
      ,
      • Rantamäki A.H.
      • Seppanen-Laakso T.
      • Oresic M.
      • Jauhiainen M.
      • Holopainen J.M.
      Human tear fluid lipidome: From composition to function.
      ,
      • Wollensak G.
      • Mur E.
      • Mayr A.
      • Baier G.
      • Gottinger W.
      • Stoffler G.
      Effective methods for the investigation of human tear film proteins and lipids.
      ,
      • Nagyová B.
      • Tiffany J.M.
      Components responsible for the surface tension of human tears.
      ,
      • Nakayasu E.S.
      • Nicora C.D.
      • Sims A.C.
      • Burnum-Johnson K.E.
      • Kim Y.M.
      • Kyle J.E.
      • Matzke M.M.
      • Shukla A.K.
      • Chu R.K.
      • Schepmoes A.A.
      • et al.
      MPLEx: a robust and universal protocol for single-sample integrative proteomic, metabolomic, and lipidomic analyses.
      ,
      • Dean A.W.
      • Glasgow B.J.
      Mass spectrometric identification of phospholipids in human tears and tear lipocalin.
      ,
      • Acera A.
      • Pereiro X.
      • Abad-Garcia B.
      • Rueda Y.
      • Ruzafa N.
      • Santiago C.
      • Barbolla I.
      • Duran J.A.
      • Ochoa B.
      • Vecino E.
      A simple and reproducible method for quantification of human tear lipids with ultrahigh-performance liquid chromatography-mass spectrometry.
      ,
      • Chen J.
      • Green K.B.
      • Nichols K.K.
      Quantitative profiling of major neutral lipid classes in human meibum by direct infusion electrospray ionization mass spectrometry.
      ). The focus of the current review is on relationships between meibum and TFLL composition and structure using spectroscopic techniques such as Raman, infrared, and NMR spectroscopies. Spectrometric and other lipidomic techniques have been reviewed and were not reviewed in this article (
      • Green-Church K.B.
      • Butovich I.
      • Willcox M.
      • Borchman D.
      • Paulsen F.
      • Barabino S.
      • Glasgow B.
      The International Workshop on Meibomian Gland Disfunction: report of the Subcommittee on Tear Film Lipids and Lipid-Protein Interactions in Health and Disease.
      ,
      • Pucker A.D.
      • Nichols J.J.
      Analysis of meibum and tear lipids.
      ,
      • Butovich I.A.
      • Millar T.J.
      • Ham B.M.
      Understanding and analyzing meibomian lipids–a review.
      ,
      • Knop E.
      • Knop N.
      • Millar T.
      • Obata H.
      • Sullivan D.A.
      The International Workshop on Meibomian Gland Dysfunction: report of the Subcommittee on Anatomy, Physiology, and Pathophysiology of the Meibomian Gland.
      ,
      • Murube J.
      The origin of tears. III. The lipid component in the XIX and XX centuries.
      ,
      • Georgiev G.A.
      • Eftimov P.
      • Yokoi N.
      Structure-function relationship of tear film lipid layer: a contemporary perspective.
      ,
      • Foulks G.N.
      • Borchman D.
      Meibomian gland dysfunction: the past, the present, the future.
      ,
      • Foulks G.N.
      The correlation between the tear film lipid layer and dry eye disease.
      ,
      • Butovich I.A.
      Meibomian glands, meibum, and meibogenesis.
      ,
      • Borchman D.
      From bench to bedside: infrared spectroscopy and the diagnosis and treatment of dry eye and cataracts.
      ). The relationships between meibum and TFLL composition and structure are less certain than the relationships between structure and function discussed in the previous sections. The interaction of the many lipid moieties contributes to molecular structure. For instance, as discussed in the sections below, unsaturation is a major factor that contributes to hydrocarbon chain order, yet 5 mol% saturated and ordered cholesterol ester can increase the phase transition temperature of completely disordered and unsaturated WE by an extraordinary 63°C (
      • Hetman Z.A.
      • Borchman D.
      Concentration dependent cholesteryl-ester and wax-ester structural relationships and meibomian gland dysfunction.
      ). None-the-less, insights can be obtained from the spectroscopic studies discussed below. Specifically, the current review will focus on saturation, CE/WE content, hydrocarbon chain branching, and protein and squalene content and their potential contribution to meibum and TFLL structure.

      Advantages and disadvantages of using a spectroscopic approach to measure meibum and tear lipid composition

      In addition to providing hydrocarbon chain conformational and structural information as discussed in the section above, infrared, NMR fluorescence, and Raman spectroscopies have been applied to the study meibum and tear lipid composition (
      • Ivanova S.
      • Borchman D.
      • Yappert M.C.
      • Tonchev V.
      • Yokoi N.
      • Georgiev G.
      Surface properties of squalene/meibum films and NMR confirmation of squalene in tears.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      Confirmation of changes in human meibum lipid infrared spectra with age using principal component analysis.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      Changes in human meibum lipid with meibomian gland dysfunction using principal component analysis.
      ,
      • Borchman D.
      • Ramakrishnan V.
      • Henry C.
      Differences in meibum and tear lipid composition and conformation.
      ,
      • Oshima Y.
      • Sato H.
      • Zaghloul A.
      • Foulks G.N.
      • Yappert M.C.
      • Borchman D.
      Characterization of human meibum lipid using Raman spectroscopy.
      ,
      • McCulley J.P.
      • Shine W.
      A compositional based model for the tear film lipid layer.
      ,
      • Borchman D.
      • Ramasubramanian A.
      • Foulks G.N.
      Human meibum cholesteryl and wax ester variability with age, gender and Meibomian gland dysfunction.
      ,
      • Butovich I.A.
      • Arciniega J.C.
      • Lu H.
      • Molai M.
      Evaluation and quantitation of intact wax esters of human meibum by gas-liquid chromatography-ion trap mass spectrometry.
      ,
      • Robosky L.C.
      • Wade K.
      • Woolson D.
      • Baker J.D.
      • Manning M.L.
      • Gage D.A.
      • Reily M.D.
      Quantitative evaluation of sebum lipid components with nuclear magnetic resonance.
      ,
      • Borchman D.
      • Yappert M.C.
      • Milliner S.
      • Duran D.
      • Cox G.W.
      • Smith R.J.
      • Bhola R.
      13C and 1H NMR ester region resonance assignments and the composition of human infant and child meibum.
      ,
      • Borchman D.
      • Ramasubramanian A.
      Human meibum chain branching variability with age, gender and meibomian gland dysfunction.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Milliner S.E.
      Differences in human meibum lipid composition with meibomian gland dysfunction using NMR and principal component analysis.
      ,
      • Shrestha R.K.
      • Borchman D.
      • Foulks N.
      • Yappert M.C.
      Analysis of the composition of lipid in human meibum from normal infants, children, adolescents, adults and adults with meibomian gland dysfunction using 1H-NMR spectroscopy.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Milliner S.E.
      Changes in human meibum lipid composition with age using NMR spectroscopy.
      ,
      • Paugh J.R.
      • Alfonso-Garcia A.
      • Nguyen A.L.
      • Suhalim J.L.
      • Farid M.
      • Garg S.
      • Tao J.
      • Brown D.J.
      • Potma E.O.
      • Jester J.V.
      Characterization of expressed human meibum using hyperspectral stimulated Raman scattering microscopy.
      ,
      • Alfonso-García A.
      • Paugh J.
      • Farid M.
      • Garg S.
      • Jester J.V.
      • Potma E.O.
      A machine learning framework to analyze hyperspectral stimulated Raman scattering microscopy images of expressed human meibum.
      ,
      • Filik J.
      • Stone N.
      Analysis of human tear fluid by Raman spectroscopy.
      ,
      • Suhalim J.L.
      • Parfitt G.J.
      • Xie Y.
      • De Paiva C.S.
      • Pflugfelder S.C.
      • Shah T.N.
      • Potma E.O.
      • Brown D.J.
      • Jester J.V.
      Effect of desiccating stress on mouse meibomian gland function.
      ) and as a diagnostic tool (
      • Foulks G.N.
      • Borchman D.
      • Yappert M.C.
      • Sung-Hye K.
      • McKay J.W.
      Topical azithromycin therapy of meibomian gland dysfunction: clinical response and lipid alterations.
      ,
      • Foulks G.N.
      • Borchman D.
      • Yappert M.C.
      • Kakar S.
      Topical azithromycin and oral doxycycline therapy of meibomian gland dysfunction: a comparative clinical and spectroscopic pilot study.
      ,
      • Borchman D.
      • Foulks G.N.
      • Yappert M.C.
      • Milliner S.E.
      Differences in human meibum lipid composition with meibomian gland dysfunction using NMR and principal component analysis.
      ,
      • Kuo M.T.
      • Lin C.C.
      • Liu H.Y.
      • Chang H.C.
      Tear analytical model based on Raman microspectroscopy for investigation of infectious diseases of the ocular surface.
      ,
      • Lin C.Y.
      • Suhalim J.L.
      • Nien C.L.
      • Miljković M.D.
      • Diem M.
      • Jester J.V.
      • Potma E.O.
      Picosecond spectral coherent anti-Stokes Raman scattering imaging with principal component analysis of meibomian glands.
      ,
      • Ong B.L.
      • Hodson S.A.
      • Wigham T.
      • Miller F.
      • Larke J.R.
      Evidence for keratin proteins in normal and abnormal human meibomian fluids.
      ). Raman spectroscopic studies indicate that meibum lipids are modified in the central duct, suggesting postprocessing of the lipid within the ductal region of the gland (
      • Paugh J.R.
      • Alfonso-Garcia A.
      • Nguyen A.L.
      • Suhalim J.L.
      • Farid M.
      • Garg S.
      • Tao J.
      • Brown D.J.
      • Potma E.O.
      • Jester J.V.
      Characterization of expressed human meibum using hyperspectral stimulated Raman scattering microscopy.
      ,
      • Alfonso-García A.
      • Paugh J.
      • Farid M.
      • Garg S.
      • Jester J.V.
      • Potma E.O.
      A machine learning framework to analyze hyperspectral stimulated Raman scattering microscopy images of expressed human meibum.