May 2003
Volume 44, Issue 5
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Cornea  |   May 2003
Kinetic Analysis of Tear Interference Images in Aqueous Tear Deficiency Dry Eye before and after Punctal Occlusion
Author Affiliations
  • Eiki Goto
    From the Ocular Surface Center and Ocular Surface Research and Education Foundation, Miami, Florida.
  • Scheffer C. G. Tseng
    From the Ocular Surface Center and Ocular Surface Research and Education Foundation, Miami, Florida.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1897-1905. doi:10.1167/iovs.02-0818
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      Eiki Goto, Scheffer C. G. Tseng; Kinetic Analysis of Tear Interference Images in Aqueous Tear Deficiency Dry Eye before and after Punctal Occlusion. Invest. Ophthalmol. Vis. Sci. 2003;44(5):1897-1905. doi: 10.1167/iovs.02-0818.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Kinetic analysis of sequential tear interference images was used to investigate how the precorneal lipid film spread and distributed in aqueous tear deficiency (ATD) dry eye.

methods. One eye of 17 patients with ATD was randomly selected for this noncomparative case series. Twelve patients also had noninflamed meibomian gland dysfunction (MGD). Sequential images were digitized and analyzed on computer. Data were further compared in 9 of the 17 cases before and after punctal occlusion (PO). Outcome measures included speed and pattern of lipid spread and resultant lipid layer thickness in the superior, central, and inferior cornea. Intensity and red/green/blue (RGB) color spectra of the tear interference image were compared before and after PO.

results. After lid blinking, it took a longer time (2.2 ± 1.1 second) to reach a stable lipid film in all eyes with ATD compared with normal subjects (P < 0.0001). Because of this retarded spread, the thickest lipid film was located at the inferior cornea adjacent to the lid margin, with a gradient spreading toward the superior cornea (P = 0.01). As a result, the lipid film was thinner than normal on the superior cornea in 10 of 17 (59%) ATD eyes. Fifteen of 17 eyes (88%) showed vertical streaking, rather than a normal horizontal propagation pattern on the superior cornea. Such a lipid-deficient state and uneven distribution did not correlate with the presence or absence of MGD. The lipid spread time was shortened (P = 0.008), the distribution of the lipid film was more even, and the thickness approached normal in all nine eyes after PO.

conclusions. In this study, kinetic analysis of tear interference images provided evidence that retardation of lipid spread is, but MGD is not, the main reason for the increased thickness of precorneal lipid film in the inferior cornea of eyes with ATD. As a result, lipid film is deficient in the superior cornea and unevenly distributed, further destabilizing the tear film. The fact that PO significantly improves lipid spread, evenness, and thickness suggests that the performance of lipid film is also dictated by the amount of aqueous tear fluid. These findings provide new insight into the interaction between the lipid film and the aqueous tear fluid.

Ocular surface health is maintained by a stable preocular tear film, which is made of lipids (primarily derived from meibum excreted from meibomian glands), aqueous fluid (consisting of proteins, electrolytes, water secreted mainly by lacrimal glands), and mucins (secreted by ocular surface epithelial cells, including conjunctival goblet cells). 1 After a complete blink these tear components are spread over the entire ocular surface to form a stable tear film, which often breaks up, leaving dry spots before the next blink. During the interblink interval, a stable tear film ensures ocular comfort, clear vision, and effective defense against microbial infections. An unstable tear film is the hallmark of many dry eye states. 2  
One requirement for maintaining a stable tear film is that a sufficient amount of meibum lipids must spread rapidly into a thin film with appropriate thickness and even distribution. Such a lipid film helps stabilize the tear film by lowering the air-fluid surface tension and preventing aqueous tear evaporation. 3 Tear interferometry is a noninvasive method used by many to visualize and evaluate the tear lipid layer. 4 5 6 7 8 9 10 11 12 13 14 15 16 17  
It is well known that the formation of a lipid film requires lid-blinking, which is a kinetic event. Therefore, we believe that it is important to retrieve tear interference images timed with the onset of blinking, and that a random, non-time-controlled single image of tear interference taken before may not represent what exactly happens during the interblink interval. That is why we developed kinetic analysis and discovered dramatic differences between eyes of patients with lipid tear deficiency (LTD) dry eye and eyes of normal subjects. 18 Our results indicate that the normal lipid film spreads rapidly in a horizontally propagating wave pattern, whereas that of LTD slowly spreads in a vertically streaking pattern (P < 0.0001, χ2 test). Also, the normal lipid film spreads and produces a stable image within 0.36 ± 0.22 second, whereas that of LTD produces a stable image in 3.54 ± 1.86 seconds (P = 0.0003, Mann-Whitney test). Subsequent images of the normal lipid film show that it remains evenly distributed, with an average thickness of 79.1 ± 13.0 nm, whereas those of LTD show that the film remains unstable and uneven with an average thickness of 53.8 ± 20.0 nm (P = 0.02 Mann-Whitney test). 
Several studies have indicated that the precorneal tear lipid layer in dry eye shows different appearance from that of normal eyes. 19 20 21 22 23 Danjo and Hamano 8 reported that the thickness increases with the intensity of vital staining in aqueous tear deficient (ATD) dry eye with Sjögren syndrome. Yokoi et al. 9 reported that the thickness of the tear lipid film increases with severity of ATD. One explanation for such a thicker lipid film in eyes with ATD may be the increased production of meibum lipids. Yokoi et al. 24 reported that the meibum lipid level measured by meibometry is increased in the lid margin reservoir of women with ATD, speculating that this is caused by the compression of a thicker lipid film left on the lid margin. 
In this study, we used kinetic analysis of interference images to provide strong evidence supporting the hypothesis that the thicker lipid found in eyes with ATD is not due to increased meibum production but results from retardation of lipid spread, which leads to uneven distribution of lipid film and deficient lipid film on the superior cornea. The significance of this finding is further discussed. 
Methods
Patients and Study Design
This was a comparative case series based on a protocol approved by the Ocular Surface Research and Education Foundation and was conducted in accordance with the tenets of the 1975 Declaration of Helsinki. After written informed consent was obtained, 17 consecutive patients with ATD (5 men and 12 women, aged 52.1 ± 16.2 years) were enrolled (Table 1) . Because both eyes of each subject were similar in symptoms and findings, one eye was randomly chosen for analysis. The symptoms included dryness, foreign body sensation, pain, burning, blurred vision, itching, redness, and ocular fatigue, all of which were worse in the afternoon. The diagnosis of ATD was based on the fluorescein clearance test (FCT) using Schirmer strips, as previously reported, with anesthesia. 25 All 17 patients had low aqueous tear secretion as evidenced by a low wetting length of 1.1 ± 0.60 mm for 1 minute of testing. Patient 1 received a diagnosis of Sjögren syndrome, according to the established criteria. 26 Patients 11 and 12 had additional delayed tear clearance, and patients 4, 8, 9, and 12 did not show reflex tearing. 
The intensity of the rose bengal and fluorescein staining of the cornea and conjunctiva was 0.8 ± 1.2 and 1.0 ± 1.6, respectively, using a scale previously described. 27 28 Tear break-up time (TBUT)—that is, the average of three measurements using the fluorescein strip wet with nonpreserved saline—was 3.3 ± 1.6 seconds (Table 1) . Criteria for the diagnosis of noninflamed MGD included the presence of meibomian gland dropout by transillumination through the tarsus, poor meibum expression in response to digital pressure, and the lack of active inflammation. 29 30 31 Based on these criteria, we detected noninflamed MGD in 12 patients (71%, Table 1 ). 
All patients had not undergone punctal occlusion (PO), were not contact lens wearers, and did not have blepharospasm or abnormal blinking at the time of enrollment. After the baseline evaluation, patients were treated with topical medications, such as nonpreserved artificial tear eye drops hourly and preservative-free methylprednisolone eye drops three times a day for a course of 3 weeks. Nine of 17 patients remained symptomatic and subsequently underwent punctal occlusion with plug or cauterization in the lower punctum of each eye. Repeated tear interference images were taken 1 day to 52 weeks after PO. 
Instrument Setup
In an examination room set at the same light intensity (350 lux), humidity (45.2%–54.0%), and temperature (21.0–22.7°C), we used the same instrument setup as previously reported. 18 In short, an ophthalmoscope (DR-1; Kowa, Inc., Nagoya, Japan) was set at a magnification of 12×, which allowed observation of an area of the cornea 8 mm in diameter. The video output was linked with a frame grabber (FlashBus MV Lite; Integral Technologies, Indianapolis, IN) and digitized as uncompressed audio-video interleaved (AVI) format using image-management software (ImagePro 4.1; Media Cybernetics, Silver Spring, MD). The frame rate was set at 5.18 frames per second (the sequential frames spanned 0.193-second intervals) and recording was performed for 29 seconds in one session, which generated 150 frames (131-megabyte video file). In a preliminary study, we had compared the data randomly chosen among three separate sets of blinks and noted that there were no significant differences among them (P = 0.4, Wilcoxon’s matched-pairs signed rank test). Therefore, we present herein data obtained from one representative blink, which started with a complete eyelid blink and its interblink time (IBT). These sequential video images were then extracted as uncompressed tag image file format (TIFF) file, which could be made into a thumbnail composite and subjected to subsequent image analysis. 
Kinetic Analysis of Interference Images
Lipid Spread Time and Pattern of Spread.
The time interval starting from time 0 to the time of the frame that first showed a stable interference image was defined as lipid spread time, as we have reported previously. 18 The first stable image was determined by playing the images frame by frame on a liquid crystal display (LCD) screen, to see whether there was any noticeable movement between frames. When there was no noticeable movement, we defined it as the first stable image. This measurement was conducted by two nonmasked observers, and the longer time of the two was chosen in the event of a disagreement. Throughout their analyses there was complete agreement between the two observers 65% of the time. Among the 35% in which there was a discrepancy, the difference was limited to two frames in 80% of the cases and to three frames in 20%. If the image did not achieve a stable pattern throughout the entire IBT, the entire IBT was used to calculate the spread time. 
The spread pattern was recorded as horizontally propagating, vertically streaking, or mixed when judged by three masked observers, as previously reported. 18  
Distribution of the Lipid Film Thickness.
Using the first stable frame taken from the sequential images, we analyzed four spots along the vertical meridian in an 8-mm diameter image: spot A, located at 2 mm above the center; spot B at the center; spot C, 2 mm below the center; and spot D, 4 mm below the center (Fig. 1A) . Each spot consisted of 14 pixels, from which an average of image intensity was obtained by the imaging software (Fig. 1A , for spots A and D; depicted in the horizontal axis). Using the look-up simulated color chart (LUT) showing the reflectance of thin film interference generated by a white light source 32 (Fig. 1B) and the red/green/blue (RGB) spectra of that spot by the image software (Fig. 1C 1D 1E 1F) , which helped determine the interference order, we translated the average intensity value into the thin film thickness (see legend of Fig. 1 for an example). The differences among the thicknesses measured at spots A, B, C, and D were used to quantify the distribution, or evenness, of the spread of lipid film. To demonstrate the changes in the distribution of thickness before and after PO, we also measured the RGB color spectra in the superior and inferior corneas, and compared these spectra before and after PO. 
Statistical Analysis
Data were collected from prospectively completed data forms. The comparison of the spread time between all 17 patients with ATD and 11 normal subjects previously reported 18 was performed by Mann-Whitney test. The comparison of categorical scales between 12 patients with ATD with MGD and 5 patients without MGD was performed by χ2 test and that of the interval scales by Mann-Whitney test. The comparison of categorical scales between nine patients with ATD before and after PO was performed by χ2 test and that of interval scales by Wilcoxon matched-pairs signed rank test. To analyze the distribution of the lipid thickness, we applied the Kruskal-Wallis test. The statistical tests were performed on computer (Instat 3.0; GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered statistically significant. 
Results
Figure 2 shows sequential digitized interference images of the eye of a representative patient with ATD (case 15) before (Fig. 2A) and after PO (Fig. 2B) . Each figure includes 16 consecutive frames encompassing a period of 2.9 seconds (i.e., the frame interval of 0.19 second). The first frame marked by the asterisk is the last frame of the blink time (BT), and the second frame is the first frame of IBT, set as time 0 (marked by 0). The frame marked with X is the first stable image when all subsequent images were similar from then on until the next blink. The spread time, defined by the interval necessary for the lipid film to reach a stable interference image, was 2.51 seconds (Fig. 2A) , and was shortened to 0.58 second after PO (Fig. 2B)
When each frame of the above composite was carefully analyzed in all patients with ATD, we observed that the lipid film spread upward from the lower lid margin after a blink. The average spread time in all eyes with ATD was 2.17 ± 1.09 seconds (Table 2) , which was significantly slower than the 0.36 ± 0.22 second of the normal eyes previously reported 18 (P < 0.0001). The spread time in the 12 eyes with ATD with MGD was 2.00 ± 1.00 seconds, which was not significantly different from the 2.55 ± 1.28 seconds in the 5 eyes without MGD (P = 0.1; Table 3 ). The spread time after PO in nine eyes was 0.79 ± 0.51 second, which was significantly shorter than the pre-PO time of 2.34 ± 1.13 seconds (P = 0.008; Table 4 ). 
Because of the slower spread in eyes with ATD, the resultant lipid film was found to be thicker on the inferior cornea than the superior cornea. This uneven distribution was clearly demonstrated when four different spots along the vertical meridian (i.e., 12 o’clock to 6 o’clock) were compared in each eye. As shown in Table 2 , the thicknesses at spots A, B, C, and D were 74.1 ± 50.0, 84.7 ± 60.8, 105 ± 85.0, and 150 ± 83.6 nm, respectively, in all eyes. These thicknesses reflected an uneven distribution of the lipid film along this axis (P = 0.01; the significance of the difference between spots A and D was P < 0.01). Such an uneven distribution of the lipid film thickness was noted in 12 eyes with ATD with MGD as well as in 5 eyes with ATD without MGD (Tables 1 3) . Nevertheless, the distribution of the lipid film became more uniform after PO, because the thicknesses at spots A, B, C, and D became 80.0 ± 17.3, 77.8 ± 19.9, 62.2 ± 17.2, and 87.8 ± 35.3 nm, respectively (Table 4 ; P = 0.2; compare Fig. 3A with Fig. 3B ). To illustrate these dramatic changes further, we measured the RGB color spectra in superior and inferior corneas and compared these spectra before and after PO. As shown in Figure 4 , the distribution of RGB spectra was wide before PO (Fig. 4 , left column), but became narrower after PO (Fig. 4 , right column). Furthermore, there was a difference in the spectra distribution between superior and inferior corneas before PO (Fig. 4 , left), whereas such a difference was not present after PO (Fig. 4 , right). 
Based on the tear interference image taken at the time when a stable image was first reached, we recognized three groups among these 17 patients. As shown in Figure 3A and Table 2 , group A included cases 1 to 10, in which the superior cornea was predominantly covered by a thin lipid film in a color of dark gray, indicating the average thickness of 50.0 ± 11.5 nm. They all had a vertical streaking pattern in the superior cornea. In eyes in cases 1 and 2, the same dark gray color extended to the entire cornea, resulting in a pattern not different from that previously defined as the LTD pattern. 18 In cases 3 to 10, the interference image of the inferior cornea showed a color of white-brown to blue or green, indicating the presence of a thicker lipid, of which the average thickness was measured to be 138 ± 52.3 nm. Besides more rapid spread, the resultant lipid film became thicker in the superior cornea, and the entire lipid film became more uniform in cases 1, 3, 6, and 7 of group A after PO (Fig. 3B) . The eye in case 3 is an example of the color change in the lipid film from dark gray (60 nm) to white (90 nm) in the superior cornea after PO (Table 4)
Group B consisted of cases 11 to 13, in which the lipid film was thicker on the superior cornea than that in group A, giving rise to a color of bright gray to white, indicating the average thickness of 76.7 ± 20.8 nm. Such a thickness was within the normal range (79.1 ± 13.0 nm). 18 However, a vertical streaking pattern, which was not noted in normal eyes, 18 was clearly observed in this region (Fig. 3A) . Group C included cases 14 to 17, in which the lipid film on the superior cornea became more colorful, yielding an average thickness of 133 ± 78.5 nm, thicker than normal. Even if the lipid thickness was increased in group C, a vertical streaking pattern was still visible on the superior cornea in three of four cases (cases 14, 16, and 17). After PO, besides the aforementioned changes of thickness and distribution, we also noted that the vertical streaking pattern on the superior cornea was changed to a horizontally propagating pattern, similar to that in normal eyes, 18 in three of nine cases (cases 3, 15, and 16). 
Discussion
Using the kinetic analysis of tear interference images, our results confirmed that the lipid film of eyes with ATD was in general thicker than that of normal subjects (Table 2) . This finding is consistent with those previously reported by Danjo and Hamano, 8 who used a modified specular microscope, and by Yokoi et al. 8 using the equipment we used (DR-1; Kowa). It should be noted that both Danjo and Hamano and Yokoi et al. reported a thicker lipid film in the central cornea. Our studies, however, showed that the thickest lipid film was actually located at the inferior cornea close to the lower lid margin and that there was a gradient of a thicker lipid film of the inferior cornea toward a thinner lipid film of the superior cornea. Such a gradient was not only noted in the frame taken when a stable image was first reached (Fig. 3A) , but was also seen in sequential images taken during the entire IBT (Fig. 2A) . In contrast to these two earlier reports, 8 9 in which a thicker lipid film was invariably detected in eyes with ATD, we noted that some (cases 1–6) actually showed marked lipid tear deficiency (discussed further later). Furthermore, unlike their findings in which a non-time-controlled still image was randomly taken, the kinetic analysis allowed us to examine all images within the entire IBT. As a result, we have discovered that the reason a thicker lipid film develops in eyes of patients with ATD is because of the retardation of lipid spread. 
The first evidence supporting our assertion was derived from the measurement of the lipid spread time, defined by the interval before a stable image is first reached. Unlike a rather short lipid spread time of 0.36 ± 0.22 second reported in normal eyes, 18 all 17 eyes with ATD had a significantly slower spread time of 2.17 ± 1.09 seconds (P < 0.0001). Due to the retarded lipid spread, a lipid film with a gradient of decreasing thickness was generated from the inferior cornea to the superior cornea, and an uneven distribution resulted. This was demonstrated qualitatively by interference images taken at the time when a stable image was reached (Fig. 3A) , as well as quantitatively by measuring the thickness at the four different spots (Table 2 , Fig. 1 ). The uneven distribution of the lipid film also resulted in nonuniformity of the entire lipid distribution, which was best illustrated quantitatively by the distribution of RGB spectra in superior and inferior corneas (Fig. 4 , middle and bottom left). 
Based on the frame taken when a stable image was first reached, we noted heterogeneity in the lipid film thickness among the three groups in these 17 patients with ATD (Fig. 3A , Table 2 ). Group A had a lipid film thinner than normal (i.e., dark gray color on the superior cornea). This pattern was also observed by Danjo and Hamano 8 and Mathers et al. 10 Nevertheless, groups B and C had a lipid film on the superior cornea similar to or thicker than normal. Unlike the normal lipid film, which spreads in a horizontal propagating wave pattern, 18 nearly all except two eyes (cases 15 and 16) showed a vertical streaking pattern on the superior cornea where the lipid film was deficient. Danjo and Hamano 8 noted a similar vertical pattern (though not described as such) in eyes of 49% of their patients with Sjögren syndrome. We have previously ascribed such a vertical streaking pattern as one of the major characteristics of the lipid film of LTD eyes. 18 Therefore, we speculate that uneven distribution of the lipid film may further destabilize the tear film, and that the vertical streaking, which reflects mechanical movement of eyelid upward excursion, may traumatize the ocular surface in patients with ATD. If this interpretation were correct, we believe that future ATD therapy should also include the restoration of the lipid film to achieve effective results. 
The aforementioned gradient of lipid film distribution and the heterogeneity of lipid film thickness did not correlate with the presence or absence of MGD (Table 3) , supporting that the thicker lipid film noted in eyes of patients with ATD is not caused by increasing meibum production. Although there was a tendency suggesting the increasing severity of ATD from group A to (combined) groups B and C with respect to positive staining and absence of reflex tearing, this trend did not reach a statistical significance. In group A, the patient in case 1 had Sjögren syndrome and the one in case 6 had positive superficial punctate keratopathy on the inferior cornea, where a thick lipid was also present, with black granules (Fig. 3A) . The latter finding resembled the “oil droplets” described by Danjo and Hamano. 8 Future studies with a larger sample size will help resolve whether kinetic analysis of tear interference images may also help better correlate the severity of ATD. 
The second line of evidence supporting that a thicker lipid in ATD is caused by retardation of lipid spread is the comparison of thickness data before and after PO. As summarized in Table 4 , the lipid spread time was significantly shortened in nine patients with ATD after PO. As a result, the pattern of spread changed from vertical streaking to horizontal wave patterns, the distribution became much more uniform and even, and the overall thickness approached normal (Fig. 3B) . It should also be noted that such a dramatic change could occur as early as 1 day and last for as long as 52 weeks, so long as the volume of the aqueous tear fluid was increased by PO. Because the same improvement of tear interference by PO was observed in patients with ATD, with or without MGD, and in both group A and groups B and C, we further believe that the most important element that affects the lipid film spread time and distribution in patients with ATD is the amount of the aqueous tear fluid. 
Because PO could make such a dramatic improvement in the quality of the lipid film, even in patients with severe lipid tear deficiency (group A), we also concur with the findings of Yokoi et al. 24 that meibum lipids are actually not absent in patients with ATD and could have been stagnated at the lid margin. If this interpretation is accurate, we also speculate that increasing the amount of aqueous tear fluid is important for effective lipid spread in patients with ATD. An intriguing finding was that in groups B and C, PO also resulted in the return to a much thicker lipid film, rather close to normal (Fig. 3B) . This finding prompts us to speculate that heterogeneity in the severity of ATD or in the production of various lipid species, especially polar versus nonpolar lipids, may influence lipid spread. Future studies are needed to delineate the exact mechanism by which lipid spread is retarded when the aqueous tear fluid is deficient in ATD. 
 
Table 1.
 
Profile of Patients with ATD Dry Eye
Table 1.
 
Profile of Patients with ATD Dry Eye
Case Age (years) Sex Symptom Fluo (0–9) RB (0–9) TBUT (sec) Gland Dropout (0–2) Meibum Expression (0–3) Wetting Length (mm) DTC Reflex Tearing
Group A
1 41 F Dryness, pain 5 3 2 2 3 0 No +
2 27 M Burning, dryness 0 0 1 0 2 2 No +
3 65 F Dryness, foreign body 1 0 6 0 1 1 No +
4 64 M Blurred vision 0 1 3 1 3 1 No
5 57 F Dryness 0 0 1 1 2 2 No +
6 50 M Dryness 5 4 3 0 1 1 No +
7 30 F Dryness 0 0 6 0 0 1 No +
8 77 F Pain 2 2 5 1 2 2 No
9 55 F Dryness, FBS 1 1 5 1 3 1 No
10 40 F Ocular fatigue 1 0 3 1 2 2 No +
Group B
11 78 F Discomfort, dryness 0 2* 3 1 3 1 Yes +
12 62 F DES 0 0 1 1 3 1 Yes
13 58 F Foreign body 1 0 3 1 2 1 No +
Group C
14 35 M Dryness 0 0 3 0 0 1 No +
15 29 F Burning, itching 0 0 5 1 3 1 No +
16 66 M Burning, redness 0 0 4 1 3 1 No +
17 51 F Pain 1 1 2 0 1 0 No +
Mean±SD 52.1 ± 16.2 1.0 ± 1.6 0.8 ± 1.2 3.3 ± 1.6 0.71 ± 0.59 2.0 ± 1.1 1.1 ± 0.6
Figure 1.
 
Method of measuring lipid film thickness at different spots. (A) In this representative image (case 11, Table 2 ) spot A (2 mm superior to the cornea center; pixel position 320, 120), spot B (at the center; pixel position 320, 240), spot C (2 mm below the center; pixel position 320, 360), and spot D (almost the lowest possible spot, pixel position 320, 475) are presented. The intensity histograms of spots A and D are shown in (C) and (E), respectively, and the RGB intensity is shown in (D) and (F), respectively. Spot A exhibited a gray dominant color with the intensity of 159, red 179 ± 4.3 SD, green 155 ± 3.6, and blue 144 ± 3.62. This gives rise to the interference order of 1/4 < m < 1/2 (based on the interference simulated color chart shown in (B) and translates to 60 nm of thickness. Spot D exhibited a blue-green dominant color with the intensity of 150, red 133 ± 9.28, green 157 ± 6.6, and blue 161 ± 8.2. This gives rise to the interference order of 1 < m < 3/2, and translates to 240 nm of thickness. The interference simulated color chart demonstrated in (B) was reproduced, with permission, from the King-Smith et al., Three interferometric methods for measuring the thickness of layers of the tear film. Optom Vis Sci. 1999;76:19–32. 227 Lippincott Williams & Wilkins.
Figure 1.
 
Method of measuring lipid film thickness at different spots. (A) In this representative image (case 11, Table 2 ) spot A (2 mm superior to the cornea center; pixel position 320, 120), spot B (at the center; pixel position 320, 240), spot C (2 mm below the center; pixel position 320, 360), and spot D (almost the lowest possible spot, pixel position 320, 475) are presented. The intensity histograms of spots A and D are shown in (C) and (E), respectively, and the RGB intensity is shown in (D) and (F), respectively. Spot A exhibited a gray dominant color with the intensity of 159, red 179 ± 4.3 SD, green 155 ± 3.6, and blue 144 ± 3.62. This gives rise to the interference order of 1/4 < m < 1/2 (based on the interference simulated color chart shown in (B) and translates to 60 nm of thickness. Spot D exhibited a blue-green dominant color with the intensity of 150, red 133 ± 9.28, green 157 ± 6.6, and blue 161 ± 8.2. This gives rise to the interference order of 1 < m < 3/2, and translates to 240 nm of thickness. The interference simulated color chart demonstrated in (B) was reproduced, with permission, from the King-Smith et al., Three interferometric methods for measuring the thickness of layers of the tear film. Optom Vis Sci. 1999;76:19–32. 227 Lippincott Williams & Wilkins.
Figure 2.
 
Representative sequential images of an ATD-affected eye before (A) and after (B) PO. A representative sequential image of an ATD eye (case 15, Table 2 ) showed a slow spread in an irregular horizontal pattern. Minor differences in sequential interference images were checked by playing the video frame by frame. The spread started at time 0 and reached stability at frame X, and the spread time was measured to be 2.51 seconds before a stable image was obtained (A). After PO, the spread time was shortened to 0.58 second in a horizontal pattern, with more uniform distribution (B). (✶) Last frame before IBT.
Figure 2.
 
Representative sequential images of an ATD-affected eye before (A) and after (B) PO. A representative sequential image of an ATD eye (case 15, Table 2 ) showed a slow spread in an irregular horizontal pattern. Minor differences in sequential interference images were checked by playing the video frame by frame. The spread started at time 0 and reached stability at frame X, and the spread time was measured to be 2.51 seconds before a stable image was obtained (A). After PO, the spread time was shortened to 0.58 second in a horizontal pattern, with more uniform distribution (B). (✶) Last frame before IBT.
Table 2.
 
Summary of Kinetic Analyses of Tear Interference Image in Patients with ATD
Table 2.
 
Summary of Kinetic Analyses of Tear Interference Image in Patients with ATD
Case Pattern of Spread Spread Time (sec) Thickness (nm)
A B C D
Group A
1 * * 40 30 30 30
2 V 4.2, † 60 60 30 20
3 V 4.8, † 60 50 60 130
4 V 1.5 60 70 50 100
5 V 3.5, † 50 40 40 170
6 V 1.9 40 40 30 90
7 M 1.7 60 60 100 170
8 M 1.4 40 60 60 100
9 M 1.4 30 60 40 240
10 M 1.4 60 90 100 100
Group B
11 M 1.5 60 90 120 240
12 V 1.5 100 110 120 120
13 M 1.2 70 70 100 110
Group C
14 M 2.1 90 100 110 120
15 H 2.5 100 110 240 240
16 M 1.9 90 100 240 240
17 M 2.1 250 300 320 330
Mean±SD 2.2 ± 1.1 74.2 ± 50.0 84.7 ± 60.8 105 ± 85.0 150 ± 83.6
Table 3.
 
Summary of Kinetic Analysis of Tear Interference Image in ATD Eyes, with and without MGD
Table 3.
 
Summary of Kinetic Analysis of Tear Interference Image in ATD Eyes, with and without MGD
Pattern of Spread Spread Time (sec) Thickness (nm)
A B C D
With MGD V (n = 4)*
M (n = 6) 2.0 ± 1.0 74.2 ± 26.1
H (n = 1) 63.3 ± 23.1 97.5 ± 74.5 143 ± 81.7
Without MGD V (n = 2) 2.5 ± 1.3 110 ± 109
M (n = 3) 100 ± 85.7 124 ± 114 168 ± 95.0
P 0.8 0.1 0.6 0.9 0.7 0.6
Table 4.
 
Summary of Kinetic Analysis of Tear Interference Images in Nine Patients with ATD before and after Punctal Occlusion
Table 4.
 
Summary of Kinetic Analysis of Tear Interference Images in Nine Patients with ATD before and after Punctal Occlusion
Pattern of Spread Spread Time (sec) Thickness (nm)
A B C D
Pre-PO V (n = 3)*
M (n = 4) 2.2 ± 1.1 96.7 ± 81.9
H (n = 1) 90.0 ± 64.2 138 ± 104 162 ± 92.6
Post-PO V (n = 1)
M (n = 4) 0.8 ± 0.5 77.8 ± 19.9
H (n = 3) 80.0 ± 17.3 62.2 ± 17.2 87.8 ± 35.3
P 0.4 0.008 >0.99 0.95 0.04 0.1
Figure 3.
 
Representative static images of all 17 ATD-affected eyes before PO (A) and of nine after PO (B). (A) Representative image of each ATD eye (with case numbers at lower left) selected when a stable image was reached (Table 2) . Eyes in group A (cases 1–10) showed dark gray coloration with a vertical streaking pattern on the superior cornea. This lipid deficiency pattern was observed on the entire cornea in cases 1 and 2. The remaining eight eyes showed a thicker lipid on the inferior cornea. Eyes in group B (cases 11–13) showed normal coloration and thickness on the superior cornea but with a vertical pattern. Eyes in group C (cases 14–17) showed a colorful interference image on the entire cornea. (B) Similar representative images of ATD eyes after PO (corresponds to cases shown in Table 2 ). In general, the lipid film became more uniform after PO.
Figure 3.
 
Representative static images of all 17 ATD-affected eyes before PO (A) and of nine after PO (B). (A) Representative image of each ATD eye (with case numbers at lower left) selected when a stable image was reached (Table 2) . Eyes in group A (cases 1–10) showed dark gray coloration with a vertical streaking pattern on the superior cornea. This lipid deficiency pattern was observed on the entire cornea in cases 1 and 2. The remaining eight eyes showed a thicker lipid on the inferior cornea. Eyes in group B (cases 11–13) showed normal coloration and thickness on the superior cornea but with a vertical pattern. Eyes in group C (cases 14–17) showed a colorful interference image on the entire cornea. (B) Similar representative images of ATD eyes after PO (corresponds to cases shown in Table 2 ). In general, the lipid film became more uniform after PO.
Figure 4.
 
Color spectra analysis between superior and inferior cornea before and after PO. RGB intensity was measured in superior (S) and inferior (I) zones and plotted before (left, Pre-PO) and after (right, Post-PO) PO (case 16). The overall RGB distribution was wider before than after PO, indicating the presence of a more colorful interference image. In contrast, the RGB distribution became narrower after PO.
Figure 4.
 
Color spectra analysis between superior and inferior cornea before and after PO. RGB intensity was measured in superior (S) and inferior (I) zones and plotted before (left, Pre-PO) and after (right, Post-PO) PO (case 16). The overall RGB distribution was wider before than after PO, indicating the presence of a more colorful interference image. In contrast, the RGB distribution became narrower after PO.
The authors thank Edgar M. España, MD, (Ocular Surface Center, Miami, FL) for acting as a masked observer to measure the lipid spread pattern. 
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Figure 1.
 
Method of measuring lipid film thickness at different spots. (A) In this representative image (case 11, Table 2 ) spot A (2 mm superior to the cornea center; pixel position 320, 120), spot B (at the center; pixel position 320, 240), spot C (2 mm below the center; pixel position 320, 360), and spot D (almost the lowest possible spot, pixel position 320, 475) are presented. The intensity histograms of spots A and D are shown in (C) and (E), respectively, and the RGB intensity is shown in (D) and (F), respectively. Spot A exhibited a gray dominant color with the intensity of 159, red 179 ± 4.3 SD, green 155 ± 3.6, and blue 144 ± 3.62. This gives rise to the interference order of 1/4 < m < 1/2 (based on the interference simulated color chart shown in (B) and translates to 60 nm of thickness. Spot D exhibited a blue-green dominant color with the intensity of 150, red 133 ± 9.28, green 157 ± 6.6, and blue 161 ± 8.2. This gives rise to the interference order of 1 < m < 3/2, and translates to 240 nm of thickness. The interference simulated color chart demonstrated in (B) was reproduced, with permission, from the King-Smith et al., Three interferometric methods for measuring the thickness of layers of the tear film. Optom Vis Sci. 1999;76:19–32. 227 Lippincott Williams & Wilkins.
Figure 1.
 
Method of measuring lipid film thickness at different spots. (A) In this representative image (case 11, Table 2 ) spot A (2 mm superior to the cornea center; pixel position 320, 120), spot B (at the center; pixel position 320, 240), spot C (2 mm below the center; pixel position 320, 360), and spot D (almost the lowest possible spot, pixel position 320, 475) are presented. The intensity histograms of spots A and D are shown in (C) and (E), respectively, and the RGB intensity is shown in (D) and (F), respectively. Spot A exhibited a gray dominant color with the intensity of 159, red 179 ± 4.3 SD, green 155 ± 3.6, and blue 144 ± 3.62. This gives rise to the interference order of 1/4 < m < 1/2 (based on the interference simulated color chart shown in (B) and translates to 60 nm of thickness. Spot D exhibited a blue-green dominant color with the intensity of 150, red 133 ± 9.28, green 157 ± 6.6, and blue 161 ± 8.2. This gives rise to the interference order of 1 < m < 3/2, and translates to 240 nm of thickness. The interference simulated color chart demonstrated in (B) was reproduced, with permission, from the King-Smith et al., Three interferometric methods for measuring the thickness of layers of the tear film. Optom Vis Sci. 1999;76:19–32. 227 Lippincott Williams & Wilkins.
Figure 2.
 
Representative sequential images of an ATD-affected eye before (A) and after (B) PO. A representative sequential image of an ATD eye (case 15, Table 2 ) showed a slow spread in an irregular horizontal pattern. Minor differences in sequential interference images were checked by playing the video frame by frame. The spread started at time 0 and reached stability at frame X, and the spread time was measured to be 2.51 seconds before a stable image was obtained (A). After PO, the spread time was shortened to 0.58 second in a horizontal pattern, with more uniform distribution (B). (✶) Last frame before IBT.
Figure 2.
 
Representative sequential images of an ATD-affected eye before (A) and after (B) PO. A representative sequential image of an ATD eye (case 15, Table 2 ) showed a slow spread in an irregular horizontal pattern. Minor differences in sequential interference images were checked by playing the video frame by frame. The spread started at time 0 and reached stability at frame X, and the spread time was measured to be 2.51 seconds before a stable image was obtained (A). After PO, the spread time was shortened to 0.58 second in a horizontal pattern, with more uniform distribution (B). (✶) Last frame before IBT.
Figure 3.
 
Representative static images of all 17 ATD-affected eyes before PO (A) and of nine after PO (B). (A) Representative image of each ATD eye (with case numbers at lower left) selected when a stable image was reached (Table 2) . Eyes in group A (cases 1–10) showed dark gray coloration with a vertical streaking pattern on the superior cornea. This lipid deficiency pattern was observed on the entire cornea in cases 1 and 2. The remaining eight eyes showed a thicker lipid on the inferior cornea. Eyes in group B (cases 11–13) showed normal coloration and thickness on the superior cornea but with a vertical pattern. Eyes in group C (cases 14–17) showed a colorful interference image on the entire cornea. (B) Similar representative images of ATD eyes after PO (corresponds to cases shown in Table 2 ). In general, the lipid film became more uniform after PO.
Figure 3.
 
Representative static images of all 17 ATD-affected eyes before PO (A) and of nine after PO (B). (A) Representative image of each ATD eye (with case numbers at lower left) selected when a stable image was reached (Table 2) . Eyes in group A (cases 1–10) showed dark gray coloration with a vertical streaking pattern on the superior cornea. This lipid deficiency pattern was observed on the entire cornea in cases 1 and 2. The remaining eight eyes showed a thicker lipid on the inferior cornea. Eyes in group B (cases 11–13) showed normal coloration and thickness on the superior cornea but with a vertical pattern. Eyes in group C (cases 14–17) showed a colorful interference image on the entire cornea. (B) Similar representative images of ATD eyes after PO (corresponds to cases shown in Table 2 ). In general, the lipid film became more uniform after PO.
Figure 4.
 
Color spectra analysis between superior and inferior cornea before and after PO. RGB intensity was measured in superior (S) and inferior (I) zones and plotted before (left, Pre-PO) and after (right, Post-PO) PO (case 16). The overall RGB distribution was wider before than after PO, indicating the presence of a more colorful interference image. In contrast, the RGB distribution became narrower after PO.
Figure 4.
 
Color spectra analysis between superior and inferior cornea before and after PO. RGB intensity was measured in superior (S) and inferior (I) zones and plotted before (left, Pre-PO) and after (right, Post-PO) PO (case 16). The overall RGB distribution was wider before than after PO, indicating the presence of a more colorful interference image. In contrast, the RGB distribution became narrower after PO.
Table 1.
 
Profile of Patients with ATD Dry Eye
Table 1.
 
Profile of Patients with ATD Dry Eye
Case Age (years) Sex Symptom Fluo (0–9) RB (0–9) TBUT (sec) Gland Dropout (0–2) Meibum Expression (0–3) Wetting Length (mm) DTC Reflex Tearing
Group A
1 41 F Dryness, pain 5 3 2 2 3 0 No +
2 27 M Burning, dryness 0 0 1 0 2 2 No +
3 65 F Dryness, foreign body 1 0 6 0 1 1 No +
4 64 M Blurred vision 0 1 3 1 3 1 No
5 57 F Dryness 0 0 1 1 2 2 No +
6 50 M Dryness 5 4 3 0 1 1 No +
7 30 F Dryness 0 0 6 0 0 1 No +
8 77 F Pain 2 2 5 1 2 2 No
9 55 F Dryness, FBS 1 1 5 1 3 1 No
10 40 F Ocular fatigue 1 0 3 1 2 2 No +
Group B
11 78 F Discomfort, dryness 0 2* 3 1 3 1 Yes +
12 62 F DES 0 0 1 1 3 1 Yes
13 58 F Foreign body 1 0 3 1 2 1 No +
Group C
14 35 M Dryness 0 0 3 0 0 1 No +
15 29 F Burning, itching 0 0 5 1 3 1 No +
16 66 M Burning, redness 0 0 4 1 3 1 No +
17 51 F Pain 1 1 2 0 1 0 No +
Mean±SD 52.1 ± 16.2 1.0 ± 1.6 0.8 ± 1.2 3.3 ± 1.6 0.71 ± 0.59 2.0 ± 1.1 1.1 ± 0.6
Table 2.
 
Summary of Kinetic Analyses of Tear Interference Image in Patients with ATD
Table 2.
 
Summary of Kinetic Analyses of Tear Interference Image in Patients with ATD
Case Pattern of Spread Spread Time (sec) Thickness (nm)
A B C D
Group A
1 * * 40 30 30 30
2 V 4.2, † 60 60 30 20
3 V 4.8, † 60 50 60 130
4 V 1.5 60 70 50 100
5 V 3.5, † 50 40 40 170
6 V 1.9 40 40 30 90
7 M 1.7 60 60 100 170
8 M 1.4 40 60 60 100
9 M 1.4 30 60 40 240
10 M 1.4 60 90 100 100
Group B
11 M 1.5 60 90 120 240
12 V 1.5 100 110 120 120
13 M 1.2 70 70 100 110
Group C
14 M 2.1 90 100 110 120
15 H 2.5 100 110 240 240
16 M 1.9 90 100 240 240
17 M 2.1 250 300 320 330
Mean±SD 2.2 ± 1.1 74.2 ± 50.0 84.7 ± 60.8 105 ± 85.0 150 ± 83.6
Table 3.
 
Summary of Kinetic Analysis of Tear Interference Image in ATD Eyes, with and without MGD
Table 3.
 
Summary of Kinetic Analysis of Tear Interference Image in ATD Eyes, with and without MGD
Pattern of Spread Spread Time (sec) Thickness (nm)
A B C D
With MGD V (n = 4)*
M (n = 6) 2.0 ± 1.0 74.2 ± 26.1
H (n = 1) 63.3 ± 23.1 97.5 ± 74.5 143 ± 81.7
Without MGD V (n = 2) 2.5 ± 1.3 110 ± 109
M (n = 3) 100 ± 85.7 124 ± 114 168 ± 95.0
P 0.8 0.1 0.6 0.9 0.7 0.6
Table 4.
 
Summary of Kinetic Analysis of Tear Interference Images in Nine Patients with ATD before and after Punctal Occlusion
Table 4.
 
Summary of Kinetic Analysis of Tear Interference Images in Nine Patients with ATD before and after Punctal Occlusion
Pattern of Spread Spread Time (sec) Thickness (nm)
A B C D
Pre-PO V (n = 3)*
M (n = 4) 2.2 ± 1.1 96.7 ± 81.9
H (n = 1) 90.0 ± 64.2 138 ± 104 162 ± 92.6
Post-PO V (n = 1)
M (n = 4) 0.8 ± 0.5 77.8 ± 19.9
H (n = 3) 80.0 ± 17.3 62.2 ± 17.2 87.8 ± 35.3
P 0.4 0.008 >0.99 0.95 0.04 0.1
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