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Raman point mapping of tear ferning patterns

Article in Proceedings of SPIE - The International Society for Optical Engineering · March 2008
DOI: 10.1117/12.786441

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 Raman Point Mapping of Tear Ferning Patterns
Jacob Filik and Nicholas Stone*
Biophotonics Research, Gloucester Royal Hospital, Great Western Road, Gloucester, UK GL1 3NN

ABSTRACT

This study attempts to combine the tear ferning test and the drop coating deposition Raman spectroscopy (DCDRS)
technique to analyze the biochemical composition of human tear fluid from healthy volunteers. DCDRS has been shown
to be a highly reproducible and sensitive method of obtaining Raman spectra from low concentration protein solutions
making it ideal for the analysis of tear fluid. On drying, tear samples were found to produce ring-shaped patterns, which
are characteristic of the DCDRS technique, with additional fern-like structures produced inside the rings. The
biochemical composition of the each drying pattern was studied by Raman point mapping and principal components
analysis. Assignment of high-signal-to-noise tear spectra showed that tear proteins, urea, bicarbonate and lipid
components were all present in the dried tear drop. Comparing an image time series of the drying process with the
biochemical distributions from the Raman point map revealed the order of biochemical deposition in the drying pattern.
The combination of DCDRS and the tear ferning test shows enough promise to be further studied as a near-patient
technique for assisting the diagnosis of ocular infection, but further work is required to validate the technique.
Keywords: Raman, Tears, Lacrimal , Drop Coating Deposition, DCDRS, Tear Fern, mucus Fern.

1. INTRODUCTION

Tear fluid is a complex mixture of proteins, lipids, metabolites and electrolytes.1 A change in the concentration of these
components can impair the ability of tear fluid to perform its key functions and is often an important indication of the
onset of disease. The main functions of tear fluid are to create a smooth surface for light to pass through, keep the eye
moist and lubricated, provide nutrients, remove waste and particulates, and to protect the eye from injury and infection.1
It is the complex composition of tear fluid that allows it to simultaneously perform all these functions.
While spread across the ocular surface, the tear film is composed of three layers; an upper lipid layer, a middle aqueous
layer and a lower mucin layer.1 The lipid layer is secreted by the meibomian glands at the rim of the eyelid. This
secretion (meibum) is a complex fluid containing hydrocarbons, wax esters, triglycerides, diesters, free sterols, sterol
esters, free fatty acids, and polar lipids.2 The main functions of this layer are to slow the evaporation rate of the aqueous
layer, and provide an airtight seal when the eyelids close. The aqueous layer contains many of the defensive proteins and
antibodies which protect the eye from infection.1 Most of these proteins are secreted by the lacrimal gland but a certain
proportion enters the tear fluid by serum leakage. The relative proportions of lacrimal to serum proteins in an individual
sample of tear fluid depend on how invasive the collection method is. More invasive methods stimulate more serum
leakage and hence the tear fluid has a greater serum protein concentration .1 The mucous layer is secreted by
conjunctival goblet cells and enhances the spread and stability of the aqueous layer, lubricates and protects the ocular
surface.1
When deposited onto a glass surface, tear fluid dries to form characteristic ferning patterns.3 These mucus, or tear, ferns
are thought to arise from the balance of macromolecules and inorganic salts in the tear fluid, although it has been
suggested that macromolecules (proteins/mucins) are not actually present in the fern pattern.4 The specific integrity,
uniformity and branching of these ferning patterns have been suggested as an inexpensive, quick and simple test to
estimate Keratoconjunctivitis sicca in patients with Sjögren's syndrome.3
*n.stone@medical-research-centre.com

Biomedical Optical Spectroscopy, edited by Anita Mahadevan-Jansen,

Wolfgang Petrich, Robert R. Alfano, Alvin Katz, Proc. of SPIE Vol. 6853,
685309, (2008) · 1605-7422/08/$18 · doi: 10.1117/12.786441

Proc. of SPIE Vol. 6853 685309-1

2008 SPIE Digital Library -- Subscriber Archive Copy
The tear ferning test method is very similar to the drop coating deposition method that is frequently used to improve the
Raman signal from weak protein solutions. In drop coating deposition Raman spectroscopy (DCDRS), a weak, aqueous,
protein solution is spotted onto a hydrophobic substrate and the water is allowed to evaporate.5 The water predominately
evaporates at the edge of the drop causing a net flow of solution from the centre of the drop to the edge. This flow carries
solute material to the periphery, which leads to formation of a ring of excess material after all the solvent has
evaporated.6 This method preconcentrates the protein allowing spectra to be obtained from solutions that would
previously have been too weak to analyze. The sensitivity of this technique can be optimized by careful choice of
substrate. Ideal substrates should have low optical absorbance, high optical reflectance, little or no background signal
and, most importantly, have a non-wetting interaction with the analyte solution.5
The aim of this study is to derive an analytical method combining the tear-ferning test and DCDRS technique. The main
objectives are three fold; 1) observe the order of the formation of the ferning pattern during drying, 2) perform Raman
point mapping over the drying pattern, and 3) relate the biochemistry within the tear to the observed structures in the
drying pattern.

2. MATERIALS AND METHODS

Three volunteers were recruited for tear collection; all were non contact lens wearers who had been free from ocular
infection for at least three years. Informed consent was obtained from each volunteer after fully explaining all
procedures. This study was approved by the ethics subcommittee of the trust, and all procedures involving human
subjects complied with the Declaration of Helsinki.
A fine flame-polished capillary tube (0.5 mm I.D., 1 mm O.D, 7.5 mm length) was used to collect the non-reflex, basal
tear fluid. The subject’s lower eyelid was gently pulled down and the tip of the tube was placed in contact with the tear
meniscus, with care being taken not to touch the conjunctiva. A minimum of 2 µl was collected from the left eye of each
volunteer. Immediately after collection a 1.5 µl aliquot was taken from the original sample for analysis with DCDRS.
CaF2 slides (crystran) were used as substrates for the DCDRS analysis. The 1.5 µl tear samples were manually spotted
onto the substrates and usually left to dry in a temperature and humidity controlled laboratory. One sample was left to
dry under observation through the ×5 objective of a Leica microscope.
A Renishaw 1000 spectrometer with equipped with a 300 mW, 830 nm diode laser (40 mW at sample) and a 300 lines
per mm grating was used to acquire the Raman spectra. A ×50 Leica ULWD objective was used to focus the laser onto
the sample and collect the back-scattered radiation. Spectra were acquired in static grating mode, centered at 1150 cm-1,
which covered the range 70–2000 cm-1. The X-H stretching region of the spectrum (ca. 2800–3200 cm-1) was not
included in this study due to the low efficiency of the CCD in this region. Data were analyzed using Matlab R2007a and
the PLS_Toolbox of Eigenvector Research, Inc.
The 1.5 µl tear aliquots spotted on the CaF2 slides dried to form deposits approximately 2 mm in diameter. The point
mapping spectra were obtained from a grid pattern with 4 um spacing in the x and y directions. Using one second
exposures each map took around 21 hrs in which time an average of ~25,000 spectra over the ~0.4 mm2 area were
obtained. Data maps were analyzed by performing Principal Components Analysis (PCA) on the cropped (400-1850 cm-
1
), standard normal variate (SNV) corrected7, mean centered data.

3. RESULTS AND DISCUSSION

3.1 The Drying Process

To better understand the formation of the tear fenring pattern, images of the drop-drying process were recorded though
the ×5 objective of a microscope. A selection of these images is shown in Fig 1. along with a higher resolution white
light image montage of the entire dried drop taken with a Perkin-Elmer Spotlight. The first image in the time series

Proc. of SPIE Vol. 6853 685309-2

(Fig. 1a) was taken 4 mins 45 secs after the deposition of the drop. In this image it is possible to see the onset of the
formation of the outer amorphous ring of the drying pattern as well as the presence of debris within the tear sample. As
the evaporation continues, the ring becomes visible (Fig. 1b) and the ferning pattern starts to nucleate and grow at the
inner edge of the ring (Fig. 1c and d). As the ferning pattern grows in from the edges, more ferns start to nucleate and
grow within the ring (Fig. 1e). Once the fern patterns start to grow they spread rapidly and it takes around a minute for
the patterning to complete. Fig. 1g shows that the fern branching is relatively uniform throughout the inner-part of the
drying pattern.

tfr
Figure 1: Still images taken using a ×5 objective at these times from deposition of the tear drop (a) 4 mins 45 s, (b) 6 mins
19 s, (c) 6 mins 36 s, (d) 6 mins 40 s, (e) 6 mins 47 s, (f) 7 mins 02 s, and (g) a white light montage of dried drop. Scale
bar shows 100 um.

3.2 Raman Point Mapping

The area observed drying in fig 1. was then mapped as described in the materials and methods section. Fig. 2 shows the
white light image, PC scores images and associated loadings for the first four principal components. To reduce the
influence of outlying data points on the contrast of the scores images an image enhancement operation was performed.7
In this operation the top and bottom 5% of the scores values from each PC were set to the highest and lowest values from
the remaining 90%. The scores values were then displayed scaled onto a 64-element grayscale colormap. No
interpolation was performed on the scores images, each pixel in the images relates to one spectrum. The scores maps
from PCs higher than four (20 PCs were calculated) did not appear to give any extra information on the spatial
distribution of components in the drying pattern so were not included. The color bar on the lower left of the image
shows that the white areas of the images relate to high scores values and black areas relate to lower values. The high
scores values (white) are associated with positive peaks in the loadings vectors and the low scores values (black) are
associated with negative peaks.
The spatial information in the scores maps and the spectral information in the loadings vectors can now be used to
determine the spatial distribution of the biochemical components in the drying pattern.
Comparing the scores image for PC 1 to the white light image of the dried tear it is clear that the high scores values
(white) are associated with both the outer ring and fern patterns. The low scores values (black), on the other hand,
correlate with the CaF2 substrate and areas in the drying pattern where little material has been deposited. The loadings
vector for PC 1 resembles a typical protein Raman spectrum8 but with a skewed background. This skewing is displayed
because the loadings vector is the difference between the protein spectrum and the background from the CaF2 which is
not perfectly flat.
The loadings vector from PC 2 is dominated by two strong negative peaks at 1003 cm-1 and 1045cm-1. These peaks are
caused by vibrational modes of urea and bicarbonate respectively, both of which have previously been observed in tear
fluid. The strongest positive peak in the loadings is a broad peak at 1440 cm-1, which is commonly assigned to motions
of C-H bonds in proteins and lipids. Studying the scores image from PC 2 it is clear that the black regions,
corresponding to urea and bicarbonate, mostly occur at the edge of the fern patterns. The amorphous ring shows up as a

Proc. of SPIE Vol. 6853 685309-3

white area (region high in C-H bond containing structures) but there are also a few white patches in the centre of the
drying pattern.
These patches are also highlighted in PC 3 scores image, but in this case they are black not white. There are also black
areas at the edges of the ferning patterns, similar to those seen in the scores image for PC 2. The white areas of the PC 3
scores map correspond to the amorphous ring and the inner parts of the tear ferns. The positive signals in the loading
vectors suggest these white areas are again protein (positive tryptophan signal at 757 cm-1 and amide I at 1665 cm-1). For
the negative peaks in the loadings (relating to the outer area of the ferns and the unknown patches) one signal is again
related to bicarbonate but there are also two sharp peaks at 1297 cm-1 (unassigned) and 1437 cm-1 (C-H).

High Scores 0.15 0.1

0.1
al C 0)
0.05 0
0
0
0
0-_nob
0-0 2
0-
0'tO
0-
I

-02
Low Scores -0.1 500 1000 1500
00 1000 i0
Reman Shift I cm
500 I 000 1500
Ranian Shift I an1
500 1000
Ranian Shift'
1500
Reman Shift I cm1

Figure 2: Section from the white light montage shown with the scores and loadings from the first four principal components
of the Raman point mapping data.

These sharp features seen in the negative loadings of PC 3 are also visible in the negative loadings of PC 4. The black
areas of the PC 4 scores image are in the same position as the patches described previously. The white areas in the PC 4
scores map correspond to spectra with large background signals.

3.3 Single Point Measurements

To aid the interpretation of the PC data three high signal-to-noise single point spectra were taken from places that the PC
scores images showed to be representative of larger areas. The first spectrum was taken from the amorphous ring
(Fig.3 {1}), the second from one of the unknown patches (Fig.3 {2}), and the third from the edge of a fern (Fig.3 {3}).
The spectrum taken from the amorphous ring is very similar to the loadings vector from PC 1, i.e. a typical protein
Raman spectrum. This is unsurprising since the whole basis of the DCDRS technique is that the protein in a solution is
concentrated into the ring pattern during evaporation. The protein composition of tear fluid is complex; indeed as many
as 491 proteins have been identified in the tear fluid of a single person.9 The majority of the protein is made up of
lysozyme, lactoferrin and tear-specific prealbumin.10 The first two of these are readily available, even the human
variants, but tear-specific prealbumin is not so easily available. This makes any attempts at determining the relative
protein concentrations difficult. Even so, very good quality protein spectra can be produced by DCDRS and it may be
possible to relate changes in the protein composition to a change in ocular pathology
The spectrum from the unknown patch is very informative. Seen outside the confinement of the PC loadings the signals
are clearly due to lipids. The sharp peaks at 1062 cm-1, 1295 cm-1 and 1440 cm-1 observed together are frequently
assigned to the C-C stretching and various CHx modes of linear fatty acids.11 The weak signals at 700 cm-1 and 1740 cm-1
also suggest the presence of cholesterol and triglycerides/wax esters (C=O stretching vibrations from the ester bonds
between glycerol and the fatty acids).11 These observations clearly show that the biochemical composition of these
patches is lipid based. The origin of these lipid deposits is likely to be meibum, the substance secreted by the meibomian
glands that makes up the oily layer at the surface of tear film.

Proc. of SPIE Vol. 6853 685309-4

The spectrum from the edge of the fern is very similar to the protein ring spectrum. The only obvious differences are the
greater intensities of the signals at 1003 cm-1 (urea and phenylalanine in the protein) and 1045 cm-1 (bicarbonate).

U)
[ AA
400 600 800 1000 1200 1400 1600 1800
Raman Shift / cm1

Figure 3: Point spectra acquired for 60 s from the positions marked on the white light image.

3.4 Relationship between the Drying Process and Biochemical Distribution

Now that the distribution of the main biochemical components within the drying pattern has been determined, it can be
related to the processes that occur during evaporation of the water.
Comparing the white light still image in Fig. 1(a) with the PC scores images there seems to be a strong correlation
between the debris visible in the solution with the areas of meibum (lipid) in the scores images (especially clear for
PC 4). Assuming that these pieces of debris are meibum, the observation and recording of these areas during drying
would allow the location of these areas to be quickly identified and probed. The fact that these particles do not appear to
move during evaporation might suggest that they sit on the surface of the drying drop away from the flow of solvent.
As has been previously observed, the protein, being the least soluble component of the tear fluid (not including meibum),
drops out of solution first producing the ring pattern. Unlike the study of Pearce et al,4 this work has also detected the
presence of protein in the ferning pattern, suggesting that this component is important in the formation of the ferns.
Another component likely to be important in fern formation is NaCl. Strong EDXA signals from sodium and chlorine
were detected in the fern pattern by Pearce et al4 but Raman spectroscopy is insensitive to NaCl.
The final observation to be accounted for is the regions of high urea and bicarbonate concentration at the edge of the
ferning pattern. Zhang et al.12 used DCDRS to study the Raman spectra of proteins in buffer solution. On drying these
solutions it was found that again that the protein formed a ring where the edge of the drop had been but the buffer
crystallized in the centre of this ring. From this they concluded that the different solubilities of the components may play
a role in their segregation during drying.12 The protein is relatively insoluble so drops out of solution early in the drying
process. More soluble compounds, such as salts and urea, and those in a very low concentration, may remain in solution
longer and not be deposited until after the formation of the protein ring. The time series of images in Fig. 1 shows that
the edges of the fern pattern are usually the last part of the drying pattern to form and hence it is unsurprising to find high
concentrations of the soluble, low-initial-concentration analytes at these locations.

4. CONCLUSIONS

Although the results of this study look promising, further work is required on larger groups of volunteers to validate the
procedure. Only the data from one volunteer was shown in this study, but tears have been collected from a total of three
volunteers and all tear maps have shown similar biochemical distrubutions.

Proc. of SPIE Vol. 6853 685309-5

 The combination of DCDRS and the tear ferning test shows promise as a simple, fast, near-patient technique for assisting
the diagnosis of ocular infection, especially considering the minute volumes required for analysis. As well as the
standard information obtained from the ferning pattern, this technique allows qualitative analysis of the tear protein
composition (which is known to vary with infection) and the possibly lipid composition of meibum.

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E. Vaikoussis, R. Georgiou and D. Nomicarios, Documenta Ophthalmologica, (1994), 87, 145.
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E. I. Pearce and A. Tomlinson, Ophthal. Physiol. Opt., (2000), 20, 306.
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D. Zhang, Y. Xie, M. F. Mrozek, C. Ortiz, V. J. Davisson, and D. Ben-Amotz, Anal. Chem. (2003), 75, 5703
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R. D. Deegan, O. Bakajin, T. F. Dupont, G. Huber, S. R. Nagel and Thomas A. Witten, Nature, (1997), 389, 827.
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V. Ng and P. Cho, Graefe’s Arch. Clin. Exp. Ophthalmol., (2000), 238, 571.
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D. Zhang, M. F. Mrozek, Y. Xie, and D. Ben-Amotz, Appl. Spectrosc., (2004), 58, 929.

Proc. of SPIE Vol. 6853 685309-6

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