Open Access
Cornea  |   February 2017
Loss of Tryptophan Fluorescence Correlates With Mechanical Stiffness Following Photo-Crosslinking Treatment of Rabbit Cornea
Author Affiliations & Notes
  • Maura Williams
    Wellman Center for Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • William Lewis
    Wellman Center for Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Walfre Franco
    Wellman Center for Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Correspondence: Walfre Franco, Wellman Center for Photomedicine, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114, USA; wfranco@mgh.harvard.edu
Investigative Ophthalmology & Visual Science February 2017, Vol.58, 1110-1115. doi:10.1167/iovs.16-20750
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      Maura Williams, William Lewis, Walfre Franco; Loss of Tryptophan Fluorescence Correlates With Mechanical Stiffness Following Photo-Crosslinking Treatment of Rabbit Cornea. Invest. Ophthalmol. Vis. Sci. 2017;58(2):1110-1115. doi: 10.1167/iovs.16-20750.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose: A clinical treatment option for keratoconus involves the use of UV-initiated photo-crosslinking with riboflavin to increase corneal stiffness. Our study investigates whether endogenous fluorescence changes following treatment for keratoconus can be correlated to alterations in the stiffness of the cornea, thereby guiding treatment of keratoconus.

Methods: A total of 78 ex vivo rabbit eyes were treated with either riboflavin-dextran solution plus UV light, dextran solution plus UV light, or riboflavin-dextran solution only for half treatment (2.84 J/cm2), standard treatment (5.28 J/cm2), or prolonged treatment (15.84 J/cm2) times. Fluorescent spectroscopy was performed on all samples before and after treatment. The stress–strain relationship was measured for all samples using a uniaxial tensiometer following treatment.

Results: We found a dose-dependent decrease in the 290/340 nm excitation/emission fluorescence pair with increase in corneal stiffening following treatment that was significant (P < 0.01) for both standard (5.28 J/cm2 total fluence) and prolonged treatment (15.84 J/cm2) times. We did not observe a significant change in this excitation/emission pair for the dextran-plus-UV or riboflavin-only treatment groups.

Conclusions: Loss of fluorescence intensity at the 290/340 nm excitation/emission pair could offer a noninvasive, in situ measurement for guiding the photo-crosslinking treatment of keratoconus. Larger relative decreases in this pair are significantly correlated with longer treatment times and with increases in stiffness and Young's modulus.

Keratoconus is a disease that alters the shape of the cornea, reducing visual acuity and potentiating the need for corneal transplant in up to 20% of cases.1 Although the etiology of the disease is unknown, the natural progression involves a gradual thinning and change in shape of the corneal stroma with resulting distorting effects to vision.2,3 It is a disease that primarily affects young adults, with a prevalence of approximately 1 in 2000 people.4 
Until recently, the only definitive surgical treatment option available in the United States for the progressive disease was a corneal transplant, usually a full-thickness corneal transplant known as a penetrating keratoplasty or a deep anterior lamellar keratoplasty, a procedure that involves leaving the recipient corneal endothelium intact.2 Rigid contact lenses or scleral lenses can also offer some utility for vision improvement, but they may not be effective or well tolerated by all patients. Both surgical procedures are effective at slowing or stopping the progression of disease, but they are invasive and subject to negative side effects, including postoperative infection, the need for revision, and the possibility of rejection.5,6 
Corneal crosslinking (CXL), a treatment that uses UV light to activate a photosensitizer that is applied topically to the eye, was recently approved by the U.S. Food and Drug Administration for use in the United States; it has been used successfully in Europe for many years.7 It has been offered as an alternative to corneal transplant or at least as an intermediate step to delay the progression of disease, possibly abrogating the need for a corneal transplant.2 It has already significantly reduced the need for corneal transplantation in the Netherlands, with 25% fewer corneal transplants performed in the 3 years following the approval of the procedure.8 The standard procedure (known as the Dresden Protocol) involves removing or weakening the corneal epithelium and applying a topical photosensitizer (in this case, 0.1% riboflavin-5-phosphate in 20% dextran) to the underlying corneal stroma.2,9 The riboflavin diffuses into the corneal stroma and is activated under long-wave UV light (365–370 nm) for 30 minutes at an irradiance of 3mW/cm2 for a total dose of 5.4J/cm2. Previous studies have shown a consistent concentration of riboflavin in the corneal stroma up to 100 μm.10 Riboflavin acts as both a photosensitizer in the stroma and an absorber of UV light, protecting the underlying ocular structures from UV damage.7 The exact mechanism by which photo-crosslinking strengthens the cornea has not been elucidated, but a prevailing theory is that riboflavin creates reactive oxygen species within the corneal stroma, which interact with proteins present in the collagen fibrils to produce crosslinks.2 The increase in collagen crosslinks within the corneal stroma causes it to stiffen, counteracting the weakening as a result of the disease, thus helping to preserve visual acuity. There exists a need for a noninvasive, accurate, in situ measurement of the efficacy of crosslinking for use during treatment to help guide treatment parameters. In this work, we investigated whether endogenous fluorescence changes following photo-crosslinking treatment can be correlated to changes in the stiffness of the corneal samples, thereby guiding treatment parameters in clinical practice and potentially providing clinical guidelines for optimal treatment length. 
Materials and Methods
Riboflavin-5-phosphate solution was prepared as a 0.1% w/v solution containing 20% dextran (∼500kDa, Dextran 500 T, Sigma-Aldrich Corp., St. Louis, MO, USA) in phosphate buffered saline (PBS) to model the solution used clinically. A 20% dextran solution without riboflavin was prepared for use on treatment controls. A total of 78 ex vivo frozen young rabbit eyes were obtained from Pel-Freez (Pel-Freez Biologicals, Rogers, AK, USA) and stored at −20°C until use. The eyes were thawed in PBS for 20 minutes at room temperature without submerging the cornea to prevent corneal edema prior to the procedure. Corneal epithelia were removed by brief submersion in ethanol and then by gentle mechanical debridement prior to application of the riboflavin or dextran solutions. The eyes were discarded if significant corneal discoloration or a loss of transparency in the cornea was present. 
Light Source
A black ray UV lamp (Blak-Ray Lamp, Model B-100A; Ted Pella, Inc., Redding, CA, USA) centered at 365 ± 3 nm as measured with a spectroradiometer (SPR-01, 235–850 nm; Luzchem Research, Inc., Gloucester, Ontario, Canada) was used as a source of UV irradiation. The intensity at the distance of the eyes from the source was 2.2 mW/cm2. During irradiation, the samples were at a distance of 20 cm from the light source. 
Fluorescence Measurements
After the corneal epithelium was removed, fluorescent spectroscopic measurements were taken prior to treatment in direct contact with the cornea centered on the pupil using a spectrofluorimeter (SPEX SkinSkan; Horiba Jobin Yvon, Edision, NJ, USA). The spectrofluorimeter uses a monochromator to filter light produced by a 125W xenon arc lamp to excite the sample via an optical fiber. Backscattered and backgenerated fluorescent light from the tissue sample was measured via fibers of the same probe and filtered by an emission monochromator and detector. The fiber optic bundle was bifurcated and was 2 meters long, with each leg containing 31 fibers. Each fiber was 200 μm in diameter. The excitation and emission fibers in the probe were randomly arranged to avoid spatial filtering effects. The emission detector was a silicon photodiode with sensitivity over wavelengths of 200 to 980 nm. The size of the detection area was 3.14 mm2. Excitation-emission maps for each sample were generated by pairing the intensity of emission of light at a particular wavelength with the corresponding excitation wavelength. The presence of particular fluorophores could be inferred from the intensity of the peaks mapped. Each eye was measured prior to treatment to establish a baseline and to serve as an internal control, and the maps were averaged to compare peaks before and after treatment. 
Photo-Crosslinking Treatment
Following the baseline fluorescent measurements, nine eyes per group were treated with either riboflavin-dextran solution plus UV irradiation (group 1, treatment group), dextran solution plus UV (group 2, dextran control), or riboflavin-dextran solution only (group 3, riboflavin control). Topical solutions were applied both 5 minutes before and immediately prior to irradiation. In the riboflavin-dextran solution only group, the eyes were immediately covered after application of the solution to prevent any light activation of the drug. Eyes in the UV groups were then irradiated with the black ray UV light for a total fluence of 5.28 J/cm2. During irradiation, riboflavin-dextran or dextran-only solution was reapplied every 5 minutes to ensure sufficient availability of the drug in the stroma for a total of five times during the procedure. The riboflavin-dextran solution was reapplied in the same intervals for the riboflavin-only group. This procedure models a procedure commonly used in clinical practice.2,11,12 Eyes undergoing treatment in clinical practice receive a total fluence of 5.4 J/cm2 (3 mW/cm2 for 30 minutes), approximately 40 minutes at 2.2 mW/cm2 (5.28 J/cm2). Nine eyes in each group were also treated for half the dose (2.64 J/cm2) and three times the irradiation dose (15.84 J/cm2). 
Repeat Fluorescent Measurements
Following each treatment, the eyes were rinsed with PBS to remove any remaining riboflavin or dextran solution on the cornea. Each cornea was then rescanned as described previously using the spectrofluorimeter to measure fluorescent species following treatment. Between measurements, each cornea was kept covered with lightly moistened gauze to maintain corneal hydration. 
Uniaxial Tensiometry
Following treatment, corneolimbal sections were excised from each sample. The orientation of each cornea was marked prior to excision to ensure the proper orientation of samples, and the axis was determined by anatomical landmarks, specifically the optic nerve and extraocular muscle attachments. The 2-millimeter strips were cut across the horizontal access using parallel blades, and the strips were placed on lightly moistened gauze and covered until stiffness measurements were taken. The samples were tested in batches of two or three at a time per treatment group. The thickness of the central cornea was measured with a spring-loaded micrometer with (0.025 mm gradations) just prior to tensiometer measurements. Tensiometer measurements were taken within an hour of excision of the cornea. Each strip was loaded onto the clamps of the testing machine (Micro EP Miniature; Admet, Norwood, MA, USA) with a 10 N load cell with a distance of 0.5 cm between the clamps. Corneal samples were conditioned by three cycles (load up to 0.03 N) before loading until more than 10% displacement. Stiffness was calculated as force divided by displacement at 10% strain. Zero displacement was defined as the value at 0.1 N tensile force applied to the sample. Young's modulus was calculated as stress (force divided by cross-sectional area) divided by strain at 10% extension. 
Results
Fluorescence Changes
For the remainder of the discussion, we refer to the riboflavin-dextran solution plus UV group, the dextran solution plus UV group, and the riboflavin-dextran solution only group as riboflavin plus UV, dextran plus UV, and riboflavin only. Average excitation-emission maps for all groups are shown in Figure 1 for excitation wavelengths 240 to 350 nm and emission wavelengths 290 to 400 nm. In group 1 (riboflavin plus UV), the band at 290/340 nm shows a dose-dependent decrease with increasing treatment times. In group 2 (dextran plus UV), this peak was stable, with a small decrease in the longest treatment group (d), although this was not statistically significant from baseline (P = 0.07). Similarly, in group 3 (riboflavin-only controls), this peak remains stable across treatment times. 
Figure 1
 
Average fluorescence excitation-emission matrices for excitation wavelengths 240 to 350 nm and emission wavelengths 290 to 400 nm for baseline pretreatment (a, e, i), following half-dose irradiance (b, f, j), following the standard dose (c, g, k), and following the prolonged dose (d, h, l) for riboflavin plus UV, dextran plus UV, and riboflavin only, respectively. The fluorescence emission peak at 290/340 nm is reduced after all treatment lengths in the riboflavin plus UV group relative to the dextran plus UV and riboflavin-only groups. Scale bar is the same for all plots.
Figure 1
 
Average fluorescence excitation-emission matrices for excitation wavelengths 240 to 350 nm and emission wavelengths 290 to 400 nm for baseline pretreatment (a, e, i), following half-dose irradiance (b, f, j), following the standard dose (c, g, k), and following the prolonged dose (d, h, l) for riboflavin plus UV, dextran plus UV, and riboflavin only, respectively. The fluorescence emission peak at 290/340 nm is reduced after all treatment lengths in the riboflavin plus UV group relative to the dextran plus UV and riboflavin-only groups. Scale bar is the same for all plots.
Prior to treatment, all eyes showed characteristic fluorescence at the 290/340 nm band (see Fig. 2). In the riboflavin-plus-UV treatment groups, fluorescence intensity at the 290/340 nm band decreased significantly when compared with both the riboflavin and dextran controls for a given treatment time as well as to the baseline control before treatment. On average, this peak decreased 75% (average before treatment 2.98 ± 0.81 average fluorescence intensity [A.U.] to 0.78 ± 0.47 arbitrary units [A.U.] after treatment) relative to its baseline value for the standard treatment dose (5.28 J/cm2, p <0.0001). In contrast, this peak in the riboflavin-only group decreased 21% (average before treatment 3.97 ± 1.56 A.U. to 3.12 ± 1.42 after treatment), and the dextran-plus-UV group increased 44% (average before treatment 3.0 ± 1.05 to 3.99 ± 1.34 after treatment), although these results were not significant when compared with baseline pretreatment (P = 0.24 and P = 0.099, respectively). In the 15.84 J/cm2 total fluence treatment group (three times the standard treatment dose), this peak decreased 97% in the riboflavin-plus-UV treatment group when compared with baseline (4.62 ± 0.71 to 0.15 ± 0.034), relative to a 30% and 17% decrease in the dextran-plus-UV and riboflavin-only treatment groups, respectively (3.87 ± 1.14 to 2.48 ± 0.71 and 3.88 ± 0.67 to 3.22 ± 1.16). In the half-fluence treatment group (2.64 J/cm2), this peak decreased 60% in the riboflavin-UV group (average before treatment 3.63 ± 1.05 to 1.42 ± 0.39 after treatment). 
Figure 2
 
Average fluorescence intensity (A.U.) of the 290/340 excitation-emission band with standard error bars grouped by treatment parameter and increasing treatment time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
Figure 2
 
Average fluorescence intensity (A.U.) of the 290/340 excitation-emission band with standard error bars grouped by treatment parameter and increasing treatment time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
The average peaks associated with riboflavin (namely, 370/520 and 450/520) increased significantly more in the riboflavin-only treatment relative to the riboflavin-plus-UV group over each time group (results not shown). 
Tensiometry Results
The average stiffness (in Newtons/mm) according to treatment time at 10% strain is shown in Figure 3. For the standard treatment protocol (5.28 J/cm2 UV irradiation), the riboflavin-plus-UV treatment group increased in stiffness relative to controls by a factor of 1.85 when compared with riboflavin only and 1.74 when compared with dextran plus UV (P = 0.0001 and P = 0.0013, respectively). For the prolonged treatment groups (15.84 J/cm2), stiffness in the riboflavin-plus-UV group increased by a factor of 1.8 relative to the dextran-plus-UV group and a factor of 2 relative to the riboflavin-only group (P = 0.0067 and 0.003, respectively). There was no significant difference in stiffness between the riboflavin-UV and dextran-UV or the riboflavin-UV and riboflavin-only groups in the half-dose irradiation treatment group (P = 0.62 and 0.31, respectively). There was no significant difference in stiffness between the riboflavin-only and the dextran-plus-UV groups for all treatment parameters (P = 0.58, P = 0.72, P = 0.48 for the half-dose, standard, and prolonged treatment groups, respectively). 
Figure 3
 
Stiffness (Newtons divided by displacement) by treatment time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
Figure 3
 
Stiffness (Newtons divided by displacement) by treatment time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
The average Young's modulus by treatment group and treatment length is shown in Figure 4. In the standard treatment group, Young's modulus increased 80% relative to the dextran-plus-UV group (P = 0.002) and doubled relative to the riboflavin-only group (P < 0.0001). 
Figure 4
 
Average Young's modulus with error bars by treatment group and time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
Figure 4
 
Average Young's modulus with error bars by treatment group and time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
Discussion
The intensity of fluorescence in the 290/340 nm band was negatively correlated with both the length of treatment time and the increase in stiffness in the riboflavin-plus-UV irradiation treatment group. There was a dose-dependent change in the relative intensity of this peak and the increased stiffness of the corneal sample, although for the shortest treatment groups, our results were not statistically significant, likely reflecting limitations in the sensitivity of the tensiometer. The values reported here for Young's modulus are similar to the results reported in the literature.1214 There was no statistical difference in this peak or in Young's modulus between treatment times for the dextran-plus-UV or riboflavin-solution-only groups. 
The peak at 290/340 nm has been previously associated with tryptophan.15 Maximum tryptophan absorption is at wavelengths less than 295 nm,15,16 although it has been reported that riboflavin mediates the photo-oxidation of tryptophan at longer wavelengths (320–400 nm).1720 One possible explanation for the decreased fluorescent signal from tryptophan is its oxidation in the presence of riboflavin and riboflavin degradation products. Riboflavin is subject to photodegradation under UV irradiation, but its degradation products, specifically lumichrome and lumiflavin, have also been shown to similarly photosensitize tryptophan.20,21 In the dextran-plus-UV control group, this decrease in the 290/340 signal is not seen in either the half or standard treatment times. In the half-dose group, the dextran group signal at 290/340 excitation/emission increased, although this difference was not statistically different from controls. 
The oxidation of tryptophan in the presence of riboflavin generates multiple reactive oxygen species, including 1O2, OH*, H2O2, and O2*.17,20,22,23 We hypothesize that the photo-oxidation of tryptophan in the riboflavin solution generates at least some of these reactive oxygen species (ROS) that then in turn contribute to the protein crosslink formation that is the basis for photo-crosslinking treatment. Products of tryptophan photo-oxidation, namely, kynurenine and N-formyl kynurenine, are also known fluorophores fluorescing at the excitation emission pairs 365/480 nm and 325/435 nm, respectively.24,25 We did not detect increases at these wavelength pairs with our setup; whether this is a result of limitations of our detection system, poor quantum yield of the fluorescent species, or another mechanism is unclear. 
An alternate explanation for this decrease in the fluorescence intensity at the 290/340 nm peak is that riboflavin degradation products effectively quench fluorescence. Riboflavin itself absorbs at 290 nm and 2,3-butanedione, an additional photo-degradation product of riboflavin, does have absorption at both 290 nm and 340 nm.26,27 It is possible riboflavin degradation products are contributing to fluorescence quenching at these wavelengths, although the molar extinction coefficient for 2,3-butanedione at 290 nm is negligible relative to the molar extinction coefficient for riboflavin at that wavelength. Similarly, riboflavin absorbs considerably more than 2,3-butanedione at 340 nm. Lumiflavin has a similar absorption spectrum to riboflavin at these wavelengths.28 
Changes to the fluorescence intensity of other amino acids, specifically histidine and tyrosine, which fluoresce at the excitation/emission pairs 220/360 nm and 260/300 nm, respectively, were not detected with our system.29,30 In the case of histidine, our fluorescence measurement was limited to excitation wavelengths equal to or greater than 240 nm. At the characteristic fluorescence peak for tyrosine, we did not see a significant change following treatment. 
Various crosslinks have been identified in diabetic corneas, namely, pentosidine and carboxy-methyl lysine, of which pentosidine is fluorescent with a peak at 330/390 nm excitation-emission.31,32 We did not see a substantial increase at this band for any of our samples (results not shown). 
Prior studies have tried techniques such as optical coherence elastography and an ocular response analyzer to measure corneal elastography. The stability of vision can also serve as a proxy for the increase in the strength of the cornea following the photo-crosslinking procedure. Optical coherence elastography uses optical coherence tomography to assess corneal biomechanics, but has not yet been validated in clinical practice.33 The Ocular Response Analyzer (Reichert, Inc., Depew, New York, NY, USA) uses an airjet to measure corneal hysteresis to monitor disease progression by direct measurement of in vivo corneal stiffness.34 Measurements of corneal hysteresis can be dependent on corneal hydration, thickness, and intraocular pressure,35,36 thereby limiting the utility of the device in some patients. A new technique described by Vinciguerra and colleagues37 using the Corvis ST (Oculus Optikgeräte GmbH, Wetzlar, Germany) was shown to be accurate in identifying keratoconic eyes from normal eyes through a new biomechanical measure called the Corvis Biomechanical Index. The index combines corneal thickness measurements with corneal deformation data, with good specificity in identifying keratoconic eyes.37 Brillouin microscopy also has been used successfully to measure mechanical changes to the cornea following CXL, but its clinical utility is limited by expense and prolonged signal acquisition time.38,39 The potential advantages to the technique described here include faster acquisition of signal and reduced error as a result of corneal thickness and hydration. The imaging of fluorescence changes to tryptophan could possibly be adapted for wide-field imaging, an additional benefit that would not require contact with the surface of the cornea.40,41 
Conclusions
We have shown that a decrease in the excitation-emission pair at 290/340 nm can be correlated to an increase in the Young's modulus of the cornea. This peak perhaps could be useful clinically in inferring corneal stiffness immediately following photo-crosslinking treatment. This method may also be less susceptible to corneal thickness or intraocular pressure, as this excitation wavelength does not significantly penetrate beyond the cornea.42 Because healing following CXL treatment is an important contributor to treatment efficacy, a better understanding of fluorescence recovery following treatment is needed to determine whether this tool could aid in measuring corneal stiffness over time. 
Additional studies could investigate spatially resolved measurements of fluorescence changes that might provide useful information about the depth of crosslinking to assess efficacy of treatment at various depths within the cornea. Further work will incorporate an incubator to better control experimental conditions because corneal thickness can be sensitive to room temperature and ambient humidity. It would also be interesting to add a reference wavelength to future experiments to further evaluate the sensitivity of the fluorescent probe described here. A better understanding of the day-to-day fluctuations in the fluorescence of eyes in vivo would be helpful to assess the sensitivity of the probe. 
The current standard of care in the United States uses a one-size-fits-all approach, for a total treatment dose of 5.4 J/cm2, although there are benefits to tailoring treatment according to patients' needs. Fluorescence spectroscopy could offer a noninvasive, in situ measurement for guiding the photo-crosslinking treatment of keratoconus. 
Acknowledgments
The authors thank R. Rox Anderson for his continued support and Irene Kochevar for her helpful feedback on this manuscript. 
Disclosure: M. Williams, None; W. Lewis, None; W. Franco, None 
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Figure 1
 
Average fluorescence excitation-emission matrices for excitation wavelengths 240 to 350 nm and emission wavelengths 290 to 400 nm for baseline pretreatment (a, e, i), following half-dose irradiance (b, f, j), following the standard dose (c, g, k), and following the prolonged dose (d, h, l) for riboflavin plus UV, dextran plus UV, and riboflavin only, respectively. The fluorescence emission peak at 290/340 nm is reduced after all treatment lengths in the riboflavin plus UV group relative to the dextran plus UV and riboflavin-only groups. Scale bar is the same for all plots.
Figure 1
 
Average fluorescence excitation-emission matrices for excitation wavelengths 240 to 350 nm and emission wavelengths 290 to 400 nm for baseline pretreatment (a, e, i), following half-dose irradiance (b, f, j), following the standard dose (c, g, k), and following the prolonged dose (d, h, l) for riboflavin plus UV, dextran plus UV, and riboflavin only, respectively. The fluorescence emission peak at 290/340 nm is reduced after all treatment lengths in the riboflavin plus UV group relative to the dextran plus UV and riboflavin-only groups. Scale bar is the same for all plots.
Figure 2
 
Average fluorescence intensity (A.U.) of the 290/340 excitation-emission band with standard error bars grouped by treatment parameter and increasing treatment time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
Figure 2
 
Average fluorescence intensity (A.U.) of the 290/340 excitation-emission band with standard error bars grouped by treatment parameter and increasing treatment time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
Figure 3
 
Stiffness (Newtons divided by displacement) by treatment time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
Figure 3
 
Stiffness (Newtons divided by displacement) by treatment time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
Figure 4
 
Average Young's modulus with error bars by treatment group and time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
Figure 4
 
Average Young's modulus with error bars by treatment group and time. The 20-minute treatment time corresponds to the half dose (2.64 J/cm2), 40-minute treatment time to the standard irradiance dose (5.28 J/cm2), and 120-minute treatment time to the prolonged dose (15.84 J/cm2).
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