# Towards an objective assessment of corneal grafts quality using back-scattered light measurements

Adrien Sarrade, Julien Fade, Virginie Ferrera, Lucie Molet, Carole Deumié, Jacques Chiaroni, Thierry David, Christophe Picard, Laure Siozade Lamoine

► To cite this version:

Adrien Sarrade, Julien Fade, Virginie Ferrera, Lucie Molet, Carole Deumié, et al. Towards an objective assessment of corneal grafts quality using back-scattered light measurements. 2026. hal-05491080

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37 **Abstract**

38 **Purpose:** Qualification process of corneal grafts in tissue banks includes two optical inspections
39 and corresponding criteria on: the transparency and the endothelial cell density (ECD). However,
40 these two analyses are prone to subjectivity and variability with the operator. Currently, the
41 overall qualification process rejects about half of all harvested corneas. This study examines the
42 potential of an alternative or complementary optical remote sensing technique for quantitatively
43 assessing the transparency of corneal grafts. In addition, the proposed deswelling kinetics
44 monitoring could provide information on the quality of the endothelium.
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46 **Methods:** Thirty-two human corneas deemed unsuitable for transplantation (sourced from 20
donors with average age: 74 ± 11 years; initial ECD: 2310 ± 525 cells/mm²) were analysed using
a laser-light scatterometry instrument which precisely quantifies the total integrated scatter (TIS)
of backscattered light. Repeated measurements were conducted over a 24-hour deswelling period
in standardized medium, and TIS values were jointly analysed with the evolution of corneal
thickness and compared among corneas with varying ECD.
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53 **Results:** Higher sensitivity of scatterometry over pachymetry was confirmed to monitor
deswelling kinetics. Optically-monitored deswelling kinetics revealed that repeated deswelling
cycles significantly accelerate the deswelling kinetics, suggesting altered or adaptive endothelial
function upon repeated swelling/deswelling cycles. Results show tissue conservation time as a
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key influencing factor of the kinetics, and that corneas with higher ECD tend to deswell faster,
in agreement with previous findings.

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**Conclusions:** These results demonstrate that optical scatterometry measurements offer an objective, reproducible and contact-free tool for evaluating corneal transparency and, potentially, hydration dynamics of the tissue.

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**Introduction**

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Approximately 185,000 corneal transplants are performed annually worldwide¹. Despite variations in the detailed graft selection protocols among different countries, after retrieval, a cornea harvested for grafting undergoes several qualification steps before being released for transplant. The main steps involve microscopically counting endothelial cells density (ECD) and evaluating cell mortality, along with a visual evaluation of stromal transparency. This process is repeated before transferring the cornea to the deswelling medium, once it has been allocated to a patient for transplantation, to ensure that cell loss did not exceed 1% per day of conservation since the initial ECD count. Currently, excluding virological factors, french tissue banks reject a cornea from the grafting process as soon as at least one of the following criteria is met:

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* An ECD²,³,⁴ below 2000 cells/mm².

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* A dead cell percentage exceeding 2%.

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* An irregular appearance of the endothelial cell mosaic.
78 • Transverse folds of dead endothelial cells.

79 • Evidence of diffuse stromal opacity.

80 The first three criteria are assessed using optical microscopy⁵. The endothelium is stained
81 with Trypan blue⁶,⁷, and cell density is measured via optical microscopy. As the graft is immersed
82 in the preservation fluid, the transparency of this fluid affects the ease and quality of the cell
83 count⁸,⁹. Drawbacks of this counting method include the potential toxicity¹⁰ of the vital dye and
84 the precision of the count, which ideally should be performed in the central corneal zone.

85 All of the above criteria (and more specifically the last three ones) are prone to operator's
86 subjectivity, making inter-bank comparisons challenging. Furthermore, these structural analyses
87 do not always detect localized defects or stromal scars (particularly those following refractive
88 surgery) in the graft. Currently, many harvested corneas fail to meet these criteria and are
89 therefore rejected from the transplant circuit¹¹,¹².

90 In this context, the use of non-contact, staining-free, non-invasive optical devices to
91 characterize corneal tissues has been a research topic for many years¹³⁻²⁰ aiming to secure the
92 qualifications of corneal grafts. Some techniques could also be used as complementary
93 assessment in in-vivo diagnosis of corneal pathologies. Several research teams have focused on
94 the study and characterization of corneal graft transparency²¹, though routine application has yet
95 to be achieved. For instance, Ventura et al.²² proposed a simple device for in vitro corneal
96 transmission measurement, validated by correlating it with classifications based on a
97 practitioner's visual impressions. A similar device was developed by Zeggari et al²³. The
98 objective of Zeggari's study was to link transparency to ECD, to eliminate the need for Trypan
99 blue staining for viable endothelial cells counting. However, while the device provides reliable
100 wavelength-dependent transmission measurements, correlating them with ECD remains
101 challenging.

102 Other works have proposed to evaluate the optical quality of the eye's anterior segment.
103 Weale et al.²⁴ introduced a system to measure the deformation of a grid of broken lines with
104 different periods. The eye response is measured for each spatial frequency represented by a
105 broken line. In an eye with poor optical quality, the contrast jump is not perfectly discontinuous.
106 This work was further developed along with an improved transparency analysis by Plamann et
107 al.²⁵. They used an optical target comprising alternately clear and dark angular sectors arranged
108 radially from a central point. Analyzing the resulting image was shown to provide quantitative
109 information on both transmitted and scattered light.

110 For many years, we have been developing tools for studying scattered light and
111 determining the origin of scattering (surface, volume, or localized defects)²⁶⁻³⁰. This expertise has
112 focused on understanding the origins of transparency loss in the cornea. Hydration variation,
113 trauma, or dystrophy disorganize the collagen fiber, composing the corneal stroma, inducing
114 haze and a blurry sight. Using a home-made prototype instrument, the performance of angularly
115 resolved measurements of the backscattered light from the graft has been notably demonstrated
116 for early detection of corneal edema³¹. The loss of sight is due to a decrease in transmitted light
117 and an increase in scattered light, and this device has proven to be significantly more sensitive
118 than instruments currently used in hospital³¹ which makes it fit for early diagnosis. More
119 recently, a possible link between the kinetics of graft deturgescence and endothelial cells
120 activity³² has been suggested. In this work, angularly resolved back-scattering measurements,
121 ECD counting and pachymetry measurements were conducted hourly over a 24-hour period on a
122 few graft samples, and showed a correlation between the decrease kinetics of the quantitatively-
123 assessed total backscattered light by the sample and the ECD. In line with this previous work, the
124 aim of the present study is to confirm such results on a much larger number of grafts samples and
125 evaluate the added value of backscattered light measurements on corneal grafts as a
126 complementary tool to standard corneal tissue qualification procedures in eye banks.

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128 Methods

129 *Experimental corneas*

130 Corneas used in this study were obtained from donors either presenting contraindications
131 for clinical transplantation or with an ECD below 2000 cells/mm², the common criterion used in
132 France for rejecting a corneal graft from the grafting process², ³, ⁴. The only exclusion criteria
133 used for this study were any known corneal disease or any previous corneal or intraocular
134 surgery. Donor eyes were collected from hospitals across the Provence-Alpes-Côte d'Azur-Corse
135 area and transported to the Tissue Bank in Marseille (Etablissement Français du Sang) for
136 processing and storage via organ culture.

137 All procedures conformed to the principles of the Declaration of Helsinki for biomedical
138 research involving human tissue. Corneal procurement for this preclinical study was authorized
139 by the French Biomedicine Agency (PFS 23-003). All corneas used in this study were obtained
140 from donors not opposed to scientific use of their tissue.

141 *Cornea preparation*

142 Upon arrival in the tissue bank, corneas were immediately immersed in CorneaPrep II
143 medium (Eurobio®, Les Ulis, France) and maintained at room temperature for a maximum of
144 three days. They were then transferred to CorneaMax medium (Eurobio®) and stored at
145 31 ± 2°C. ECD was assessed by optical microscopy following trypan blue staining (0.04%
146 dilution). Endothelial cells were visualized and manually counted under a Nikon optical
147 microscope. For the optical experimental procedures detailed below, corneas were transferred
148 into a deswelling medium (CorneaJet medium, Eurobio®) at room temperature.

149 ECD Estimation

150 Several days may elapse between graft disqualification and experimental analysis. During
151 this interval, ECD gradually declines due to storage in preservation medium. To account for this
152 time-dependent decrease, ECD at the time of measurement was estimated using a geometric
153 ECD decay model assuming a daily loss of 1% in storage, as previously proposed by Filev et al.³³

154 Scatterometer for Scattered Light Measurement

155 The scatterometry instrument used in this study is a home-made designed setup with
156 limited bulk size (97.5 × 68 × 34.5 cm). It includes a 640 nm illuminating laser beam (BioRay,
157 Coherent, Saxonburg, PA, USA), directed vertically at the center of the corneal graft (Figure 1a),
158 with a collimated beam diameter of 1 mm (Figure 1b). The intensity of the light scattered by the
159 cornea is measured using a series of identical photodiodes (SM1PD1A, Thorlabs, Newton, NJ,
160 USA) distributed along a meridian arc of the reflecting hemisphere as a function of the scattering
161 angle (θ), with an angular sampling of 5°, producing measurement of the bidirectional
162 reflectance distribution function (BRDF) (Figure 2a). Each BRDF measurement takes 30
163 seconds. Full details about the instrument, its calibration, repeatability, detection limits,
164 accuracy/precision, etc. can be found in Ref³⁴.
<figure><img src="image_2.png" /><figcaption>Figure 1. Diagram of the scatterometer (a) and picture of a cornea in the scatterometer (b).</figcaption></figure>

Analysis focused on scattering angles between 35° and 70°, as angles below 35° primarily account for surface scattering properties which are strongly affected by epithelial roughness. Our experimental configuration also excluded angles above 70° to avoid any occlusion issue. Given the assumed radial symmetry of the cornea around the visual axis, BRDF was treated as rotationally invariant³⁴. To extract a single global metric from the BRDF measurements, we compute the Total Integrated Scattering (TIS) defined as³⁴:

$$TIS = \iint_{\theta, \varphi} \mathrm{BRDF}(\theta, \varphi) \cos(\theta) \sin(\theta) d\theta d\varphi \quad (1)$$

The BRDF measure being calibrated by a reference measurement on a Lambertian diffuser calibration target, the TIS quantities that will be handled in the following are dimensionless, and allow quantitative comparisons across all measurements performed in this experimental campaign. Every BRDF measured on corneas ranged between ground detectivity level and calibration Lambertian signal³⁴.

As assessed through careful characterization of the instrument, the TIS has a dynamic range of 0.0068–0.585, with a measurement precision of +/- 0.001. The main limitation to
accuracy arises from the precision of corneal apex alignment with the laser beam³⁴. The repeatability and alignment reproducibility of the instrument has been experimentally checked in this study, showing a coefficient of variation <2% on average, as reported in Section 2 of Supplementary Document, in agreement with its specifications³⁴.

<figure><img src="image_3.png" /><figcaption>**Figure 2.** BRDF(θ,φ) x cos(θ) of cornea 19R (a). Evolution of the TIS over time during deswelling (b) for cornea 19R.</figcaption></figure>

Previous studies have demonstrated the potential interest of using TIS to characterize corneal deswelling kinetics³². In the present study, the TIS of a corneal graft was continuously monitored over a 24-hour deswelling period. The early deswelling phase (first 6 hours) was fitted with a mono-exponential decay function to quantify the TIS evolution with deswelling time (Figure 2.b) as

$$TIS(t) = Ae^{-t/\tau} + B. \quad (2)$$

In the above equation, parameter A stands for the range of variation of TIS across deswelling, B is the final floor scattering level and τ is the characteristic deswelling time.
<figure><img src="image_4.png" /><figcaption>Figure 3. Top view picture of cornea 19R for qualitative assessment of transparency (a). Cross-section view of cornea 19R through OCT measure (b).</figcaption></figure>

OCT-based pachymetry

Pachymetry, consisting in evaluating the corneal central thickness (CCT), was measured using an OCT device (OQ LabScope 2.0, Lumedica, Durham, NC, USA). The system comprises an embedded computer (33 × 19 × 15 cm) and a scanner head (11 × 6.4 × 4.8 cm) mounted vertically. The scanner was placed 2 cm above the cornea. The OCT resolution was 512 × 512 pixels, with an air-based field of view of 7 mm (width) × 2.2 mm (depth). The in-air pixel resolution was 12.3 µm (horizontal) and 3.9 µm (vertical). Accounting for an average refractive index for corneal tissues³⁵ of 1.37, the in-tissue axial resolution was 3.2 µm.

Each cornea was positioned endothelial side down in air in an empty, flat-bottomed, open Petri dish (50 mm diameter, 9 mm height, Corning-Falcon, Corning, NY, USA) without any medium. The cornea was centered in the field of view, with both air/epithelium and endothelium/air interfaces visible. OCT acquisition took ~30 seconds (Figure 3.b). Images were post-processed to calculate the CCT. Images were horizontally rescaled to an orthonormal grid (5 µm resolution). Using a home-made semi-automated image processing pipeline, corneal thickness was measured 15 times around the apex to reliably estimate the thickness of each sample across the experimental process. Thickness measurement precision using this method was
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estimated to be of ± 35 µm³. In the remainder of this article, we shall denote by E0 the initial

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thickness of a cornea at the beginning of a deswelling cycle.

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*Transparency evaluation*

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An additional qualitative transparency assessment through black and white picture on a

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cross-test pattern (see Fig. 3.a) has been performed to be able to detect any visible abnormalities

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of the grafts.

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*Optical measurements procedure*

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Corneas suspended in preservation or deswelling medium were removed and placed

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epithelial side up in a Petri dish. No further manipulation of the cornea occurred. The dish was

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manually transferred between devices successively for transparency imaging, TIS measurement,

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and pachymetry. The cornea was then returned to its preservation or deswelling medium. The full

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procedure lasted approximately 2.5 minutes per cornea. Corneas stored in CorneaMax

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(Eurobio®) underwent deswelling in CorneaJet (Eurobio®). During the first two hours (t = 0–2

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h) in the deswelling medium, measurements were taken every 30 minutes. Between 2 and 6

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hours of the deswelling period, measurements were taken hourly. When feasible, a final

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measurement was performed after 24 hours of deswelling, before the cornea could be stored

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again in the preservation medium for later measurements.

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*Statistical analysis*

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Linear relationships were evaluated using Pearson's correlation coefficient analysis.

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Statistical significance was set at p < 0.05. Prior to analysis, data were assessed for linearity,

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normality, and homoscedasticity. A sample size of n = 31 corneas was confirmed to provide
sufficient power to detect significant correlations with $|r| > 0.5$. From those 31 samples we
measured 48 deswelling series.

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## 240 **Results**

241 *Corneas cohort*

Thirty-one corneas from 19 donors were tested (see Table 1). The donors had a mean age
of 74 ± 12 years. Corneal procurement was performed post-mortem in 18 cases and as part of a
multi-organ retrieval in 1 case. The primary cause of death was cardiac arrest. The initial ECD
count ranged from 1260 to 3340 cells/mm² and averaged 2310 ± 525 cells/mm² at the time of
their arrival in the tissue bank. However, corneas were only directed to this scientific
experimental study after a period of preservation in the tissue bank (and were hence finally
excluded from the grafting circuit), and consequently the ECD was significantly lower when they
were made available. Therefore, the final ECD count performed at the date they were given to us
for this study rather averaged 1680 ± 420 cells/mm².

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251 *Comparison of scatterometry and pachymetry*

Across the 31 corneas analyzed in this study, 535 TIS measurements were taken in total.

The TIS values ranged from ground detectivity level (0.0068) to 0.071. Over the 535
measurements, only four were below the detectivity level. The corresponding CCT values for the
same dataset ranged from 440 µm to 1500 µm. Each scattering measurement was associated with
the sample's CCT at the time of measurement. The paired scattering/CCT data for every cornea at
each time point are plotted in Figure 4, showing a significant dispersion of TIS values for a given
thickness.

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<figure><img src="image_5.png" /><figcaption>**Figure 4.** Relation between TIS and central corneal thickness (CCT (µm)), regardless of cornea index or deswelling time (left axis). Standard deviation (σ) of TIS for a given pachymetry (right axis). Example photographs of low thickness (left) and high-thickness corneas (right) showing loss of transparency with edema.</figcaption></figure>

For a given CCT range, the standard deviation of TIS below 1100 µm remains indeed globally stable and significantly greater than the TIS measurement error (± 0.001), confirming that the observed variability reflects true biological or structural differences rather than instrumental noise.

*Deswelling kinetics observed through TIS measurements*

As reported in Table S.1 of the supplementary document, from the 31 corneas considered, we successfully performed at least one 6-hour deswelling on 21 of them (representing 34 deswelling series). Among these 21 corneas, full 24-hour deswelling was completed for 20
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samples (25 deswelling series). Furthermore, for 5 of these corneas, we obtained two 24h deswelling series.

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For each deswelling measurement, the following data are reported in Table S.1 of the Supplementary Document: E0, final CCT, estimated ECD at the time of experiment, number of days since harvest, number of days since the last medium change (representing time in conservation medium since harvest or since the previous deswelling process), initial TIS, TIS after 6 hours of deswelling, TIS after 24 hours of deswelling, and the estimated characteristic time of deswelling $τ$, estimated over the first 6 hours of deswelling.

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## Variation of TIS over time upon successive deswelling cycles

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From the TIS values at $t = 0h$, $t = 6h$, and $t = 24h$, it appeared interesting to represent the absolute variation of TIS during the initial 6 hours ($\Delta TIS_{0-6h} = TIS(6h) - TIS(0h)$) and over the subsequent 18 hours ($\Delta TIS_{6-24h} = TIS(24h) - TIS(6h)$), depending on the deswelling cycle (first, or second when applicable). Figure 5. (left) compares the TIS variation during the first deswelling process for each cornea after harvest. For 10 out of 16 corneas, $\Delta TIS_{0-6h}$ is approximately equal to $\Delta TIS_{6-24h}$, and both are less than 0. This indicates a slow decrease in TIS over time, with a total TIS variation ranging between -0.01 and -0.04. On the other hand, Figure 5. (right) compares TIS variations during the second deswelling process for each available cornea, and shows a different trend. For every cornea, the absolute change in the first 6 hours is at least twice greater than in the subsequent 18 hours ($|\Delta TIS_{0-6h}| > |\Delta TIS_{6-24h}|$), and $\Delta TIS_{6-24h}$ is greater than 0, demonstrating that most of the deswelling occurs within the first 6 hours in this case. Furthermore, TIS systematically increased between $t = 6h$ and $t = 24h$. As a result, the observations reported here exhibited a significant difference in behavior between the first and
second deswelling of a cornea graft stored in preservation medium, regardless of the swelling time (2 - 19 days), the ECD (900 - 2000 cells/mm²) and E0, (850 – 1600 µm).

<figure><img src="image_6.png" /></figure>

**Figure 5.** Variation of TIS during deswelling across consecutive deswelling cycles: experiment 1 (left) and experiment 2 (right). The y-axis shows the change in TIS final state and initial state, respectively TIS(6h) - TIS(0h) and TIS(24h) - TIS(6h).

### Driving parameter of TIS evolution

We aimed at identifying the origin of such an unexpected result among the various experimental parameters at stake, including those concerning conservation and processing of the samples in the tissue bank. In order to rely on a relative (normalized) quantity, we analyzed the temporal TIS evolution during a deswelling cycle through the following quantity: $\Delta\text{TIS}_{6h-0h}/\Delta\text{TIS}_{24h-0h}$, allowing to compare the initial versus total deswelling dynamics across the deswelling cycle.

We identified ECD, E0, time from harvest, and conservation time as potential explanatory parameters for the observed differences in TIS behavior during a deswelling cycle. Conservation time is defined here as the time spent by the tissue in preservation medium since its last medium changes, or in other words, since the last time it was showing its normal physiological thickness.
As shown in Section 3 of the Supplementary Document (Fig. S.1 and Table S.3), these parameters have been tested, and conservation time has been identified as the relevant explanatory parameter to understand the differences observed in Figure 5. The relation between deswelling kinetics and conservation time is shown in Figure 6, exhibiting a clear negative correlation (Pearson corr. coef PCC=-0.65, p-value<0.0005) between the evolution of ΔTIS<sub>6h-0h</sub>/ΔTIS<sub>24h-0h</sub> and the duration in conservation medium.

<figure><img src="image_7.png" /><figcaption>**Figure 6:** Relationship between deswelling kinetics - assessed as the relative change in TIS during the first 6 hours of deswelling, normalized to the total TIS change over 24 hours – and the conservation time.</figcaption></figure>

TIS kinetics and ECD

After having identified the key parameter influencing TIS variations, we can now consider it to examine the potential relationship between deswelling kinetics and ECD. Figure 6 suggests that we basically categorize the tested corneas into two conservation time groups: below
and above 30 days. Again, in our experiments, the same cornea can be associated with two
deswelling procedures and hence be associated with two different conservation times.

Within each of these conservation time groups, we sorted the corneas into two ECD
groups: below and above 1600 cells/mm². For each cornea, we determined the TIS deswelling
characteristic time (τ) by fitting a decreasing exponential model on the TIS evolution data as
sketched in Eq. (2). We considered a τ value to be outside the model's validity range if it was
below 30 minutes or if the estimation error³⁴ on τ was comparable to τ (indicating a poor quality
of fit). A τ value of less than 30 minutes indicates a system faster than our study's temporal
resolution (the smallest interval between two TIS measurements was 30 minutes).

Table 2 reports the number of corneas considered for each combination of ECD, and the
conservation time τ (along with its corresponding estimated precision), where τ falling within the
above defined validity range. Notably, most deswelling measurements from corneas with a
conservation time exceeding 30 days had a τ value that was classified as too high. In contrast, the
majority of deswelling measurements from corneas with a conservation time below 30 days had
τ values within the retained validity range.

For each group defined by conservation time and ECD, the average characteristic
deswelling time (τ) is presented in Table 2. The groups with conservation times above 30 days
exhibited higher τ values compared to the group below 30 days – which means slower
deswelling than the group below 30 days.

Furthermore, within the group with conservation times below 30 days, the characteristic
deswelling time tended to decrease with increasing ECD, in line with previous observations¹³.
For corneas below 30 days, τ decreased from 110 minutes (for ECD < 1600 cells/mm²) to 31 minutes (for ECD > 1600 cells/mm²).

An important observation from this experimental data analysis is that, for τ values below 120 minutes, deswelling should be at least 95% complete within the first 6 hours of deswelling. According to Fig. 6, corneas conserved for less than 20 days seem to exhibit a capacity to deswell within 6 hours, even those with lower ECDs. This may have possible implications in the process of corneal graft delivery, as will be highlighted in the Discussion section.

## Discussion

The present study intended to confirm and extend the proof-of-concept investigations conducted by Gil et al.³¹ and Georges et al.³². Initially, our objective was to explore the potential relationship between deswelling kinetics and endothelial cell density (ECD) in grafts of human corneas stored in preservation medium in tissue banks. Additionally, we aimed at determining whether repeated deswelling cycles would lead to deteriorated deswelling kinetics, potentially due to progressive endothelial cells loss during storage in preservation medium.

As shown in previous studies³⁰⁻³², stromal transparency can be quantitatively assessed by measuring the TIS. When TIS is compared with CCT, a higher sensitivity of scatterometry over pachymetry is confirmed. Therefore, TIS appears to be a more precise quantitative and repeatable indicator for grading corneal transparency than CCT, and seems to be an interesting and quantitative tool, complementary to usual visual inspection, in tissue banks to identify very low levels of transparency loss in corneal grafts. Moreover, the repeatability of TIS measurements was evaluated, and its behavior was found to align with predictions from light scattering theory. Existing models³⁶,³⁷ accurately predict TIS for stacks of homogeneous layers
371 with known thickness and refractive indices. At low thicknesses (<1100 µm), deviations from 372 theoretical TIS values may result from stromal collagen inhomogeneity, delamination, or 373 edema³⁸⁻⁴⁰.

374 Another outcome of this experimental study is the following: when two deswelling 375 procedures were performed on the same cornea - separated by storage intervals between 2 to 19 376 days – the initial TIS (evaluating transparency level) during the second deswelling was 377 consistently lower than that of the first. This may be explained by progressive ECD loss, during 378 storage and deswelling, as already reported³³.

379 Furthermore, and quite counter-intuitively, the TIS variation during the second deswelling 380 was greater, and the deswelling rate was at least three times faster in average. This pattern was 381 highly reproducible across samples. This result would tend to suggest that, despite ECD loss, 382 remaining endothelial cells may exhibit increased activity during the second deswelling cycle. A 383 finer data analysis presented above showed however that storage duration in preservation 384 medium (also termed conservation time) appears to be the primary factor influencing this altered 385 behavior. While the cornea remains fully immersed in the preservation medium, homeostatic 386 mechanisms⁴¹,⁴² regulating hydration become saturated, leading to swelling. Two hypotheses 387 might explain the observed differences in deswelling kinetics between the first and second 388 procedures (and hence differences in kinetics as a function of conservation time):

389 • Reduced Homeostatic Function Over Time: Endothelial function may diminish during 390 prolonged storage. The first deswelling would therefore be abnormally slow due to 391 suppressed endothelial activity, requiring a reactivation or "awakening" phase. The faster 392 second deswelling (i.e., after a smaller conservation time) could reflect restored function, 393 with increased overall TIS levels possibly indicating apoptosis of some cells.
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* Over-activation Following Adaptation: the first deswelling would occur at a typical rate, but endothelial cells would adapt to the deswelling medium and become overactivated during the second deswelling procedure, leading to rapid deswelling before stabilization.

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To distinguish between these hypotheses, future studies should focus on characterizing endothelial cell activity immediately after retrieval. A more detailed analysis of the second deswelling phase is also needed to determine whether overactivation or apoptosis predominates. If endothelial function is indeed compromised during storage, identifying the reactivation period could lead to improvements in graft preservation protocols.

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Lastly, comparative analysis of corneas with similar conservation times showed that those with higher ECD deswelled faster, corroborating initial findings by Georges et al.³². Corneas with ECD >1000 cells/mm² consistently exhibited characteristic deswelling times below 2 hours, suggesting that full deswelling could occur within less than 6 hours. Surgical-grade corneas (ECD >2000 cells/mm²) are expected to follow similar if not faster kinetics. This outcome could have important consequences in graft delivery processes, where the deswelling time in deswelling medium before graft delivery is usually significantly longer. If these results were confirmed in forthcoming further experiments on surgical-grade corneas, this would make it possible to reduce the delivery delay for some situations of emergency grafting.

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Due to manual preparation and handling of the samples in the instrument by the operator, the current time between two measures is 30 minutes. A better sample conditioning and a full automation of the measurement process could allow to accurately capture deswelling kinetics with shorter time constant - light scattering assessments every 10 or 15 minutes should suffice.

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For that purpose, a perspective of this work is the design of a fully automated scatterometer prototype that would be suited to enter clinical rooms and sterile environments.

Another limitation holds on the study population; for more representative results, replication using numerous cornea grafts with ECD >2000 cells/mm² (surgical-grade corneas) or fresher tissues is necessary, thereby confirming the need for a faster automated instrument able to be used in routine at the tissue bank. Lastly, this study was conducted using the Eurobio preservation and deswelling media. As different media may affect endothelial activity in different ways, further comparative research with other media might be another interesting perspective to this work.

In conclusion, light scatterometry used as a kinetic assessment of corneal transparency offers a non-invasive, label-free, and quantitative evaluation of graft transparency, but also can provide relevant clues on the endothelial activity, and hence on the overall graft quality. This metric could complement conventional ECD measurements in eye banks by providing additional insights into corneal transparency and endothelial tissue viability. Future work will focus on adapting the scatterometer for routine use in eye-banks, including clean-room sterilization, automation, and measuring TIS directly through the deswelling medium. This would enable systematic TIS measurement on viable grafts, improving our understanding of endothelial cells function during organ-culture storage.

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434 Acknowledgements

This work was partially funded by the French Biomedicine Agency (Agence de la Biomédecine, ABM). The authors gratefully acknowledge Centrale Méditerranée for funding A. Sarrade's position. The authors also gratefully acknowledge the Eye Bank of EFS-PACA Corse and the European Center for Research in Medical Imaging (CERIMED) for their support. We
439 extend our sincere thanks to Valentin Espinas, formerly at Institut Fresnel, and to Stéphanie Roque, Julien Dubuisson, Pascale Isetti, Sylvie Albergne, and Paul Martin, technicians at the EFS-PACA Corse Eye Bank, for their valuable technical assistance. We also express our deepest gratitude to the donors and their families who made this study possible through their generous contribution.

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445 **References**

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<table><thead><tr><th>dono<br>r</th><th>Donation<br>(PM : post-<br>mortem,<br>MO :<br>multi-<br>organ)</th><th>pathology</th><th>age</th><th>sex</th><th>eyes</th><th>ECD<br>(cells/mm²)</th><th>sclera diameter<br>(mm)</th><th>cornea</th></tr></thead><tbody><tr><td>1</td><td>PM</td><td>malignant tumor</td><td>78</td><td>M</td><td>left</td><td>2940</td><td>18</td><td>1L</td></tr><tr><td rowspan="2">2</td><td rowspan="2">PM</td><td rowspan="2">digestive hemorrhage</td><td rowspan="2">60</td><td rowspan="2">M</td><td>right</td><td>1860</td><td>19</td><td>2R</td></tr><tr><td>left</td><td>1860</td><td>19</td><td>2L</td></tr><tr><td>3</td><td>PM</td><td>stroke</td><td>88</td><td>F</td><td>right</td><td>1920</td><td>18</td><td>3R</td></tr><tr><td rowspan="2">4</td><td rowspan="2">PM</td><td rowspan="2">traumatism</td><td rowspan="2">81</td><td rowspan="2">M</td><td>right</td><td>1360</td><td>18</td><td>4R</td></tr><tr><td>left</td><td>1480</td><td>18</td><td>4L</td></tr><tr><td>5</td><td>PM</td><td>malign tumor</td><td>84</td><td>F</td><td>right</td><td>2300</td><td>18</td><td>5R</td></tr><tr><td>6</td><td>PM</td><td>cardiac arrest</td><td>86</td><td>M</td><td>right</td><td>2400</td><td>18</td><td>6R</td></tr><tr><td>7</td><td>PM</td><td>cardiac arrest</td><td>80</td><td>M</td><td>left</td><td>1260</td><td>18</td><td>7L</td></tr><tr><td rowspan="2">8</td><td rowspan="2">PM</td><td rowspan="2">cardiac arrest</td><td rowspan="2">71</td><td rowspan="2">M</td><td>right</td><td>2000</td><td>18</td><td>8R</td></tr><tr><td>left</td><td>2560</td><td>18</td><td>8L</td></tr><tr><td rowspan="2">9</td><td rowspan="2">PM</td><td rowspan="2">cardiac arrest</td><td rowspan="2">74</td><td rowspan="2">M</td><td>right</td><td>1640</td><td>18</td><td>9R</td></tr><tr><td>left</td><td>1980</td><td>18</td><td>9L</td></tr><tr><td rowspan="2">10</td><td rowspan="2">PM</td><td rowspan="2">stroke</td><td rowspan="2">64</td><td rowspan="2">M</td><td>right</td><td>2160</td><td>18</td><td>10R</td></tr><tr><td>left</td><td>2260</td><td>21</td><td>10L</td></tr><tr><td rowspan="2">11</td><td rowspan="2">PM</td><td rowspan="2">infection</td><td rowspan="2">79</td><td rowspan="2">M</td><td>right</td><td>2780</td><td>17</td><td>11R</td></tr><tr><td>left</td><td>2540</td><td>18</td><td>11L</td></tr><tr><td rowspan="2">12</td><td rowspan="2">PM</td><td rowspan="2">stroke</td><td rowspan="2">94</td><td rowspan="2">M</td><td>right</td><td>2360</td><td>18</td><td>12R</td></tr><tr><td>left</td><td>2200</td><td>18</td><td>12L</td></tr><tr><td rowspan="2">13</td><td rowspan="2">PM</td><td rowspan="2">cardiac arrest</td><td rowspan="2">52</td><td rowspan="2">M</td><td>right</td><td>2780</td><td>18</td><td>13R</td></tr><tr><td>left</td><td>2260</td><td>19</td><td>13L</td></tr><tr><td>14</td><td>PM</td><td>stroke</td><td>75</td><td>M</td><td>left</td><td>2040</td><td>19</td><td>14L</td></tr><tr><td rowspan="2">15</td><td rowspan="2">PM</td><td rowspan="2">cardiac arrest</td><td rowspan="2">61</td><td rowspan="2">F</td><td>right</td><td>3020</td><td>17</td><td>15R</td></tr><tr><td>left</td><td>3080</td><td>17</td><td>15L</td></tr><tr><td>16</td><td>PM</td><td>infection</td><td>91</td><td>M</td><td>left</td><td>2480</td><td>18</td><td>16L</td></tr></tbody></table>
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /></figure>

626

<figure><img src="image_11.png" /></figure>

627