# Dynamic full-field optical coherence tomography for extemporaneous high-resolution imaging of adrenal glands

Irene A Spiridon, Michel Vix, Didier Mutter, Barbara Seeliger

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Irene A Spiridon, Michel Vix, Didier Mutter, Barbara Seeliger. Dynamic full-field optical coherence tomography for extemporaneous high-resolution imaging of adrenal glands. Experimental and Molecular Pathology, 2025, 141, pp.104958. 10.1016/j.yexmp.2025.104958. hal-04962363

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# Dynamic full-field optical coherence tomography for extemporaneous high-resolution imaging of adrenal glands

Irene A. Spiridon<sup>a,b,c</sup>, Michel Vix<sup>d</sup>, Didier Mutter<sup>a,d</sup>, Barbara Seeliger<sup>a,d,e,*</sup>

## ARTICLE INFO

**Keywords:**

Real-time histopathology
Functional tissue analysis
Adrenal surgery
Image-guided surgery

## ABSTRACT

**Background:** There is an interindividual variance in postoperative adrenocortical capacity, and the minimal functional remnant size is unknown. New imaging technologies may allow for improved intraoperative assessment of adrenal tissue morphology, cell activity, and surgery-related changes. The aim of this experimental study was to provide a pilot assessment of adrenal gland architecture with dynamic full-field optical coherence tomography (D-FF-OCT) in comparison to standard hematoxylin-eosin (HE) examination.

**Methods:** D-FF-OCT was performed on freshly resected porcine adrenal glands to simultaneously assess both morphology and metabolic activity in real time, and for comparison with standard histopathology. Left and right adrenal glands were assessed from 8 pigs (1 M, 7 F, mean weight 43.9 ± 8.3 kg).

**Results:** The evaluation with D-FF-OCT proved fast in terms of acquisition time, averaging 32 min/specimen. The technique required a relatively short learning curve and provided morphological details similar to standard microscopy. In the comparative analysis of both methods, D-FF-OCT scans allowed easy identification of normal adrenal morphology and facilitated differentiation of the structural components of the cortical and medullary areas based on architectural and vascular patterns. Furthermore, it was possible to distinguish more accurately between cell subpopulations based on their metabolic activity.

**Conclusion:** While HE examination remains the gold standard for morphological evaluation, time weighs heavy on this ancillary technique. In our study, we prove that D-FF-OCT is effective in achieving a comparable level of morphological details, with added metabolic activity of the cells, a combination which can prove useful in the real-time assessment of various diseases requiring adrenal surgery.

## 1. Introduction

Surgical removal of adrenal lesions via adrenalectomy or cortical-sparing approaches reduces the functional capacity of adrenal glands. On the one hand, there is a correlation between residual volume and adrenocortical stress capacity (Brauckhoff et al., 2008), and in general, at least 15–30 % remaining adrenal tissue is required for adequate function (Brauckhoff et al., 2003; Kaye et al., 2010). On the other hand, there is interindividual variability – an intact stress capacity was observed in a patient where only 10 % of adrenal tissue was preserved

(Brauckhoff et al., 2008), whereas other patients with 50 % remaining adrenal tissue (after unilateral adrenalectomy) did not respond to stress situations as normal controls do (Kaye et al., 2010; Nakada et al., 1995). To avoid adrenal insufficiency after complete removal of adrenal lesions, preservation of functional adrenal tissue may be considered for unilateral disease and must be pursued for bilateral tumors (Lorenz et al., 2019). There is no definitive data to intraoperatively determine individual adrenal remnant function and minimal functional size (Brauckhoff et al., 2008; Nagaraja et al., 2015).

Ultrastructural analysis in real time has the potential to provide an

**Abbreviations:** 3D, three-dimensional; CLE, confocal laser endomicroscopy; DCI, dynamic cell imaging; D-FF-OCT, dynamic full-field optical coherence tomography; DICOM, digital imaging and communications in medicine; ECM, extracellular matrix; EM, electron microscopy; FF-OCT, full-field optical coherence tomography; HE, hematoxylin-eosin; LED, light-emitting diode; OCT, optical coherence tomography; RGB, red, green, blue.

<footnote><sup>a</sup> Institute of Image-Guided Surgery, IHU Strasbourg, Strasbourg, France</footnote>

<footnote><sup>b</sup> "Grigore T. Popa" University of Medicine and Pharmacy, Department of Morpho-functional Sciences I - Pathology, Iasi, Romania</footnote>

<footnote><sup>c</sup> Department of Pathology, University Hospitals of Strasbourg, Strasbourg, France</footnote>

<footnote><sup>d</sup> Department of Digestive and Endocrine Surgery, University Hospitals of Strasbourg, Strasbourg, France</footnote>

<footnote><sup>e</sup> ICube, UMR 7357, CNRS, INSERM U1328 RODIN, University of Strasbourg, Strasbourg, France</footnote>

<footnote>* Corresponding author at: IHU Strasbourg, 1 Place de l'Hôpital, 67091 Strasbourg, France.</footnote>

<footnote>E-mail address: [barbara.seeliger@ihu-strasbourg.eu](mailto:barbara.seeliger@ihu-strasbourg.eu) (B. Seeliger).</footnote>
assessment of adrenal gland architecture, structural changes, and potential cellular cortisol-producing capacity. Novel intraoperative imaging technologies provide valuable information about microcirculation and real-time *in vivo* histopathology of adrenal tissue (Mascagni et al., 2018; Seeliger et al., 2020a). Among these *in vivo* imaging modalities with microscopic resolution, probe-based confocal laser endomicroscopy (CLE) is based on the systemic intravenous injection of a fluorescent dye, requires experienced probe handling, and an interdisciplinary learning curve for image interpretation by a surgeon / pathologist team (Ignat et al., 2019). In contrast, optical coherence tomography (OCT) does not require specific tissue fixation or staining (Mascagni et al., 2018; Pavone, 2025). For parathyroid glands, the probe-based *in vivo* OCT had lower sensitivity and specificity, with probe handling compromised by artifacts, however, a high correlation between *ex vivo* OCT images and conventional histology was reported (Abmaci et al., 2018).

In the medical field, OCT has outgrown its well-established use in ophthalmology to other clinical applications such as gastroenterology and oncology (Pavone, 2025; Wang et al., 2017). By use of near-infrared light and interferometry, OCT allows an in-depth scanning of fresh tissue that provides a level of detail comparable to standard histopathological examination (Zheng et al., 2021). Due to its increased sensitivity and speed in image acquisition, termed high-resolution full-field OCT (FF-OCT), a novel derivative technique that also records rapid temporal variation of the light signal has been developed for freshly excised tissue analysis (Azzollini et al., 2023). The cell activity is translated to a color scale, rendering a high-resolution image of the tissue where the likeability of identifying certain cell subtypes is thus higher, a technique called dynamic cell imaging (DCI) (Azzollini et al., 2023). Thus, FF-OCT and DCI distinguish themselves as alternatives for extemporaneous high-resolution imaging and can provide rapid intraoperative diagnosis beyond classic morphology (Yang et al., 2020).

Consequently, an *ex vivo* OCT approach suitable for FF-OCT and DCI was chosen for immediate specimen analysis. In absence of prior reports on such dynamic full-field OCT (D-FF-OCT) of adrenal glands in the literature, the present preclinical pilot study was conducted to determine the feasibility and diagnostic utility of D-FF-OCT in the adrenal glands for visualization of adrenal morphology, cell activity, and surgery-related changes. The secondary objective was a comparative description of adrenal tissue architecture in D-FF-OCT and the gold standard histopathological examination using optical microscopy.

## 2. Materials and methods

### 2.1. Animal model and ethical statement

All animals in the experimental laboratory were managed according to French laws for animal use and care and according to European Community Council directives (2010/63/EU) and ARRIVE Guidelines (Kilkenny et al., 2010). The animals on which the open adrenalectomies were performed upon euthanasia were used in experiments approved by the French Ministry of Research (project authorizations APAFIS #28479/#28599/#38189), after having been evaluated by the Ethics committee No. 35 in accordance with the National Charter on Ethics of Animal Experimentation. Left and right adrenalectomy specimens from 8 pigs (1 M, 7 F, mean weight 43.9 ± 8.3 kg) were analyzed in this experimental study. The adrenalectomy and subsequent imaging were performed jointly by a surgeon and a pathologist for optimal specimen handling and immediate processing in the laboratory within the same building.

### 2.2. Specimen preparation and dynamic full-field optical coherence tomography (D-FF-OCT)

The gross aspect of the porcine adrenal gland shows a dense connective tissue capsule surrounding the parenchyma, with a clear

distinction between the cortical area (reddish-brown) and the medullary area (tan white), the latter presenting a central vessel (central vein). The morphology of the porcine adrenal gland is similar to that of humans with the typical division into the two distinct areas, the cortex and the medulla. Corresponding to the human adrenocortical zones (zona glomerulosa, zona fasciculata, zona reticularis) the porcine adrenal cortex is subdivided as follows: the outer layer is called zona arcuata (mineralocorticoids), followed by the zona fasciculata (glucocorticoids) and zona reticularis (androgens), located at the interface with the medullary area.

Fresh adrenalectomy specimens were longitudinally transected with a microtome blade for an even section, so that the cross-section included the adrenal capsule, cortex, and medulla over the entire length. The samples were then cut to fit the sample container with 27 mm diameter and 5 mm height, and put in the container in a saline environment, with the cut surface on top. A cover glass was placed over the sample and tightly fit by use of a retaining ring and gradual compression. The Cel-Tivity Biopsy System (Aquyre Biosciences, Boston, USA) was used for FF-OCT and DCI imaging with the proprietary software, mounted on an anti-vibration table (TS-150, HWL Scientific instruments GmbH, Switzerland). The system is based on the low coherence interference microscopy technique (FF-OCT) in a configuration that is referred to as a Linnik interferometer, and the recording of the interferometric variations over time provides intracellular dynamics (DCI) (Assayag et al., 2014). In short, the system is equipped with a white light LED source illumination with low spatial coherence that evenly irradiates the whole tissue surface. The tissue section is placed in a dedicated sample holder that ensures a perpendicular axis to the imaging plane. A camera collects the interferograms of different phases at the focusing depth of the sample by a planar detector, rendering images in a greyscale spectrum. The DCI technology uses short movies of 512 frames of the interferogram pattern that are recorded and processed to extract variations in cell movement and render them in a color spectrum. In DCI imaging, the degree of intracellular activity is translated into a pseudo-colored RGB image where each color channel is built on the Fourier-domain analysis of the micromotions integrated on three different frequency ranges, with the highest frequencies [5.4–25 Hz] represented in the red, medium frequencies [0.6–5.4 Hz] in the green, and low frequencies [0–0.6 Hz] in the blue channel (Durand et al., 2023).

For subsequent FF-OCT and DCI imaging, an optical fluid (Aquyre Biosciences, Boston, USA) ensured contact between the objective lens and the cover glass. For the analysis of freshly excised adrenal tissue with D-FF-OCT, a macro image of the tissue in the specimen container is captured first with the integrated wide-field camera. The software interface then allows the sample to be explored at different depth levels from the surface (0-100 µm) to determine the optimal section of tomographic imaging, with a slice thickness set to 1 µm corresponding to the axial resolution of the device. By manually dragging a 1.25 × 1.25 mm red square over the macro field, the corresponding area is shown in a live view, where cell movement can be observed in real time (Video 1). At this stage, centripetal movement of red blood cells was observed in the capillaries/sinusoids of the corticomedullary junction, towards the central vein. In acquisition mode, either FF-OCT (high-resolution full-field optical coherence tomography) or DCI (dynamic cell imaging) can be selected, and either the black-and-white FF-OCT or the color-coded DCI acquisition preview is displayed accordingly in the live view. After the individual selection of the region of interest, the imaging depth, number and distance of slices, the size of the acquisition surface in mm and the total acquisition duration are displayed before the settings are confirmed (Video 1).

In standard histopathology, tissues are examined at different magnifications with a field of view in the order of cm down to µm. In order for these parameters to be reproduced by the D-FF-OCT system, the sample is automatically moved on the integrated motorized platform to create a mosaic of image tiles measuring 1mm² that have an overlap with the neighboring tiles and are stitched together to display a larger
field of view to enable comparison with histological sections (Assayag et al., 2014).

## 2.3. Comparative analysis of adrenal gland architecture

For comparison with FF-OCT and DCI scans, microscopic imaging was used from prior studies on the same animal model as previously reported, which included *in vivo* Confocal Laser Endomicroscopy of normal adrenal parenchyma, as well as *ex vivo* histology after hematoxylin-eosin (HE) stain, and electron microscopy (Seeliger et al., 2020a; Seeliger et al., 2020b).

# 3. Results

All adrenalectomy specimens were analyzed with D-FF-OCT. The samples tailored to the size of the specimen containers were entirely scanned with FF-OCT within a matter of seconds. The increased acquisition time of DCI per frame led to a choice of smaller areas for the complete D-FF-OCT imaging (15–20 % of the sample size). The software shows the expected duration of image acquisition for the chosen area, so that this strategy for combination of both modalities was tailored to an optimal mean acquisition time of 32 min per specimen.

In the black-and-white FF-OCT images, the adrenal capsule made of dense fibrous connective tissue is identified by the scatter produced by the collagen fibers which appear white and display a laminar structure (Fig. 1A, 2B). Other elements such as blood vessels (through content of elastic fibers in their wall) and extracapsular adipose tissue can occasionally also be observed. Fatty tissue is typically hypo-scattering, hence the adipocytes appear black, with a fine grey cellular membrane generating the classic honeycomb pattern known from HE staining. The

characteristic subdivisions of the cortex are less apparent by cellular details in FF-OCT, yet more by the background, i.e. the extracellular matrix (ECM), which shows different degrees of scatter in the white spectrum, making tissue architecture easily identifiable. On the other hand, the epithelial-derived cells in the cortical area show ill-defined limits, with grey granular cytoplasm and inconspicuous nuclear details.

With DCI, the morphology of the tissue becomes more apparent, the cells in the zona arcuata displaying a lesser volume, but more intracellular activity (bright yellow color) when compared to the cells in the zona fasciculata (dark green) (Fig. 1B). The vascular network can be identified by the presence of delicate vascular lumina which contain red blood cells, apparent in an orange-red shade, albeit the endothelial lining is not clearly visible and does not allow discrimination between vessel types (Fig. 2C, 3B).

In standard histopathological examination with optical microscopy, the adrenocortical zones in HE staining can be recognized based on structural characteristics, with different morphology corresponding to the type of hormonal secretion. Histologically, the zona arcuata has more intense affinity for staining pigment and is composed of columnar cells that are arranged in elongated islands and separated by delicate septa that are in close juxtaposition with a dense vascular network (Fig. 1C). In the zona fasciculata, the cells are plump, with abundant vacuolated cytoplasm and oval nuclei with open chromatin. The architecture is trabecular, with cords of cells typically measuring one cell thickness; the trabeculae display a rectilinear arrangement and show close contact with the accompanying vasculature (Fig. 1D). The zona reticularis shows polyhedral cells with foamy cytoplasm and variable size, forming irregular anastomosing cords with intervening sinusoidal capillaries.

In the medullary area, the chromaffin cells show nested and

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** A) In full-field optical coherence tomography (FF-OCT), the porcine adrenal capsule (stars) shows a thick fibrous structure, with laminar disposition (right), followed by cortical parenchyma (left). The zonation of the adrenal cortex is apparent, through observation of architecture and differences in composition of the extracellular matrix. The thickness of the zona arcuata (131 µm) corresponds to the diameter measured in standard histopathology; B) In dynamic cell imaging (DCI), collagen rich areas such as the capsule display a bright blue signal, while the zonation of the cortical area can also be observed by metabolic activity of the cells – zona arcuata showing a bright yellow signal compared to the green signal in the zona fasciculata, indicating increased cell motricity (D-FF-OCT). C) In hematoxylin-eosin (HE) staining with a 200 µm scale, the porcine adrenal gland is shown with its fibrous capsule and cortical areas composed of zona arcuata and zona fasciculata (predominant in this image); D) The zona arcuata is an area with low thickness, composed of epithelial cells organized in small groups and increased pigment affinity, while the zona fasciculata displays larger cells with vacuolated pale cytoplasm and trabecular arrangement. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Macroscopic image of the porcine adrenal gland after longitudinal section in the specimen container with selected area (yellow box) for full-field OCT analysis. The area contains the fine capsule (left), followed by the cortical and medullary parenchyma, centered by a rich vascular network draining into the central vein (diamonds) (A); FF-OCT (B) and dynamic cell imaging (DCI) (C) illustrate the normal anatomy. DCI highlights the different intracellular activities according to the zonation, with the highest activity being observed in cells of the cortical zona arcuata (subcapsular, left) and the adrenal medulla (right, adjacent to the central vein). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** A) In full-field optical coherence tomography (FF-OCT), the cortico-medullary junction is identifiable through the difference in architecture and composition of the extracellular matrix (ECM). The medullary cells display a nested and trabecular organization; B) In dynamic full-field OCT, the cortico-medullary junction becomes even more apparent as the rich morphological details and differences in cell motricity are captured by dynamic cell imaging (DCI). The central vein (diamond) is highlighted as the strong fluctuations of red blood cells appear in an orange-red hue. The scale for A and B is the same; C) In hematoxylin-eosin (HE) staining with a 400 µm scale, the medullary area of the porcine adrenal gland has a parenchymatous component composed of chromaffin cells and the central vein (diamond); D) In optical microscopy with a 200 µm scale, the columnar medullary cells form trabecular and occasional cystic spaces, and polyhedral cells show pale to clear cytoplasm and a nested arrangement. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

trabecular architecture, as well as the presence of the central vein can also be visualized with the help of FF-OCT, with ease in identifying the cortico-medullary junction. The variation in scatter of their cytoplasm can be correlated with the presence of preformed granules, which are best visible with the DCI. The two distinct cell populations (adrenaline and noradrenaline-producing) that are indistinguishable in standard optical microscopy display different motion patterns, as can be seen

accentuated by D-FF-OCT (Fig. 3A,B), a feature that could allow identification of each cell type, to be confirmed by immunohistochemical study.

In standard histopathological examination with optical microscopy, the zonation of the gland is microscopically evident, the medullary area showing distinctly different morphology. The medulla consists of two populations of cells: one population of cells with larger size, columnar
appearance and basophilic cytoplasm, forming short trabeculae and occasional pseudoglandular spaces, while the other cell population displays a polygonal shape, smaller size, pale granular cytoplasm and insular arrangement (Fig. 3C,D). Occasional tracts, forming an invagination of the capsule, displayed a fibrovascular core surrounded by cells of the zona arcuata extending towards the medulla, generating an almost lobulated appearance of the adrenal gland (Fig. 4).

Imaging performed after the recommended acquisition times, at a one-hour interval post-surgical removal of the adrenal gland shows the immediate/acute and post-acute effects of anoxia on the tissue. As observed in Fig. 5, the effects on the macroscopic aspect of the adrenal gland are minimal (palour), while the FF-OCT highlights the morphological integrity of the tissue (Fig. 5B). It is only in DCI that we observe a shift in color spectrum to dark green and blue, corresponding to decreased cell movement and metabolic activity until complete arrest of cells (blue color) (Fig. 5C).

## 4. Discussion

Intraoperative tissue analysis provides valuable information to tailor the surgical strategy both in cases of suspected malignancy and in benign and potentially hormone-secreting adrenal neoplasms. In adrenal surgery, the gold standard remains histopathological analysis, and frozen section examination in selected cases, to provide a diagnosis and orient intraoperative and post-surgical management of the patients, particularly in an oncological setting. However, given the delays caused by transport, processing, and interpretative challenges pertaining to the technique of specimen analysis in a context of sometimes limited manpower, the quest for intraoperative histopathological diagnosis

remains highly relevant (Namdar et al., 2021). Novel methods that are faster and do not alter tissue quality are needed for rapid assessment and intraoperative decision making. New-generation OCT systems were reported to have the potential of providing intraoperative diagnosis in neck endocrine surgery without tissue staining (Conti de Freitas et al., 2014; Ignat et al., 2019), and extemporaneous distinction between normal and metastatic lymph nodes in FF-OCT (Pavone et al., 2023).

In the present pilot study, the feasibility and diagnostic utility of real-time D-FF-OCT were demonstrated for the first time on adrenal glands. The present study characterizes the structure of healthy porcine adrenal glands in D-FF-OCT, a tool capable of assessing both cellular morphology and dynamics in real time, immediately ex vivo. By comparative analysis, the FF-OCT and DCI scans allowed for an easy identification of normal adrenal gland morphology, differentiating the structural components of the cortical and the medullary areas via architectural and vasculature patterns. In the preclinical model, the characteristic adrenal tissue architecture within the three adrenocortical zones and the medullary area was congruent between all modalities assessed. The adrenal morphology in the optical coherence tomography imaging modalities FF-OCT and DCI corresponded to the one we previously reported in standard histopathology and electron microscopy (Seeliger et al., 2020a), at their respective resolutions. The sample size was deemed sufficient for confident analysis of the tissue architecture in normal adrenal glands prior to a clinical study, since the characteristic adrenal tissue architecture within the three adrenocortical zones and the medullary area was congruent between all modalities assessed.

Classic histopathological examination is limited to static ex vivo analysis after fixation and staining. Similarly, fixed tissue shows no motion signals in D-FF-OCT, whereas fresh specimens exhibit a degradation over time, leading to a loss of signal at varying intervals for different tissues (Apelian et al., 2016). However, immediate specimen analysis with D-FF-OCT prior to time-induced cell death can allow an assessment as close as possible to an in situ examination (Queneherve et al., 2021), while avoiding the motion artifacts of surrounding organs and structures observed in probe-based OCT for in vivo imaging (Abbaci et al., 2018). The chosen ex vivo assessment on an anti-vibration table prevented motion artifacts and thus enabled dynamic analysis on a cellular and sub-cellular level. The ability to detect subcellular motricity with DCI is an indicator of the cell status and D-FF-OCT thus has the potential to reflect cell changes due to metabolic, hormonal or even ischemic stimuli. The use of both modalities (FF-OCT and DCI) can attribute unique signatures to normal tissue and to disease, as has been validated in a study on breast cancer (Yang et al., 2020). Although CelTivity Biopsy System's software interface for sample exploration with a live view (Video 1) primarily serves the role of identification of target areas for analysis, its live visualization of cell dynamics in the fresh ex vivo specimen provided fascinating insight into the complex network of capillaries and their distribution within the different areas of the adrenal gland (Figs. 2 and 3). This ability to visualize the vascular network in real time with the help of DCI can bring further intraoperative advantages, a recent study mentioned the ability to discriminate the type of blood vessel based on its content - red blood cells or immune cells, which are characterized by different motricity profiles (Thouvenin et al., 2017). In adrenal surgery, D-FF-OCT could prove to be a useful tool to assess ischemia-related damage and tissue viability in cortical-sparing surgery, or even to assess the functional status in terms of cortisol-producing capacity of the remaining adrenal cortex, which may be downregulated in the vicinity of an adrenal adenoma with excessive cortisol secretion. Adrenal insufficiency has been reported even after unilateral adrenalectomy for non-cortisol secreting tumors, so it is crucial to identify patients with limited adrenal reserve, while a standard protocol to detect patients at risk for developing postoperative adrenal insufficiency is still not available (Pinto and Parameswaran, 2023).

In the present study, D-FF-OCT created an opportunity for rapid assessment of fresh tissue samples, using a stepwise approach. A fast

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Occasional invaginations of the capsule (stars) give the adrenal gland a lobulated appearance. These correlating aspects are visible in FF-OCT (A), D-FF-OCT with dynamic cell imaging (B), and classic optical microscopy with HE staining (C). The dense fibrous connective tissue appears white on FF-OCT due to scattering and has low cellular activity, which is why it appears blue in DCI. This can be observed at the level of the adrenal capsule (lower right area in each image) up to the upper edge of images A and B with fibrous tissue in continuity with the invagination of the capsule. The scale bar corresponds to 300 µm in all images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Macroscopic image of the same porcine adrenal gland as in Fig. 2 in the specimen container after transversal section (A). After the previous imaging sequences for Fig. 2, the timepoint of image acquisitions was 1 h after specimen extraction. The acquisition surface for (B) and (C) is highlighted in the macro view (A) and measured 5.2 × 12.2 mm. The acquisition duration for FF-OCT (B) of this 63.4 mm² surface was 2.5 min, and 29 min for DCI (C). FF-OCT (B) shows a preserved cellular architecture. However, DCI (C) reveals that the color code representing the intracellular activity shifted towards the dark green and blue nuances, indicating a gradual descent up to a complete halt of cell movement (decrease in metabolic activity) and progression towards cell death. Only in the central vein and some capillaries can a yellow or orange hue be observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

scan of the tissue with FF-OCT allows for identification of areas of concern and assessment of respective target areas, with subsequent DCI analysis. On the other hand, the entire specimen can technically be scanned with DCI and in several layers of depth, a large-scale and 3D imaging would have provided additional insight at the price of considerably increasing individual specimen imaging duration. However, a delay in the acquisitions of the following samples would have led to cell death-related changes in DCI. With our balanced approach tailored to an exploratory (pilot) study, the average sample analysis took 32 min per tissue specimen. This time can be narrowed down further when limiting DCI to specific areas of interest, with similar or even improved processing time when compared to frozen section examination.

Static FF-OCT imaging was not time-sensitive, and even worked for formalin-fixed samples in our tests prior to the present study. In contrast, our experience has also shown that an increasing time interval between resection and D-FF-OCT imaging had a negative impact on observed cellular metabolism, as previously remarked by other authors (Yang et al., 2020). In hypovolemic shock, a contrast-enhanced dynamic computed tomography study demonstrated preserved enhancement and perfusion of the adrenal glands (Ito et al., 2010). Consequently, we assumed that there was no degradation of adrenal architecture and activity within the first minutes after cardiocirculatory arrest (animal sacrifice and immediate adrenalectomy). However, sequential specimen imaging has demonstrated a markedly lower level of cellular dynamics one hour after adrenalectomy when compared to immediate imaging. A strict protocol is thus mandatory when examining *ex vivo* tissue with the use of D-FF-OCT. This could be considered a limitation of the technique and a challenge in generating reproducible results.

As essential organs, the adrenal glands are part of both the sympathoadrenomedullary system (key actor in the body's response to stress) and of the hypothalamic-pituitary-adrenal axis (Kanczkowski et al., 2017). Porcine models have been an efficient way to study disease models in cardiovascular, neurodegenerative and metabolic disease, allowing for a better understanding of the pathogenesis of disease, development of diagnostic tools and refinement of currently existing treatment techniques (Hou et al., 2022). Considered among the most vascularized organs in mammals, the blood is supplied from the aorta *via* multiple

arteries that give rise to branches opening into the adrenal subcapsular space, where an arteriolar plexus expands into a network of numerous sinusoids that supply all areas of the gland; drainage of blood takes place *via* the main adrenal vein that is centrally located in the medulla and provides outflow into the inferior vena cava (*via* the renal vein on the left, and directly on the right side) (Abdellatif et al., 2022; Suzuki et al., 1985). Current approaches to obtain a timely and accurate assessment of tissue vascularity and viability with quantitative fluorescence imaging are complex and mostly performed in preclinical and clinical research protocols (Hardy et al., 2023; Seeliger et al., 2020a). The porcine animal model was previously validated as suitable to demonstrate the effect of cortical-sparing approaches on adrenal remnant perfusion and viability, with intraoperative imaging technology and immediate specimen analysis, and by assessment of the arterial and venous adrenal vascular supply *via* digital subtraction angiography (Seeliger et al., 2020a; Seeliger et al., 2020b). In this regard, D-FF-OCT can provide rapid morphological details not only regarding tissue parenchyma, but also of blood vessels. As previously mentioned in the literature, the presence of red blood cells inside the vascular lumen generates an orange/red signal in DCI which is characteristic due to the strong membrane fluctuations of these cells (Thouvenin et al., 2017). Therefore, changes such as congestion, microhemorrhage and hypoxia become more easily apparent, while also allowing a real time visualization of the consequences of these changes on the tissue.

As demonstrated by studies on tissue cultures, evaluation of cell activity in D-FF-OCT is subject to a number of intracellular factors (Monfort et al., 2023; Scholler et al., 2020). The different cell types in the adrenal gland are subject to intense intracellular transit and exocytosis of vesicles under homeostatic and pathogenic conditions, as demonstrated by EM studies (Altayeb and Salem, 2017; Pihlajoki et al., 2015). Despite the capacity of EM to reveal the cellular and mitochondrial architecture of healthy and necrotic adrenal cells (Seeliger et al., 2020a), it cannot provide the dynamic profile of D-FF-OCT, which allows to even distinguish cells undergoing apoptosis or necrosis as they are characterized by a higher subcellular activity (Groux et al., 2022). While it was known that the movement of intracellular organelles is a major contributor to the D-FF-OCT signal, a recent study with
immunohistochemistry validation revealed that mitochondria and lysosomes were the main actors in this process. By exposing the mitochondria in porcine retinal pigment epithelium to oxidative stress, the dynamic profile observed in D-FF-OCT was brighter and faster, and the mitochondrial aspect in immunohistochemistry and live fluorescence imaging changed considerably from unstressed cells with mitochondria organized in a network towards uncoupled and damaged mitochondria after 4 h of oxidative stress (Groux et al., 2022). The authors further demonstrated that stress and triggering of apoptosis directly reflected in cell dynamics (Groux et al., 2022), underlining the potential of D-FF-OCT to illustrate the effects of ischemia and other microenvironment changes also on adrenal tissue.

Novel techniques present the challenge of a learning curve, which was quite short in the present case of OCT, in line with reported experience (Yang et al., 2020). The similarity of tissue morphology illustration when compared to standard HE histopathological examination was an advantage, with regard to the morphological evaluation in the black-and-white FF-OCT images of fresh tissue specimens in our study. Despite these similarities, there were some particularities such as the loss of intracellular details and enhanced visualization of the ECM that require additional training of specialists for accurate interpretation by constant comparison with the gold standard of classical histopathology examination (Scholler et al., 2023). The interdisciplinary surgeon / pathologist approach for resection and imaging provided valuable information regarding potential tissue degeneration (hematoma, etc.). Joint analysis was mutually beneficial, as in a prior study comparing CLE with standard histopathology (Ignat et al., 2019). While the image acquisition technique is easy to learn, image interpretation regarding detailed tissue characteristics and currently novel information in terms of adrenal cellular activity in the different compartments of the gland will require further studies for comparative assessment of DCI in adrenal disease. Should surgeons perform sample processing and image analysis themselves, in a dedicated lab within or near the operating room suite, they would need specific training to recognize detailed tissue characteristics in such new imaging modalities for real-time diagnosis at the moment of surgery. Here, the advantage of data acquisition in a digital format facilitates the exchange with a specialist pathologist via teleconsultation in the era of digital pathology, and training of machine learning algorithms can provide additional support for automation of tissue recognition and classification (Spiridon and Seeliger, 2023). The application of deep learning algorithms to support the analysis of two- and three-dimensional OCT data is highly anticipated to support automatic tissue classification, as recently demonstrated for the distinction between normal and abnormal thyroid tissue (Tampu et al., 2023).

Full-size 3D reconstruction of serial histological sections is challenging and time-consuming. After staining with specific fixatives to distinguish between adrenaline- and noradrenaline-storing cells in the adrenal medulla with identification of nerve and vascular tissue and subsequent software reconstruction in 3D, the spatial organization of the respective cell populations was recently revealed in rat adrenal glands (Kemoklidze et al., 2021). In our case, DCI allows for easy visualization of the two cell populations due to their intrinsic variability in motility (Fig. 4). In contrast to HE-based 3D models of normal tissue architecture and its alteration in disease, D-FF-OCT acquisition creates digital data in a standardized DICOM format amenable to 3D reconstruction of multiple, in-depth scans of the tissue. Numerous slices can be acquired per specimen, which results in longer acquisition times, but more detailed information for further research studies. Standardization of this approach could provide a large amount of spatial details, relevant to assess both non-tumor and tumor-related changes or even treatment response or progression in case of the latter (Yang et al., 2022).

The valuable insights gained in the present pilot study providing first D-FF-OCT data on normal porcine adrenal parenchyma show that the chosen animal model could provide more insight into adrenal function in health and disease. Given the reuse of animals under other protocols, the present model was limited to normal adrenal glands. Especially in

adrenal disease, which sometimes requires postoperative hormone replacement therapy to avoid adrenal insufficiency (Lowery et al., 2017), assessment of adrenal remnant functional activity and capacity at the time of surgery - in relation to remnant volume and perfusion in case of partial adrenalectomy - would be a great accomplishment to guide postoperative corticosteroid supplementation. In hypercortisolism, metabolic activity assessment in the resected adenoma as well as in the adjacent normal adrenal parenchyma could provide valuable insight in future clinical studies.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.yexmp.2025.104958.

## Funding

This work was supported by French state funds managed by the ANR within the 'Programme d'investissements d'avenir' France 2030 (reference ANR-10-IAHU-02), and by the French National Agency for Research (Agence Nationale de la Recherche) under the references ANR-22-CE17-0019-01 and ANR-21-CE17-0024-01.

## CRediT authorship contribution statement

**Irene A. Spiridon:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Michel Vix:** Writing – review & editing, Validation. **Didier Mutter:** Writing – review & editing, Validation, Supervision, Resources, Methodology, Funding acquisition. **Barbara Seeliger:** Writing – review & editing, Writing – original draft, Visualization, Validation, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Declaration of competing interest

B.S. declares that she is the recipient of a grant from the French National Agency for Research (Agence Nationale de la Recherche (ANR), 86 rue Regnault, 75,013 Paris, France) within the framework of the project AI-DIAL—Diagnostic Imaging of Adrenal Lesions (ANR-22-CE17-0019-01) and has a consultant agreement with Intuitive Surgical unrelated to the present study. I.A.S., M.V. and D.M. have no conflicts of interest to declare.

## Acknowledgements

The authors would like to express their gratitude to Bertrand Le Conte de Poly, Claude Boccara, François Grammont and Emilie Benoît from Aquyre Biosciences, and Morgane Gilg and Jean-Luc Dimarcq at IHU Strasbourg for their technical support and advice for implementation of the CelTivity Biopsy System at IHU Strasbourg Institute of Image-Guided Surgery.

This study was selected for oral presentation at the 41. Arbeitstagung der Chirurgischen Arbeitsgemeinschaft Endokrinologie (CAEK) der Deutschen Gesellschaft für Allgemein- und Viszeralchirurgie (DGAV) (41st Working Conference of the Surgical Working Group on Endocrinology of the German Society for General and Visceral Surgery), Vienna, Austria, November 2023, and the International Society of Innovative Technologies for Endocrine Surgery (ISITES) symposium, Nashville, USA, March 2024.

## Data availability

Data will be made available on request.
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