# Experimental evaluation of laparoscopic laser speckle contrast imaging to visualize perfusion deficits during intestinal surgery

Wido Heeman, Aurelia C L Wildeboer, Mahdi Al-Taher, Joost E M Calon, Laurents P S Stassen, Michele Diana, Joep P M Derikx, Gooitzen M van Dam, E. Christiaan Boerma, Nicole D Bouvy

► To cite this version:

Wido Heeman, Aurelia C L Wildeboer, Mahdi Al-Taher, Joost E M Calon, Laurents P S Stassen, et al. Experimental evaluation of laparoscopic laser speckle contrast imaging to visualize perfusion deficits during intestinal surgery. Surgical Endoscopy, 2023, 37 (2), pp.950 - 957. 10.1007/s00464-022-09536-9. hal-05156132

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<figure><img src="image_1.png" /></figure>

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# Experimental evaluation of laparoscopic laser speckle contrast imaging to visualize perfusion deficits during intestinal surgery

Wido Heeman<sup>1,2,3</sup> · Aurelia C. L. Wildeboer<sup>4,5</sup> · Mahdi Al-Taher<sup>6,7</sup> · Joost E. M. Calon<sup>8</sup> · Laurents P. S. Stassen<sup>7,9</sup> · Michele Diana<sup>6</sup> · Joep P. M. Derikx<sup>4</sup> · Gooitzen M. van Dam<sup>2</sup> · E. Christiaan Boerma<sup>10</sup> · Nicole D. Bouvy<sup>7,9</sup>

Received: 14 February 2022 / Accepted: 5 August 2022 / Published online: 6 September 2022
© The Author(s) 2022

## Abstract

**Background** Ischemia at the site of an intestinal anastomosis is one of the most important risk factors for anastomotic leakage (AL). Consequently, adequate intestinal microperfusion is essential for optimal tissue oxygenation and anastomotic healing. As visual inspection of tissue viability does not guarantee an adequate objective evaluation of intestinal microperfusion, surgeons are in dire need of supportive tools to decrease anastomotic leakage after colorectal surgery.

**Methods** In this feasibility study, laparoscopic laser speckle contrast imaging (LSCI) was used to evaluate intestinal micro-perfusion in an experimental ischemic bowel loop model. Both large and small ischemic loops were created from the small intestine of a pig; each loop was divided into 5 regions of interest (ROI) with varying levels of ischemia. Speckle contrast and local capillary lactate (LCL) was measured in all ROIs.

**Results** Both real-time visualization of intestinal microperfusion and induced perfusion deficits was achieved in all bowel loops. As a result, the emergence of regions of intestinal ischemia could be predicted directly after iatrogenic perfusion limitation, whereas without LSCI signs of decreased intestinal viability could only be seen after 30 minutes. Additionally, a significant relation was found between LCL and LSCI.

**Conclusion** In conclusion, LSCI can achieve real-time intraoperative visualization of intestinal microperfusion deficits, allowing for accurate prediction of long-term postoperative ischemic complications. With this revealing capacity, LSCI could potentially facilitate surgical decision-making when constructing intestinal anastomoses in order to mitigate ischemia-related complications such as AL.

**Keywords** Intestinal perfusion · Anastomosis · Ischemia · Perfusion assessment · Anastomotic leakage · Bowel viability

Wido Heeman and Aurelia C. L. Wildeboer both the authors have been contributed equally.

☑ Wido Heeman
w.t.heeman@umcg.nl

☑ Aurelia C. L. Wildeboer
a.wildeboer@maastrichtuniversity.nl

☑ Mahdi Al-Taher
mahdi.altaher@mumc.nl

1 Faculty Campus Fryslân, University of Groningen,
Leeuwarden 8911 CE, The Netherlands

2 Department of Surgery, Optical Molecular Imaging
Groningen, University Medical Centre Groningen,
Groningen 9713 GZ, The Netherlands

3 LIMIS Development BV, Leeuwarden 8934 AD,
The Netherlands

4 Department of Pediatric Surgery, Amsterdam UMC,
1105 AZ Amsterdam UMC, The Netherlands

5 GROW, School for Oncology and Developmental Biology,
Maastricht University, Maastricht 6229 ER, The Netherlands

6 IRCAD, Research Institute Againstgainst Digestive Cancer,
67000 Strasbourg, France

7 Department of Surgery, Maastricht University Medical
Center, Maastricht 6200 MD, The Netherlands

8 ZiuZ Visual Intelligence BV, Gorredijk 8401 DK,
The Netherlands

9 NUTRIM School of Nutrition and Translational Research
in Metabolism, Maastricht University Medical Center,
Maastricht 6229 HX, The Netherlands

10 Medical Centre Leeuwarden, Department of Intensive Care,
Leeuwarden 8934 AD, The Netherlands
Anastomotic leakage (AL) remains one of the most frequently occurring and feared complications in gastrointestinal surgery. In AL, the enteric content contaminates the peritoneal cavity and causes high morbidity and mortality rates. Despite evolving surgical techniques and improved perioperative care, the average reported incidence of AL is 10%, which has not significantly declined over the last decades [1–4]. Although the exact etiology of AL remains a difficult conundrum, adequate anastomotic perfusion has been identified as one of the most important prerequisites for optimal anastomotic healing [5]. When inadequate anastomotic perfusion is left unaltered, an improvement in anastomotic perfusion from increased collateral circulation is unlikely to develop within the first five postoperative days [6], compromising anastomotic regeneration. Having an accurate indication of the local intestinal perfusion quality can help the surgeon to identify the optimal anastomotic site in order to prevent AL. As a result, intraoperative assessment of bowel perfusion is a vital element in surgical decision-making when performing restorative intestinal resections [6]. However, the traditional direct visual inspection of the intestinal tissue for signs of viability (e.g., mucosal color, pulsatile bleeding from marginal arteries) is highly subjective and does not allow for an adequate evaluation of intestinal microperfusion [6–8]. For this reason, numerous techniques, such as near-infrared fluorescence angiography and the assessment of various parameters of intestinal viability have recently been developed. Unfortunately, due to a lack of convincing evidence for the use of these existing techniques, there is still no general consensus regarding their clinical impact [6], leaving surgeons with a dire need for alternative tools. Horgan and Gorey defined 5 criteria for an ideal bowel viability test, namely (1) ready-to-use availability in every operating room, (2) the technique should require no other specialist personnel other than the surgeon, (3) maximum accuracy with a minimum of false-negative and, more importantly, few false positive results, (4) objectivity and reproducibility, and (5) cost-effectiveness (6).

We hypothesize that laser speckle contrast imaging (LSCI) can give an accurate indication of local intestinal perfusion quality. LSCI is a dye-free, non-contact, non-invasive, and full-field perfusion imaging technique which can deliver 2D perfusion images in real time [9]. The technique uses laser light to generate a so-called speckle pattern which changes due to motion of scatterers, or red blood cells in this case. In this study, we tested the feasibility and potential of laparoscopic LSCI for real-time intraoperative visualization of intestinal perfusion.

## Materials and methods

This study was performed at the central animal facility of Maastricht University (Maastricht, The Netherlands). The animal was handled in compliance with the regulations of the Dutch legislation for animal research and the ARRIVE guidelines [10] following a protocol approved by the Experimental Animal Committee of Maastricht University (DEC-UM) under working protocol number 2017-021-001.

## Animal experiment

One female Dutch landrace pig, weighing 35.5 kg, was used for the current experiment. Following an acclimatization period in the animal keeping facility, an intramuscular injection of Zolazepam/Tiletamine 6 mg/kg (Virbac, Barneveld, The Netherlands) and Thiopental 10 mg/kg (Panpharma SA, Trittau, Germany) was given as premedication. Anesthesia was induced through an intravenous injection of Sufentanyl 0.01 mg/kg/h (Hameln Pharma GmbH, Hameln, Germany), Propofol 9 mg/kg/h (B. Braun Melsungen AG, Melsungen, Germany), and Midazolam 1 mg/kg/h (Aurobindo, Baarn, The Netherlands). After intubation, the pig was continuously mechanically ventilated and anesthesia was deepened with an additional administration of Sufentanyl and Propofol when deemed necessary. At the end of the procedure, the animal was sacrificed with a lethal dose of Pentobarbital 200 mg/kg (AST Farma, Oudewater, The Netherlands).

## Surgical procedure

Access to the peritoneum and the small bowel was obtained through a midline laparotomy. Four intestinal loops, with a length of approximately 15 cm, were randomly selected. At the mesenteric side of the first two loops, tissue perfusion was compromised by clipping ~7 peripheral arteries and veins using a vessel-clipping device (Endo Clip 10 mm Pistol Grip single use clip applier, Medtronic, Dublin, Ireland). To investigate the ability of LSCI to distinguish very subtle changes in perfusion, an additional set of two smaller loops was created. In these loops, tissue perfusion was compromised by the clipping of ~4 peripheral arteries and veins. In each bowel loop, 5 regions of interest (ROIs) were identified and marked with a surgical marker pen (Fig. 1). The regions were located as follows: (1 and 5) the two outer regions, located at the lateral borders of the loops, and (2 and 4) the two inner regions adjacent to 1 and 5, flanking the central region of the ischemic loop (3). This ischemic bowel loop model was a simulation of a model which was previously described [11, 12]. Images were recorded right
<figure><img src="image_2.png" /><figcaption>**Fig. 1** Graphic representation of the experimental setup. (a) Local capillary lactate analyzer, (b) the PerfusiX-Imaging laparoscopic laser speckle contrast imaging setup, (c) mounted laparoscope set at 15 cm above the intestine, and (d) marked positions of the regions of interest</figcaption></figure>

on the bowel serosa together with a dotted line indicating the vascular clipping site at the base of the central ischemic region (ROI 3). *Illustration made by Sieben Medical Art, © 2022 Sieben Medical Art*

before perfusion limitation (baseline), right after perfusion limitation (T=0), and after 30 (T=30) or 45 min (T=45), respectively.

## Local capillary lactate levels

Local capillary lactate (LCL) levels were measured in chosen ROI by puncturing bowel serosa with a 23 Gauge needle (BD Switzerland Sarl, Eysins, Switzerland) and analyzing this blood with an EDGE lactate analyzer (ApexBio, Taipei, Taiwan, People’s Republic of China). LCL levels were obtained during all intestinal perfusion assessments.

## Laparoscopic laser speckle imaging set-up and data acquisition

PerfusiX-Imaging (LIMIS Development BV, Leeuwarden, The Netherlands) device was used to acquire laparoscopic LSCI images [13]. PerfusiX-Imaging is a laparoscopic perfusion

imager which works with standard laparoscopic equipment, namely an Olympus laparoscopic video system (OTV-S200, Olympus, Hamburg, Germany) and a 30-degree laparoscope (EndoEye, Olympus, Hamburg, Germany) in this case. It provides real-time 2D-perfusion maps instantaneously and continuously. LSCI requires a laser light source to illuminate the tissue of interest. The random interference pattern then forms an interference pattern on the camera sensor, i.e., the so-called speckle pattern. This pattern changes with movement of underlying red blood cells, in a rate which corresponds to the amount of blood flow. Hence, it is this blurring or loss in contrast which is quantified as laser speckle perfusion units (LSPU) [9]. Higher LSPU values correspond to better tissue perfusion compared to lower LSPU values. The LSPU are defined as the ratio of standard deviation divided by the mean intensity (Eq. 1).

$$LSPU = \frac{\sigma}{<I>} \qquad (1)$$
<table><thead><tr><th></th><th>Perfusion maps</th><th>Annotated perfusion maps</th><th>White light</th><th></th></tr></thead><tbody><tr><td>a</td><td></td><td></td><td></td><td>b</td></tr><tr><td></td><td></td><td></td><td></td><td>Laser speckle perfusion units</td></tr><tr><td></td><td></td><td></td><td></td><td><table><thead><tr><th>Ischemic time (min)</th><th>ROI 1 (LSPU)</th><th>ROI 2 (LSPU)</th><th>ROI 3 (LSPU)</th><th>ROI 4 (LSPU)</th><th>ROI 5 (LSPU)</th></tr></thead><tbody><tr><td>Baseline</td><td>43.1</td><td>48.5</td><td>43.4</td><td>48.2</td><td>47.0</td></tr><tr><td>0</td><td>42.1</td><td>26.0</td><td>25.0</td><td>24.4</td><td>19.7</td></tr><tr><td>30</td><td>29.4</td><td>18.7</td><td>16.5</td><td>24.3</td><td>21.5</td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td></td><td>c</td></tr><tr><td></td><td></td><td></td><td></td><td>Local capillary lactate</td></tr><tr><td></td><td></td><td></td><td></td><td><table><thead><tr><th>Ischemic time (min)</th><th>Systemic lactate (mmol/L)</th><th>ROI 1</th><th>ROI 2</th><th>ROI 3</th><th>ROI 4</th><th>ROI 5</th></tr></thead><tbody><tr><td>30</td><td>15</td><td>18</td><td>37</td><td>71</td><td>50</td><td>14</td></tr></tbody></table></td></tr><tr><td>d</td><td></td><td></td><td></td><td>e</td></tr><tr><td></td><td></td><td></td><td></td><td>Laser speckle perfusion units</td></tr><tr><td></td><td></td><td></td><td></td><td><table><thead><tr><th>Ischemic time (min)</th><th>ROI 1 (LSPU)</th><th>ROI 2 (LSPU)</th><th>ROI 3 (LSPU)</th><th>ROI 4 (LSPU)</th><th>ROI 5 (LSPU)</th></tr></thead><tbody><tr><td>Baseline</td><td>51.4</td><td>55.6</td><td>51.7</td><td>40.0</td><td>37.9</td></tr><tr><td>0</td><td>34.4</td><td>30.0</td><td>29.9</td><td>28.9</td><td>27.0</td></tr><tr><td>45</td><td>30.4</td><td>24.7</td><td>19.5</td><td>18.0</td><td>35.1</td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td></td><td>f</td></tr><tr><td></td><td></td><td></td><td></td><td>Local capillary lactate</td></tr><tr><td></td><td></td><td></td><td></td><td><table><thead><tr><th>Ischemic time (min)</th><th>Systemic lactate (mmol/L)</th><th>ROI 1</th><th>ROI 2</th><th>ROI 3</th><th>ROI 4</th><th>ROI 5</th></tr></thead><tbody><tr><td>45</td><td>13</td><td>13</td><td>12</td><td>47</td><td>13</td><td>14</td></tr></tbody></table></td></tr><tr><td>g</td><td></td><td></td><td></td><td>h</td></tr><tr><td></td><td></td><td></td><td></td><td>Laser speckle perfusion units</td></tr><tr><td></td><td></td><td></td><td></td><td><table><thead><tr><th>Ischemic time (min)</th><th>ROI 1 (LSPU)</th><th>ROI 2 (LSPU)</th><th>ROI 3 (LSPU)</th><th>ROI 4 (LSPU)</th><th>ROI 5 (LSPU)</th></tr></thead><tbody><tr><td>Baseline</td><td>49.1</td><td>53.6</td><td>48.0</td><td>46.7</td><td>46.4</td></tr><tr><td>0</td><td>45.2</td><td>34.5</td><td>31.6</td><td>30.9</td><td>39.0</td></tr><tr><td>30</td><td>40.84</td><td>49.1</td><td>38.3</td><td>39.5</td><td>37.5</td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td></td><td>i</td></tr><tr><td></td><td></td><td></td><td></td><td>Local capillary lactate</td></tr><tr><td></td><td></td><td></td><td></td><td><table><thead><tr><th>Ischemic time (min)</th><th>Systemic lactate (mmol/L)</th><th>ROI 1</th><th>ROI 2</th><th>ROI 3</th><th>ROI 4</th><th>ROI 5</th></tr></thead><tbody><tr><td>30</td><td>1</td><td>8</td><td>13</td><td>13</td><td>9</td><td>7</td></tr></tbody></table></td></tr><tr><td>j</td><td></td><td></td><td></td><td>k</td></tr><tr><td></td><td></td><td></td><td></td><td>Laser speckle perfusion units</td></tr><tr><td></td><td></td><td></td><td></td><td><table><thead><tr><th>Ischemic time (min)</th><th>ROI 1 (LSPU)</th><th>ROI 2 (LSPU)</th><th>ROI 3 (LSPU)</th><th>ROI 4 (LSPU)</th><th>ROI 5 (LSPU)</th></tr></thead><tbody><tr><td>Baseline</td><td>57.0</td><td>54.5</td><td>55.3</td><td>55.4</td><td>45.8</td></tr><tr><td>0</td><td>55.9</td><td>53.0</td><td>47.9</td><td>51.8</td><td>44.5</td></tr><tr><td>45</td><td>53.9</td><td>53.0</td><td>47.6</td><td>52.0</td><td>54.6</td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td></td><td>l</td></tr><tr><td></td><td></td><td></td><td></td><td>Local capillary lactate</td></tr><tr><td></td><td></td><td></td><td></td><td><table><thead><tr><th>Ischemic time (min)</th><th>Systemic lactate (mmol/L)</th><th>ROI 1</th><th>ROI 2</th><th>ROI 3</th><th>ROI 4</th><th>ROI 5</th></tr></thead><tbody><tr><td>45</td><td>7</td><td>7</td><td>16</td><td>1</td><td>10</td><td>10</td></tr></tbody></table></td></tr></tbody></table>
►Fig. 2 Overview of all 2D perfusion maps, corresponding white light images and the LSPU and LCL data captured from all ROIs. a Representative perfusion maps of the large ischemic bowel loop and annotated perfusion maps with ischemia indication and the location of the ROI. And corresponding white light images at baseline, 0, and 30 min of iatrogenic perfusion limitation. b Laser speckle perfusion units (LSPU) calculated for corresponding ROIs. c LCL levels in the ROIs located in a large ischemic bowel loop after 30 min of iatrogenic perfusion limitation. d Representative perfusion maps of a large ischemic bowel loop and annotated perfusion maps with ischemia indication and the location of the ROI. And corresponding white light images at baseline, 0, and 45 min of iatrogenic perfusion limitation. e LSPU calculated for corresponding ROIs. f LCL levels in the ROIs located in a large ischemic bowel loop after 45 min of iatrogenic perfusion limitation. g Representative perfusion maps of a small ischemic bowel loop and annotated perfusion maps with ischemia indication and the location of the ROI. And corresponding white light images at baseline, 0, and 30 min of iatrogenic perfusion limitation. h LSPU calculated for corresponding ROIs. i LCL levels in the ROIs located in a small ischemic bowel loop after 30 min of iatrogenic perfusion limitation. j Representative perfusion maps of a small ischemic bowel loop and annotated perfusion maps with ischemia indication and the location of the ROI. And corresponding white light images at baseline, 0, and 45 min of iatrogenic perfusion limitation. k LSPU calculated for corresponding ROIs. l LCL levels in the ROIs located in a small ischemic bowel loop after 45 min of iatrogenic perfusion limitation

LSCI is a fast and full-field imaging technique which
can image large surfaces without the need for a contrast
agent. Non-invasive subsurface perfusion measurements
are characterized by means of high spatial and temporal
resolution. The device houses a 639 nm laser and allows
for a fast, instant switching between conventional white
light and laser light. The laser was connected to the lapa-
roscope using the unmodified optical fiber attached to the
EndoEye (Fig. 1). The 2D perfusion maps were directly
available in the operating room. The laparoscope was
mounted onto a frame directly above the operating table
at a fixed distance of 15 cm from the operating field to
standardize measurement conditions and create a static
optical fiber [14] (Fig. 1). The camera exposure time was
20 ms for all measurements. The aperture could not be
determined. However, it was kept constant with a speckle/
pixel ratio larger than one, satisfying the Nyquist criterion
[15]. Images were acquired at 50 frames per second. The
images were analyzed using the PerfusiX-Imaging soft-
ware suite (LIMIS Development BV, Leeuwarden, The
Netherlands). Images were analyzed using spatial LSCI
with a spatial window size of 7x7 pixels.

On each loop, 5 ROI were marked corresponding to
the vascularized (ROI 1&5), presumed marginally viable
(ROI 2&4) and central ischemic (ROI 3, directly above
clipped vessels) regions, respectively (Fig. 1D). LCLs
were obtained by puncturing the bowel serosa in the
same ROIs. The ROI size was ~ 1.5 × 1.5 cm. The average
LSPU values of the ROIs of 25 frames were calculated and
reported using Eq. 1.

**Statistical analysis**

Basic statistics were used to analyze the results. A loga-
rithmic curve estimation was performed to test the relation
between LSPU values and LCL levels.

Results

Animal experiment and surgical procedure

The surgical procedure was performed without any compli-
cations or adverse events.

Laser speckle perfusion assessment

Real-time visualization of intestinal microperfusion was
achieved using PerfusiX-imaging in all bowel loops result-
ing in analyzable 2D speckle contrast images. The 2D per-
fusion maps allowed us to make a clear distinction between
adequately and poorly perfused tissue regions in both large
(Fig. 2a and 2d) and small ischemic loops (Fig. 2g and 2j).
For larger loops, LSPU values (Fig. 2b and 2e) and cor-
responding perfusion maps of T=0 seemed to predict the
emergence of ischemic tissue regions, which could only be
seen in white light at T=30/T=45. In smaller loops (Fig. 2h
and 2k), visualized perfusion deficits at T=0 were much
larger than those later observed at T=30/T=45.

Local capillary lactate levels

LCL levels in all ROIs at the time of laser speckle perfusion
assessment are presented in Fig. 2c and 2f (large loops) and
Fig. 2i and 2l (small loops). LCL levels below 0.60 mmol/L
were expressed as ‘low’ using the lactate analyzer. For the
statistical analysis, all ‘low’ LCL levels were regarded as
1 mmol/L in order to standardize and prevent underestima-
tion. When comparing LCL levels measured in each of the
ROIs within each bowel loop, no significant differences in
LCL levels were found for smaller loops. However, LCL
levels for larger loops did show a steep increase in LCL.
The logarithmic curve estimation of the relationship between
measured LSPU and LCL levels revealed a significant rela-
tion (p=0.013) with an R-value of 0.55, indicating a moder-
ate correlation.

Discussion

In this animal study, we successfully acquired laser speckle
contrast images during intestinal surgery using the PerfusiX-
Imaging laparoscopic LSCI setup. With this laparoscopic
perfusion imager, we were able to detect both small and

<figure><img src="image_3.png" /></figure>
large intestinal perfusion deficits in real time. This was in
contrast to visible light, which only allowed for the detection
of compromised intestinal perfusion no sooner than 30 min.

Measurement of LCL levels was chosen as an objective
reference for LSCI findings. LCL reflects tissue oxygena-
tion status of intestinal cells, and its use is validated in both
experimental and clinical perfusion imaging studies [11, 16,
17]. In this study we observed a clear correlation between
LSPU values and lactate levels in larger ischemic loops. As
anticipated, impaired perfusion was accompanied by a rise
in lactate levels. However, this relation was less apparent
in smaller ischemic loops, in which (relative) changes in
LSPU values were not accompanied by changes in LCL.
The overall logarithmic correlation of combined small and
large areas could indicate that only below a critical level of
perfusion, tissue becomes hypoxic, causing a steep rise in
LCL. This phenomenon is commonly seen in other hemody-
namic processes (e.g., hemodilution and venous-to-arterial
carbon dioxide gradient related to dysoxygenation [18], i.e.,
an imbalance between oxygen supply and demand. Future
studies into this cut-off value may support clinical decision-
making to prevent irreversible tissue damage.

Observed characteristics of LSCI seem of particular
interest during construction of intestinal anastomoses. In
this setting, an adequate intraoperative assessment of intes-
tinal microperfusion is required to ascertain the viability of
the newly formed anastomosis to prevent ischemia-related
complications such as AL. Traditional visual inspection by
the surgeon has proven to be highly subjective and has little
predictive value [7]. This fueled development of perfusion
imaging techniques such as near-infrared fluorescence imag-
ing, a promising technique which has gained a steep increase
in popularity recent decade [19, 20]. However, a limitation
of fluorescence angiography applications lies in the need for
specific fluorescent dyes. Besides the fact that the injection
of a dye carries certain risks (e.g., allergic reactions and
toxicity), the use of dyes creates an opportunity for error
in the execution and interpretation of perfusion assessment
especially if repeated measurements are required. Indocya-
nine green (ICG) fluorescence angiography, for instance,
requires the binding of ICG to blood proteins in order to
visualize blood flow. When used incorrectly, ICG fluores-
cence angiography measures the binding of the fluorescent
dye to blood proteins rather than the flow of red blood cells
[21]. This could lead to a situation where the measured fluo-
rescence intensity is caused by a difference in dye concentra-
tion rather than by a change in blood flow. Advantages of
LSCI over fluorescence angiography include the ability to
repetitively and continuously measurement of perfusion as
there is no wash-out effect. In addition, LSCI is a full-field
imaging modality and allows for real time and direct quan-
tification of entire organ perfusion. Laparoscopic LSCI is
still an uncommon perfusion imaging technique that requires

further clinical validation. Next steps should include stud-
ies with regards to the ability to detect perfusion deficits in
colorectal surgery and ultimately the effect of laparoscopic
LSCI on anastomotic leakage rates.

Despite succeeding to detect intestinal perfusion defi-
cits using the PerfusiX-imaging laparoscopic LSCI setup,
some limitations regarding this study need to be addressed.
Although we performed multiple measurements on 4 bowel
loops, we only used one animal. Therefore, the data pre-
sented in this study should be treated with caution as they
are limited to allow for solid conclusions. As our results
contain no unreasonable outliers, we do expect to see similar
results in a larger experiment. Moreover, to respect the direc-
tives on animal experimentation and the 3 R's (Replace-
ment, Reduction, Refinement) [22], the current set up was
deemed sufficient to study the feasibility of this novel tech-
nique. The second limitation regards the choice of a small
intestinal model. AL more frequently occurs in colorectal
anastomoses than in small intestinal anastomoses [4], which
merits a higher need for an adequate perfusion evaluation
during surgery of the large intestine. Unfortunately, the
spiral-like orientation of the porcine colon is not fitting for
the creation of intestinal loops, hence the choice of our cur-
rent model. Nevertheless, the porcine model does allow for
the best translation of experimental results, as similarity to
human intestinal physiology is high [23]. Another limita-
tion is that throughout perfusion assessments in this study
the distance between the camera and the bowel loops was
kept constant to minimize data variations. Probably, in a
clinical setting, this distance cannot be maintained at a fixed
distance, which could potentially lead to altered perfusion
assessment. Therefore, the system should be further studied
in a minimally invasive setting, as this would be the clinical
application. These studies should also focus on the normali-
zation and quantification of the LSPU findings.

Altogether, the demonstrated feasibility of using laparo-
scopic LSCI to evaluate intestinal perfusion during surgery,
together with the convincing results of this experimental
study, trigger enthusiasm for a rapid clinical translation of
this technology. However, the fine-tuning of the PerfusiX-
Imaging technology, as well as additional experiments in
both animal and human studies are required to further dem-
onstrate the anticipated clinical usefulness.

Conclusion

In conclusion, laparoscopic LSCI setup can achieve a real-
time intraoperative visualization of intestinal perfusion defi-
cits, allowing for accurate prediction of long-term postopera-
tive ischemic complications. With this revealing capacity,
LSCI could potentially facilitate surgical decision-making
when constructing intestinal anastomoses in order to mitigate ischemia-related complications such as AL.

**Acknowledgements** The authors would like to thank the central animal facility of Maastricht University for their valuable assistance in conducting this animal PerfusiX-imaging experiment. The Olympus laparoscopic video system was kindly provided by Olympus. Additionally, the authors want to thank Anna Sieben for her help creating Figure 1.

**Author contribution** WH and MA designed the experimental protocol. MA, AW, and NB performed the surgical procedure and LCL sampling. WH and JC acquired imaging data. WH and JC analyzed laser speckle contrast images. WH and ECB performed statistical analysis. AW and WH wrote the main manuscript and prepared all figures and tables. All authors critically reviewed and approved the manuscript.

## Declarations

**Conflict of interest** Drs. Joost Calon is inventor of the PerfusiX-Imaging technology and drs. Wido Heeman is part of the PerfusiX-imaging project, both authors have no financial interest in the project. Drs. Autelia Wildeboer, dr. Mahdi Al-Taher, dr. Michele Diana, dr. Joep Derikx, dr. Christiaan Boerma, professor dr. Laurents Stassen, professor dr. Gooitzen van Dam and professor dr. Nicole Bouvy have no conflicts of interest or financial ties to enclose.

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