Radiol Oncol 2024; 58(3): 326-334. doi: 10.2478/raon-2024-0042 326 review Laser speckle contrast imaging of perfusion in oncological clinical applications: a literature review Rok Hren1,2,3, Simona Kranjc Brezar4, Urban Marhl2, Gregor Sersa4 1 Faculty of Mathematics and Physics, Ljubljana, Slovenia 2 Institute of Mathematics, Physics, and Mechanics, Ljubljana, Slovenia 3 Syreon Research Institute, Budapest, Hungary 4 Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2024; 58(3): 326-334. Received 16 July 2024 Accepted 26 July 2024 Correspondence to: Rok Hren, Ph.D., Faculty of Mathematics and Physics, University of Ljubljana, Jadranska ulica 19, SI-1000 Ljubljana, Slovenia. E-mail: rok.hren@fmf.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Laser speckle coherence imaging (LSCI) is an emerging imaging modality that enables noninvasive visualization and assessment of tissue perfusion and microcirculation. In this article, we evaluated LSCI in imaging per- fusion in clinical oncology through a systematic review of the literature. Methods. The inclusion criterion for the literature search in PubMed, Web of Science and Scopus electronic data- bases was the use of LSCI in clinical oncology, meaning that all animal, phantom, ex vivo, experimental, research and development, and purely methodological studies were excluded. Results. Thirty-six articles met the inclusion criteria. The anatomic locations of the neoplasms in the selected articles were brain (5 articles), breasts (2 articles), endocrine glands (4 articles), skin (12 articles), and the gastrointestinal tract (13 articles). Conclusions. While LSCI is emerging as an appealing imaging modality, it is crucial for more clinical sites to initiate clinical trials. A lack of standardized protocols and interpretation guidelines are posing the most significant challenge. Key words: laser speckle contrast imaging (LSCI); oncology; perfusion; blood flow Introduction In the cancer research and treatment, the assess- ment of tissue perfusion and microcirculation plays a pivotal role in understanding tumor physi- ology, monitoring treatment responses, and deter- mining surgical outcomes. Among the advanced visualization systems, fluorescence angiography utilizing indocyanine green (FA-ICG) has emerged as an objective tool for evaluating intraoperative perfusion.1-3 Despite its versatility, FA-ICG imaging has limitations: for example, it requires external dye injection, is constrained by pharmacokinetic factors in repeat assessments, and may potentially lead to allergic reactions to the dye.2 To overcome these shortcomings, novel imaging techniques have been explored for microvascular imaging. One such modality is laser speckle contrast imaging (LSCI), a non-invasive optical imaging technique based on the unique properties of laser light to visualize blood flow and tissue perfusion in real-time.4,5 At the core of LSCI lies the phe- nomenon of capturing the dynamic interference pattern, known as speckle, created when coherent laser light interacts with moving particles such as red blood cells, generating a real-time 2D color Radiol Oncol 2024; 58(3): 326-334. Hren R et al. / Laser speckle contrast imaging in oncology 327 heatmap of blood flow (Figure 1).6 By analyzing the temporal fluctuations in the speckle pattern, LSCI can quantitatively assess blood flow velocity, perfusion dynamics, and tissue microcirculation with high spatial and temporal resolution. LSCI is a versatile modality with its applicabil- ity ranging from material science7 to notable ap- plications in medical therapeutic segments.8 LSCI has aided, among others, in studying retinal blood flow9, cardiovascular diseases10,11 and organ perfu- sion6,12, while demonstrating potential as a valua- ble tool for assessing burns13-15 and wound healing processes16-18, and monitoring perfusion during reconstructive surgery19 and neurosurgery.20-26 The value of LSCI in quantifying blood flow dynamics within clinical oncology remains unclear, and to that end, we systematically reviewed the literature with a specific focus on studies in which LSCI was conducted on patients in a clinical oncology set- ting. Methods Authors conducted jointly—to minimize potential bias—a comprehensive literature search on April 16, 2024, through PubMed, Web of Science and Scopus electronic databases using the following search terms: “laser speckle coherence imaging tu- mors”, “laser speckle coherence imaging cancer”, “laser speckle coherence imaging carcinoma”, “la- ser speckle coherence imaging anastomosis”, and “laser speckle coherence imaging thyroid”. No re- strictions on publication date or language were im- posed. The inclusion criterion was the application of LSCI in a clinical oncological setting, meaning that all animal and phantom, ex vivo, experimen- tal, research and development, and purely meth- odological studies were excluded. Special care was taken to remove duplicates across databases and studies; for example, if the study was first pub- lished in proceedings and later in a journal, the proceedings article was considered a non-primary publication and therefore excluded. Studies were categorized with respect to the anatomical location of the tumors. Results In total, 309 articles were found to be of interest in the PubMed, Web of Science and Scopus data- bases. After excluding duplicates and applying the exclusion criteria, first considering the title and abstract and then, if necessary, reading the en- tire article, 36 articles were identified for further analysis. The anatomical locations of tumors in the selected articles were as follows: brain (5 articles), breasts (2 articles), endocrine glands (4 articles), skin (12 articles), and the gastrointestinal (GI) tract (13 articles). Brain Parthasarathy et al.21 made a pioneering effort in the evaluation of perfusion in clinical oncology using LSCI. Their pilot study focused on imaging cerebral blood flow either before (1 patient) or after (2 patients) tumor resections, across various corti- cal regions. The same group continued research on larger patient groups (10 and 8, respectively), dem- onstrating the feasibility of using LSCI to monitor blood flow during neurosurgery.22,27 Despite these promising outcomes, their research output ceased after 2017. Another research group25 highlighted the po- tential of LSCI for functional brain mapping during awake craniotomy for tumor removal. They observed a strong correlation between cor- tical microvascular blood flow, as determined by LSCI, and electrocortical stimulation mapping. Additionally, Ideguchi et al.28 emphasized the ca- pability of LSCI for noninvasive and rapid intraop- erative real-time recognition of mass lesion-related FIGURE 1. Schematic representation of the laser speckle contrast imaging (LSCI) method. (A) The technique relies on the interference of light backscattered from moving particles, creating distinct dark and bright areas (speckle pattern) captured by a camera. (B) Variations in the speckle pattern are predominantly driven by the movement of red blood cells, enabling interpretation as perfusion. (C) Analysis of speckle-pattern variations yields an image displayed on the monitor, where white and yellow depict areas with high perfusion, contrasting with darker areas indicating lower perfusion areas. Taken from Berggren et al. 19 and reprinted with permission from the publisher. A B C Radiol Oncol 2024; 58(3): 326-334. Hren R et al. / Laser speckle contrast imaging in oncology328 TABLE 1. Included articles reporting the use of laser speckle contrast imaging (LSCI) to quantify perfusion in clinical applications in oncology Reference Year of publication Number of patients Oncologic setting Brain Parthasarathy et al.21 2010 3 Tumor resection Richards et al.22 2014 10 Tumor resection Richards et al.27 2017 8 Tumor resection Klijn et al.25 2013 8 Tumor resection Ideguchi et al.28 2017 12 Tumor resection Breasts Tesselaar et al.29 2017 15 Adjuvant radiotherapy for stage I-II breast cancer Zötterman et al.30 2020 23 Deep inferior epigastric artery perforator (DIEP) flap surgery Endocrine glands de Paula et al.31 2021 42 Non-functioning adrenal incidentaloma Mannoh et al.32 2017 28 Thyroidectomy/parathyroidectomy Mannoh et al.33 2021 72 Thyroidectomy Mannoh et al.34 2023 21 Thyroidectomy/parathyroidectomy Skin Tchvialeva et al.35 2012 214 lesions Malignant melanoma, squamous cell carcinoma, basal cell carcinoma, melanocytic nevus, seborrheic keratosis Reyal et al.36 2012 12 Basal cell carcinoma Zhang et al.37 2019 12 (total 143) Facial nerve palsy due to nerve tumor (also including other etiology) Zieger et al.38 2021 9 Basal cell carcinoma Tenland et al.39 2019 13 Oculoplastic reconstructive surgery (tarsoconjunctival flaps) Berggren et al.40 2019 9 Oculoplastic reconstructive surgery (tarsoconjunctival flaps) Tenland et al.41 2021 12 Oculoplastic reconstructive surgery after squamous cell carcinoma, basal cell carcinoma, and intradermal nevus Berggren et al.42 2021 7 Oculoplastic reconstructive surgery after squamous cell carcinoma and basal cell carcinoma Berggren et al.43 2021 7 Oculoplastic reconstructive surgery after squamous cell carcinoma and basal cell carcinoma Berggren et al.44 2021 1 Oculoplastic reconstructive surgery Berggren et al.45 2022 7 Oculoplastic reconstructive surgery after squamous cell carcinoma and basal cell carcinoma Stridh et al.46 2024 1 Cutaneous angio-sarcoma Gastrointestinal tract (open surgical setting) Eriksson et al.47 2014 10 Liver resection Milstein et al.48 2016 11 Esophagectomy Ambrus et al.49 2017 45 Esophagectomy Ambrus et al. 50 2017 25 Ivor-Lewis esophagectomy Di Maria et al.51 2017 2 Colorectal resection Jansen et al.52 2018 26 Esophagectomy Kojima et al.53 2019 8 Colorectal resection Kaneko et al.54 2020 36 Colorectal resection (34 due to colorectal carcinoma) Gastrointestinal tract (laparoscopic/ thoracoscopic setting) Heeman et al.55 2019 10 Colorectal resection Kojima et al.56 2020 27 Colorectal resection Slooter et al.57 2020 24 Esophagectomy Heeman et al.58 2023 67 Hemicolectomy and sigmoid resection Nwaiwu et al.59 2023 40 Colectomy, also non-oncological interventions (Roux-en-Y gastric bypass and sleeve gastrectomy) Radiol Oncol 2024; 58(3): 326-334. Hren R et al. / Laser speckle contrast imaging in oncology 329 vasculature, which could be crucial in mitigating ischemic complications and complementing neu- rophysiological monitoring. Breasts Tesselaar et al.29 conducted a study exploring the relationship between radiation exposure and changes in microvascular perfusion in 15 women undergoing adjuvant radiation therapy for stage I-II breast cancer. Their findings suggested that LSCI holds promise as a useful tool for objectively assessing radiation-induced microvascular chang- es in the skin, even before visible changes occur, thereby aiding in the earlier prediction of potential severe reactions. In another prospective clinical pilot study con- ducted across two centers30, LSCI was employed in 23 women undergoing primary, secondary, or tertiary deep inferior epigastric artery perforator (DIEP) procedures, either unilateral or bilateral. Researchers used laser speckle patterns to calcu- late perfusion values in arbitrary units (PU), re- flecting the concentration and mean velocity of red blood cells. Categorizing patients into high (> 30) and low (< 30) PU, they found that all flaps with perfusion < 30 PU immediately after surgery had postoperative complications, necessitating revi- sion in 4 women. These results suggest potential utility of LSCI for early detection of flap necrosis, aiding surgeons in identifying viable parts of the flaps. Traditionally, assessment of flap viability relies on subjective methods like skin color, flap temperature, capillary refill time, and dermal edge bleeding. Endocrine glands Endothelial reactivity60,61 was evaluated by LSCI in patients with mostly benign non-functioning ad- renal incidentaloma.31. Mannoh et al.32 used LSCI to assess parathyroid viability post-thyroidectomy in 20 patients, achieving an accuracy of 91.5% in distinguishing between well vascularized (n = 32) and compromised (n = 27) parathyroid glands compared to visual assessment by an experienced surgeon. Ability to detect vascular compromise with LSCI was further validated in parathyroidec- tomies in 8 patients, showing that this technique could identify parathyroid gland devasculariza- tion before it became visually apparent to the sur- geon. LSCI demonstrated promise as a real-time, contrast-free, objective method to mitigate hy- poparathyroidism after thyroid surgery. Subsequently, Mannoh et al.33 expanded their research, enrolling 72 patients who underwent thyroidectomy. They established an intraoperative speckle contrast threshold of 0.186 to distinguish between normoparathyroid and hypoparathyroid groups with 87.5% sensitivity and 84.4% specific- ity. This threshold served as an indicator of ad- equate parathyroid vascularization, with glands below the value of 0.186 considered adequately perfused (Figure 2). Additionally, Mannoh et al.34 combined LSCI with ICG angiography in 21 patients undergoing thyroidectomy or parathyroidectomy. While both modalities offered similar information on parathy- roid gland blood flow, they suggested advantages of LSCI, including lower costs, non-invasiveness, absence of contraindications, and compatibility with near-infrared autofluorescence (NIRAF) de- tection, which has recently emerged as a reliable technique for intraoperative parathyroid gland lo- calization or confirmation.62-64 Skin Tchvialeva et al.35 applied LSCI to differentiate among 214 skin lesions, encompassing the three major types of skin cancers (malignant melanoma, squamous cell carcinomas, and basal cell carcino- mas – BCCs), and two benign conditions (melano- cytic nevus and seborrheic keratoses). In another FIGURE 2. Speckle contrast demonstrates lower values for well-vascularized parathyroid glands. Lower speckle contrast values indicate greater blood flow due to more blurring of the speckle pattern, while higher contrast values indicate less blood flow. The top row displays representative white light images, and the bottom row shows speckle contrast images of a well-vascularized (left), a compromised (middle), and a devascularized (right) parathyroid gland, with parathyroid glands marked with ellipses. The corresponding speckle contrast values were 0.11, 0.18, and 0.21, respectively. Taken from Mannoh et al. 33 and reprinted with permission from the publisher. Radiol Oncol 2024; 58(3): 326-334. Hren R et al. / Laser speckle contrast imaging in oncology330 early clinical study, LSCI was used to demonstrate that post-occlusive reactive hyperemia could occur in BCC as well.36 Zhang et al.37 explored differences in facial microvascular perfusion between ipsilat- eral and contralateral sides in patients with facial nerve palsy (FNP), observing significant decreases on the ipsilateral side, which improved after treat- ment. In their feasibility study, Zieger et al.38 intro- duced a compact handheld LSCI device, affirming its reliability in assessing BCC. In oculoplastics, Tenland et al.39 and Berggren et al.40 conducted studies using LSCI to monitor perfusion in patients with lower eyelid defects after post-tumor surgery large enough to require a tarsoconjunctival graft. Building on their initial work, the group continued research of employing LSCI in various oculoplastic reconstructive sur- gery procedures. First, Tenland et al.41 monitored perfusion using LSCI in a study in which free bi- lamellar eyelid grafts appeared to be an excellent alternative to the tarsoconjunctival flap procedure in the reconstruction of both upper and lower eyelid defects. Next, Berggren et al.42 noted rapid revascularization of H-plasty procedure flaps within a week postoperatively, attributing it to the pre-existing vascular network of the flap pedicle, rather than significant angiogenesis. In another study, Berggren et al.43 demonstrated complete rep- erfusion of skin grafts in the periorbital area after 7 weeks (Figure 3). Berggren et al.44 also presented a case illustrating nearly complete restoration of rep- erfusion in a rotational full-thickness lower eyelid flap within 5 weeks. Finally, they assessed blood perfusion in glabellar flaps, finding rapid reper- fusion.45 These convincing findings suggest that perioperative LSCI monitoring of perfusion in hu- man periocular flaps and during oculoplastic re- constructive surgery offers an attractive imaging modality for routine clinical use. Not surprising- ly, Stridh et al.46 recently conducted a pilot study comprehensively combining LSCI with two other emerging non-invasive medical imaging modali- ties, hyperspectral imaging 65-67 and photoacoustic imaging68 to monitor not only blood perfusion but also oxygen saturation and the molecular compo- sition of the tissue. Gastrointestinal tract (open surgical setting) The majority of clinical oncology studies with intraoperative LSCI were conducted in an open surgical setting, which we will review first. In an initial pilot clinical study, Eriksson et al.47 as- sessed liver blood perfusion by occluding the portal vein and hepatic artery in ten consecutive patients undergoing liver resection for colorectal liver metastases. This early effort was followed by Milstein et al.48, who evaluated microvascular blood flow during esophagectomy, affirming that intraoperative LSCI offered a non-contact, non-in- vasive approach for real-time analysis of potential anastomotic leakage without requiring a contrast medium. This finding was subsequently corrobo- rated by Ambrus et al. who first performed gastric microvascular perfusion measurements during es- ophagectomy in 45 patients49 and later used LSCI in Ivor-Lewis esophagectomy in 25 patients.50 Di Maria et al.51 explored the feasibility of LSCI in 2 patients undergoing colorectal surgery, while Jansen et al.52 investigated the impact of thoracic epidural anesthesia during esophagectomy, once again demonstrating that LSCI could detect subtle changes in gastric microvascular perfusion in real- time. Another group conducted an additional fea- sibility study of intraoperative LSCI in 8 patients undergoing colorectal surgery.53 Kaneko et al.54 further expanded on these feasibility studies by enrolling 36 patients undergoing colorectal resec- tion, 34 of whom had colorectal carcinoma, aiming to compare demarcation lines determined by LSCI with transection lines where marginal vessels FIGURE 3. Representative examples of laser speckle contrast images, showing the blood perfusion in the free skin grafts, immediately postoperatively (0 weeks), and at follow-up after 1, 3, and 7 weeks. It can be seen that reperfusion occurred simultaneously in the center and periphery of the graft, and that complete reperfusion was achieved after 7 weeks. Taken from Berggren et al. 43 and reprinted with permission from the publisher. Radiol Oncol 2024; 58(3): 326-334. Hren R et al. / Laser speckle contrast imaging in oncology 331 were divided. They found that 58.3% (21/36) of de- marcation lines matched transection lines, with a median distance of 0.0 mm (0.0–12.1 mm) between the demarcation line determined by LSCI and the transection line. Gastrointestinal tract (laparoscopic/ thoracoscopic setting) Heeman et al.55 reported the first intraabdominal application combining a standard laparoscopic surgical setup with LSCI in 10 patients, enabling imaging of intestinal blood flow during a vascular occlusion test. Their findings were corroborated by Kojima et al.56 in a study involving 27 patients (Figure 4). Slooter et al.57 systematically compared four different emerging optical modalities, high- lighting the clinical utility of FA-ICG as the most promising. Recently, Heeman et al.58 tested a com- mercial LSCI system in the oncological clinical set- ting, noting that the system was “non-disruptive of the surgical procedure with an average added surgical time of only 2.5 min and no change in surgical equipment”. They also observed a poten- tial clinical benefit of the LSCI system, with 17% of operating surgeons altering anastomosis loca- tions based on perfusion assessments. Nwaiwu et al.58 evaluated another commercial intraopera- tive system combining LSCI and FA-ICG in mostly non-oncological patients, demonstrating that LSCI identified the same perfusion boundaries as FA- ICG, with anastomoses and gastric remnants ap- pearing well perfused. Discussion Based on this literature review, several advantages of LSCI emerge, including its non-invasive and non-contact nature, short acquisition time, high spatial and temporal resolution, low cost of equip- ment, and simplicity of operation. In the oncologi- cal clinical setting, LSCI holds particular promise for assessing skin flap perfusion post-oculoplastic reconstructive surgery and anastomotic perfu- sion during gastrointestinal reconstruction. While LSCI offers numerous advantages in imaging blood flow dynamics, it is essential to recognize its limitations. Limited penetration depth One of the obvious limitations of LSCI in clinical oncology and medical applications, in general, is its restricted penetration depth. LSCI relies on de- tecting motion contrast generated by moving red blood cells, limiting its applicability to superficial structures. Tumors and lesions located in deeper anatomical locations, such as within organs or soft tissues, may not be adequately visualized due to this limitation, hindering comprehensive evalu- ation and monitoring of oncological conditions. However, studies like that of Stridh et al.46 demon- strate that PAI as a complementary imaging tech- nique can overcome this limitation. Another pos- sibility to potentially consider is the use of optical clearance techniques69 to enhance tissue transpar- ency and improve light penetration depth. Motion artifacts LSCI is susceptible to motion artifacts, which can arise from either involuntary movement of the subject or vibrations in the imaging setup. These artifacts can lead to image distortions and reduced image quality, compromising the accuracy and reliability of LSCI in clinical oncology. To address this, advanced post-processing algorithms are nec- essary to improve image quality. Since motion ar- tifacts are well-known sources of artifacts in LSCI, they have been extensively researched. One pos- sibility is to implement motion compensation tech- niques, such as image stabilization algorithms70 or gating strategies71, which can mitigate the effects of motion artifacts in LSCI. By minimizing motion- induced distortions in the speckle pattern, these FIGURE 4. Typical laser speckle images in two patients. High-resolution laser speckle contrast imaging (LSCI) can indicate the bowel demarcation line at the point of ligation of the marginal vessels. (A) Normal color image before ligating the marginal vessels. (B) LSCI image before ligating the marginal vessels. (C) LSCI image after ligating the marginal vessels. Taken from Kojima et al.56 and reprinted with permission from the publisher. A E F B C D Radiol Oncol 2024; 58(3): 326-334. Hren R et al. / Laser speckle contrast imaging in oncology332 techniques improve the accuracy and reliability of blood flow measurements. Inherent speckle noise The presence of inherent speckle noise in LSCI im- ages can compromise the accuracy and reliability of blood flow measurements, particularly in low- flow regions or under conditions of low contrast. Speckle noise can obscure subtle flow changes and restrict the sensitivity of LSCI in detecting small- scale perfusion variations. Advanced noise reduc- tion algorithms72 offer a solution by effectively suppressing speckle noise and enhancing the sig- nal-to-noise ratio. These algorithms filter out un- wanted noise components while retaining relevant flow information, thereby improving the sensitiv- ity and specificity of LSCI in detecting perfusion changes, even in challenging imaging conditions. Lack of standardized protocols and interpretation A significant limitation of LSCI in clinical oncology is the lack of standardized protocols and interpre- tation guidelines. Varying acquisition settings, im- age processing algorithms, or interpretation meth- odologies across different centers can yield incon- sistent and non-comparable results. Establishing standardized protocols and guidelines tailored to oncology applications would enhance the accura- cy and reproducibility of LSCI findings. Despite its potential, the clinical integration of LSCI faces obstacles, including the standardiza- tion of imaging protocols, validation of its utility in large-scale clinical trials, and integration into existing surgical workflows. Addressing these limitations requires advancements in technology, algorithm refinement, and increased participation of clinical sites in conducting trials. 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