Background: Dementia is a global public health concern, with prevalence projected to reach 78 million individuals by 2030 and 139 million by 2050. Most persons living with dementia reside in community settings and are supported by family caregivers. As caregiving demands grow, caregivers experience significant psychosocial, emotional, and financial burden, including high rates of stress, social isolation, and depressive symptoms. Access to effective support services remains limited, highlighting the urgent need for innovative and accessible caregiver interventions. Objective: This pilot study first aims to assess the feasibility, acceptability, and tolerability of VR-SIM Carers, a virtual reality (VR)-based psychoeducational training program for family caregivers or care partners of people living with dementia. Second, it will aim to provide a preliminary evaluation of potential impact on caregiver outcomes, including caregiver competence, stress, resilience, empathy, and quality of life. The study is not designed to support causal inference regarding the effectiveness of VR-SIM Carers. Methods: A mixed methods design will be used with a sample of 30 family caregivers of people living with dementia. Participants will complete 3 immersive, self-paced VR caregiving scenarios-Managing Apathy, Crisis Response, and Refusal of Care-receiving real-time feedback from simulated characters, including clinician and person living with dementia avatars. Primary outcomes (feasibility and educational impact) include recruitment, retention, adherence, usability, acceptability, and tolerability and caregiver competence (Pearlin Caregiving Competence Scale), perceived stress (Cohen Perceived Stress Scale), resilience (Connor-Davidson Resilience Scale), and empathy (Empathy Assessment Scale). Secondary outcomes (preliminary efficacy) include caregiver quality of life (Adult Carer Quality of Life Questionnaire), caregiver burden (Burden Questionnaire), and behavioral symptoms (Neuropsychiatric Inventory, Center for Epidemiologic Studies Depression Scale Short Form) assessed at baseline, postintervention, and 1-month follow-up. Feasibility and user engagement will be evaluated via the 18-item Gaming Use Engagement and Severity Scale, Igroup Presence Questionnaire, qualitative interviews, reflection notes, and open-ended feedback. Quantitative data will be analyzed using repeated-measures ANOVA and paired 2-tailed t tests, while qualitative data will be analyzed using an inductive thematic coding framework. Data analyses are descriptive and exploratory only, and no causal claims regarding intervention effectiveness will be made. Consistent with CONSORT (Consolidated Standards of Reporting Trials) guidance for pilot and feasibility studies, caregiver outcomes (eg, competence, stress, resilience, empathy) are treated as exploratory. Results: The findings from this study will inform the feasibility, acceptability, and educational value of immersive VR for caregiver training, while providing preliminary evidence regarding the efficacy of VR-SIM Carers as a training tool to improve psychoeducation outcomes for family caregivers of people living with dementia and reduce caregiving burden. Data collection commenced in March 2024 with a projected end date of March 2026. As of the submission of the manuscript (December 2025), 21 participants have been enrolled. Data analysis will be completed in April 2026, and the results are expected to be published in fall 2026. Conclusions: VR-SIM Carers represents an innovative, scalable intervention designed to enhance caregiver preparedness, psychosocial outcomes, and sustainable community-based dementia care. This pilot study will provide critical evidence to guide further development and implementation of VR-based caregiver support programs.

BACKGROUND AND OBJECTIVES:Preoperative anxiety is common before surgery and is associated with adverse outcomes, yet access to mental health support remains limited. We evaluated the feasibility and acceptability of a novel preoperative virtual reality (VR) prototype designed to reduce anxiety in patients undergoing cancer surgery. METHODS:In a multi-method feasibility and pilot trial, participants were randomized to either the VR intervention or standard of care (SoC) between 2021 and 2023. VR participants completed a simulation 1-2 weeks before surgery exploring the operating room (OR) and experiencing anesthesia induction. Intervention feasibility and acceptability (primary outcomes), and preliminary trends in anxiety and distress (secondary outcomes) were explored using measures and open-ended questions administered at four perioperative timepoints. RESULTS:Of 33 interested individuals, 27 were randomized, and 23 completed the study (VR: n = 12; SoC: n = 11). No adverse VR effects occurred. Most participants reported elevated preoperative anxiety (78.3%-87%), rated the VR as helpful (M = 80.4%), enjoyable (M = 87.7%), and all viewed it as worthwhile. Six qualitative themes emerged: preparation for the OR, psychological and emotional impact, realism, interactivity, technical issues, and supporting others. Trends suggested reduced postoperative distress and anxiety for the VR group. CONCLUSION:The VR intervention was feasible and acceptable, supporting further development and advancement to a larger clinical trial. TRIAL REGISTRATION:The clinical trial was registered at clinicaltrials.gov: https://clinicaltrials.gov/study/NCT04544618 on September 10th, 2020.

BackgroundIn collaboration with clinical domain experts, we developed a prototype of immersive virtual reality (VR) cognitive remediation for major depressive disorder (bWell-D). In the development of a new digital intervention, there is a need to determine the effective components and clinical relevance using systematic methodologies. From an implementation perspective, the effectiveness of digital intervention delivery is challenged by low uptake and high noncompliance rates. Gamification may play a role in addressing this as it can boost adherence. However, careful consideration is required in its application to promote user motivation intrinsically. ObjectiveWe aimed to address these challenges through an iterative process for development that involves co-design for developing content as well as in the application of gamification while also taking into consideration behavior change theories. This effort followed the methodological framework guidelines outlined by an international working group for development of VR therapies. MethodsIn previously reported work, we collected qualitative data from patients and care providers to understand end-user perceptions on the use of VR technologies for cognitive remediation, reveal insights on the drivers for behavior change, and obtain suggestions for changes specific to the VR program. In this study, we translated these findings into concrete representative software functionalities or features and evaluated them against behavioral theories to characterize gamification elements in terms of factors that drive behavior change and intrinsic engagement, which is of particular importance in the context of cognitive remediation. The implemented changes were formally evaluated through user trials. ResultsThe results indicated that feedback from end users centered on using gamification to add artificial challenges, personalization and customization options, and artificial assistance while focusing on capability as the behavior change driver. It was also found that, in terms of promoting intrinsic engagement, the need to meet competence was most frequently raised. In user trials, bWell-D was well tolerated, and preliminary results suggested an increase in user experience ratings with high engagement reported throughout a 4-week training program. ConclusionsIn this paper, we present a process for the application of gamification that includes characterizing what was applied in a standardized way and identifying the underlying mechanisms that are targeted. Typical gamification elements such as points and scoring and rewards and prizes target motivation in an extrinsic fashion. In this work, it was found that modifications suggested by end users resulted in the inclusion of gamification elements less commonly observed and that tend to focus more on individual ability. It was found that the incorporation of end-user feedback can lead to the application of gamification in broader ways, with the identification of elements that are potentially better suited for mental health domains.

[This corrects the article DOI: 10.7759/cureus.64082.].

BackgroundClinically elevated preoperative distress and anxiety are common among patients undergoing cancer surgery. Preoperative interventions have been developed to mitigate this distress and anxiety but are inconsistent in efficacy and feasibility for broad implementation. ObjectiveThis preliminary pilot study aims to assess the feasibility and utility of a newly developed virtual reality (VR) intervention to expose patients awaiting breast cancer surgery to the operating room environment and a simulation of anesthetic induction. MethodsPatients undergoing breast cancer surgery (N=7) were assigned to the VR intervention or control (treatment as usual) group and completed self-report measures of distress and anxiety before surgery, on the day of surgery, and after surgery (5 and 30 d postoperatively). Those in the intervention group trialed the VR simulation 1 to 2 weeks preoperatively and provided qualitative and quantitative feedback. We assessed the feasibility of recruitment capability and study design and evaluated participants’ impressions of the intervention using self-report rating scales and open-ended questions. We also descriptively examined distress and anxiety levels throughout the duration of the study. ResultsRecruitment occurred between December 2021 and December 2022 and progressed slowly (rate: 1 participant/7 wk on average; some hesitancy because of stress and being overwhelmed). All participants who consented to participate completed the entire study. All participants were female and aged 56 (SD 10.56) years on average. In total, 57% (4/7) of the participants were assigned to the intervention group. On average, intervention participants spent 12 minutes engaged in the VR simulation. In general, the intervention was rated favorably (eg, clear information, enjoyable, and attractive presentation; mean% agreement 95.00-96.25, SD 4.79-10.00) and as helpful (mean% agreement 87.50, SD 25.00). Participants described the intervention as realistic (eg, “It was realistic to my past surgical experiences”), impacting their degree of preparedness and expectations for surgery (eg, “The sounds and sights and procedures give you a test run; they prepare you for the actual day”), and having a calming or relaxing effect (eg, “You feel more relaxed for the surgery”). ConclusionsThis preoperative VR intervention demonstrated preliminary feasibility among a sample of patients undergoing breast cancer surgery. Results and participant feedback will inform modifications to the VR intervention and the study design of a large-scale randomized controlled trial to examine the efficacy of this intervention. Trial RegistrationClinicalTrials.gov NCT04544618; https://clinicaltrials.gov/study/NCT04544618

Background Preoperative state anxiety (PSA) is distress and anxiety directly associated with perioperative events. PSA is associated with negative postoperative outcomes such as longer hospital length of stay, increased pain and opioid use, and higher rates of rehospitalization. Psychological prehabilitation, such as education, exposure to hospital environments, and relaxation strategies, has been shown to mitigate PSA; however, there are limited skilled personnel to deliver such interventions in clinical practice. Immersive virtual reality (VR) has the potential for greater accessibility and enhanced integration into an immersive and interactive experience. VR is rarely used in the preoperative setting, but similar forms of stress inoculation training involving exposure to stressful events have improved psychological preparation in contexts such as military deployment. Objective This study seeks to develop and investigate a targeted PSA intervention in patients undergoing oncological surgery using a single preoperative VR exposure. The primary objectives are to (1) develop a novel VR program for patients undergoing oncological surgery with general anesthesia; (2) assess the feasibility, including acceptability, of a single exposure to this intervention; (3) assess the feasibility, including acceptability, of outcome measures of PSA; and (4) use these results to refine the VR content and outcome measures for a larger trial. A secondary objective is to preliminarily assess the clinical utility of the intervention for PSA. Methods This study comprises 3 phases. Phase 1 (completed) involved the development of a VR prototype targeting PSA, using multidisciplinary iterative input. Phase 2 (data collection completed) involves examining the feasibility aspects of the VR intervention. This randomized feasibility trial involves assessing the novel VR preoperative intervention compared to a VR control (ie, nature trek) condition and a treatment-as-usual group among patients undergoing breast cancer surgery. Phase 3 will involve refining the prototype based on feasibility findings and input from people with lived experience for a future clinical trial, using focus groups with participants from phase 2. Results This study was funded in March 2019. Phase 1 was completed in April 2020. Phase 2 data collection was completed in January 2024 and data analysis is ongoing. Focus groups were completed in February 2024. Both the feasibility study and focus groups will contribute to further refinement of the initial VR prototype (phase 3), with the final simulation to be completed by mid-2024. Conclusions The findings from this work will contribute to the limited body of research examining feasible and broadly accessible interventions for PSA. Knowledge gained from this research will contribute to the final development of a novel VR intervention to be tested in a large population of patients with cancer before surgery in a randomized clinical trial. Trial Registration ClinicalTrials.gov NCT04544618; https://www.clinicaltrials.gov/study/NCT04544618 International Registered Report Identifier (IRRID) DERR1-10.2196/55692

The VR-SIM Carers project is focused on developing a virtual reality-based simulation training platform (VRSTP) where dementia caregivers/care partners may safely practice different strategies and approaches to difficult caregiving scenarios without real-world consequences within an interactive 3D virtual world. Since no two caregiver situations are alike, it is necessary to build multiple VR scenarios. However, developing each scenario is time-consuming, requiring extensive programming knowledge. Here, we briefly outline our preliminary work developing a graphical-based, “no-code” authoring platform that allows educators and clinicians with limited, if any, programming experience to develop new, or modify existing scenarios specific to VR-SIM Carers.

This paper describes the development of a novel immersive virtual reality (VR) simulation designed to reduce preoperative state anxiety in patients undergoing breast cancer surgery. A custom interactive VR simulation allows participants to experience the setting of an operating room and the key preoperative stages, all the way through the administration of general anesthesia. Interactivity is provided through a self-avatar with which the various simulated medical personnel interact directly. We evaluate the capacity of the simulation to induce an emotional response as measured by the participants' galvanic skin response (GSR). To our knowledge, this is the first fully interactive simulation of an oncology surgery induction procedure for stress inoculation, and the first preoperative VR study to measure emotional impact using GSR. Out of a larger trial, we analyzed 6 participants who had been randomized to the simulation group and for whom baseline and intra-simulation GSR data had been successfully acquired. Three-minute samples were compared for statistical difference with a 95% confidence interval on the mean. 5 out of 6 showed a statistically significant and visually noticeable increase in GSR, and participants reported a high sense of spatial presence. Early results are encouraging, showing that the described simulation can induce a physiological response consistent with the participants' subjective evaluation of presence. While this was a limited experiment, it provides a basis for a larger trial to be conducted in the future

Background Immersive technologies like virtual reality can enable clinical care that meaningfully aligns with real-world deficits in cognitive functioning. However, options in immersive 3D environments are limited, partly because of the unique challenges presented by the development of a clinical care platform. These challenges include selecting clinically relevant features, enabling tasks that capture the full breadth of deficits, ensuring longevity in a rapidly changing technology landscape, and performing the extensive technical and clinical validation required for digital interventions. Complicating development, is the need to integrate recommendations from domain experts at all stages. Objective The Cognitive Health Technologies team at the National Research Council Canada aims to overcome these challenges with an iterative process for the development of bWell, a cognitive care platform providing multisensory cognitive tasks for adoption by treatment providers. Methods The team harnessed the affordances of immersive technologies while taking an interdisciplinary research and developmental approach, obtaining active input from domain experts with iterative deliveries of the platform. The process made use of technology readiness levels, agile software development, and human-centered design to advance four main activities: identification of basic requirements and key differentiators, prototype design and foundational research to implement components, testing and validation in lab settings, and recruitment of external clinical partners. Results bWell was implemented according to the findings from the design process. The main features of bWell include multimodal (fully, semi, or nonimmersive) and multiplatform (extended reality, mobile, and PC) implementation, configurable exercises that pair standardized assessment with adaptive and gamified variants for therapy, a therapist-facing user interface for task administration and dosing, and automated activity data logging. bWell has been designed to serve as a broadly applicable toolkit, targeting general aspects of cognition that are commonly impacted across many disorders, rather than focusing on 1 disorder or a specific cognitive domain. It comprises 8 exercises targeting different domains: states of attention (Egg), visual working memory (Theater), relaxation (Tent), inhibition and cognitive control (Mole), multitasking (Lab), self-regulation (Butterfly), sustained attention (Stroll), and visual search (Cloud). The prototype was tested and validated with healthy adults in a laboratory environment. In addition, a cognitive care network (5 sites across Canada and 1 in Japan) was established, enabling access to domain expertise and providing iterative input throughout the development process. Conclusions Implementing an interdisciplinary and iterative approach considering technology maturity brought important considerations for the development of bWell. Altogether, this harnesses the affordances of immersive technology and design for a broad range of applications, and for use in both cognitive assessment and rehabilitation. The technology has attained a maturity level of prototype implementation with preliminary validation carried out in laboratory settings, with next steps to perform the validation required for its eventual adoption as a clinical tool.

Remote patient monitoring has only become more relevant in the current pandemic context. Contactless sensing extracts vital signs (ex. heart rate) and other physiological information from signals obtained from non-contact hardware such as video cameras. Such a technology could have a significant impact on the healthcare system by enabling a physician to remotely acquire quantitative measurements of a patient. This could help prevent sudden deterioration of patient condition, improve the efficiency and support for medical decision making and diagnostics, and reduce costs and hospitalizations. In this work, a practical and fully automatic video-based contactless monitoring framework for the evaluation of patient heart rate is presented. The framework allows the calculation of heart rate in quasi real-time and with a high accuracy, using a consumer grade laptop equipped with a usb-connected webcam. The proposed heart rate assessment algorithm combines state-of-the-art methods in video- and signal-processing for contactless sensing; to attain high accuracy predictions, an additional probabilistic formulation that improves the temporal consistency is proposed and used along with live error rejection. To validate the framework, twenty-two subjects were recorded under varying physiological states (total of 111 two-minute long videos). Heart rate predictions were compared to medical-grade sensors and showed an accuracy of $-0.17\pm 1.81$ bpm with a MAE of 0.91 bpm.

Structural proteins like collagen and elastin are major constituents of the extracellular matrix (ECM). ECM degradation and remodeling in diseases significantly impact the microorganization of these structural proteins. Therefore, tracking the changes of collagen and elastin fiber morphological features within ECM impacted by disease progression could provide valuable insight into pathological processes such as tissue fibrosis and atherosclerosis. Benefiting from its intrinsic high-resolution imaging power and superior biochemical specificity, nonlinear optical microscopy (NLOM) is capable of providing information critical to the understanding of ECM remodeling. In this study, alterations of structural fibrillar proteins such as collagen and elastin in arteries excised from atherosclerotic rabbits were assessed by the combination of NLOM images and textural analysis methods such as fractal dimension (FD) and directional analysis (DA). FD and DA were tested for their performance in tracking the changes of extracellular elastin and fibrillar collagen remodeling resulting from atherosclerosis progression/aging. Although other methods of image analysis to study the organization of elastin and collagen structures have been reported, the simplified calculations of FD and DA presented in this work prove that they are viable strategies for extracting and analyzing fiber-related morphology from disease-impacted tissues. Furthermore, this study also demonstrates the potential utility of FD and DA in studying ECM remodeling caused by other pathological processes such as respiratory diseases, several skin conditions, or even cancer. NEW & NOTEWORTHY Textural analyses such as fractal dimension (FD) and directional analysis (DA) are straightforward and computationally viable strategies to extract fiber-related morphological data from optical images. Therefore, objective, quantitative, and automated characterization of protein fiber morphology in extracellular matrix can be realized by using these methods in combination with digital imaging techniques such as nonlinear optical microscopy (NLOM), a highly effective visualization tool for fibrillar collagen and elastic network. Combining FD and DA with NLOM is an innovative approach to track alterations of structural fibrillar proteins. The results illustrated in this study not only prove the effectiveness of FD and DA methods in extracellular protein characterization but also demonstrate their potential value in clinical and basic biomedical research where protein microstructure characterization is critical.

A porcine burn model was used to demonstrate the potential of using the attenuation of the optical coherence tomography signal as an indicator of the degree of burn injury.A swept-source optical coherence tomography system amenable to operating in burn trauma centers and in surgeries was designed and built in-house.A special design feature of our optical coherence tomography system is its large stand-off distance (272 mm) between the data collecting lens terminating the sample arm and the sample surface.This feature eliminates potential contact between patient and instrument, and thereby decreases the potential for infection through remote contamination.Four contact burn types, histologically confirmed to produce superficial, superficial-intermediate, deeper-intermediate and full thickness injuries, were investigated.Burn types and matching control sites were randomly placed over a predetermined grid.The burn injuries were restricted to under 1% of the total body surface area in order to minimize systemic effects and eliminate cross-talk between the measurement sites on the grid.At each grid location, the data was acquired very fast, amounting to total acquisition times of less than 3 seconds per data set, speeds practical for use in the high-stress environment characterizing burn trauma units.We used attenuation coefficients of optical coherence tomography signal in tissue as a summary parameter for burn injury determination.This simple parameter can be computed in real-time during the OCT acquisition sequence across the burn wound providing the clinician a real-time assessment of the degree of injury.

Early detection of incipient caries would allow dentists to provide more effective measures to delay or to reverse caries’ progression at earlier stage. Such earlier intervention could lead to improved oral health for the patients and reduced burden to the health system. Previously, we have demonstrated that the combination of morphological and biochemical information furnished by optical coherence tomography (OCT) and polarized Raman spectroscopy (PRS), respectively, provided a unique tool for dental caries management. In this study we will report the first pre-clinical caries detection system that includes a hand-held probe with a size slightly larger than a tooth brush. This probe presents a novel platform combining both OCT and PRS optics in a very tight space ideal for clinical practice. OCT cross-sectional images of near-surface enamel morphology are obtained with miniaturized MEMS scanning device and are processed in real-time to identify culprit regions. These regions are sequentially analyzed with polarized Raman spectroscopy for further confirmation. PRS is performed using 830nm laser line and four detection channels in order to obtain polarized Raman spectroscopic data, i.e. depolarization ratio of the hydroxyapatite Raman band at ~960 cm-1. A detailed description of this hand-held caries detector and ex-vivo/in-vivo test results will be presented.

We tested the imaging capabilities for variants of a 1550-nm swept-source fiber-based optical coherence tomography system with a telecentric system incorporated at the end of its sample arm. The system was designed for in vivo imaging of burns; therefore, we acquired images from samples located at distances greater than 24 cm from the exit of the telecentric system. Each system variation had a specific combination of diameters for the reference and sample beams. In the reference arm, we used, alternately, two collimated beams with diameters of 1.5 and 14 mm, respectively. In the sample arm, we tested collimated beams with the following diameters: 1.5, 3.5, 5.7, 8.4, and 14 mm. A galvanometric mirror system scanned the collimated sample beam across the entrance pupil of the telecentric system. The sample beam exited the telecentric system parallel with its optical axis and convergent onto the sample. Depending on the collimator used in the sample arm, images were acquired with beams focused to waist diameters ranging from 40 to 240 μm. We acquired images with the sample at different locations within a ±30 mm range centered about the sample beam waist. Furthermore, we used the signal-to-noise ratio, the detected signal intensity, and the visual appearance to compare images acquired with different sample/reference beam configurations.

Luminal atherosclerosis imaging was demonstrated by multimodal femtosecond CARS microscopy (MM-CARS). Using a myocardial infarction-prone rabbit model of atherosclerosis, this study demonstrated the utility of multimodal CARS imaging in determining atherosclerotic plaque burden through two types of image analysis procedures. Firstly, multimodal CARS images were evaluated using a signal-intensity parameter based on intensity changes derived from the multi-channel data (e. g. TPEF, SHG and CARS) to classify plaque burden within the vessel. Secondly, the SHG images that mainly correspond to collagen fibrils were evaluated using a texture analysis model based on the first-order statistical (FOS) parameters of the image histogram. Correlation between FOS parameters of collagen images with atherosclerosis plaque burden was established. A preliminary study of using spectroscopic CARS in identifying the different lipid components within the plaque was also discussed.

The composition and structure of atherosclerotic lesions can be directly related to the risk they pose to the patient. Multimodal nonlinear optical (NLO) microscopy provides a powerful means to visualize the major extracellular components of the plaque that critically determine its structure. Textural features extracted from NLO images were investigated for their utility in providing quantitative descriptors of structural and compositional changes associated with plaque development. Ten texture parameters derived from the image histogram and gray level co-occurrence matrix were examined that highlight specific structural and compositional motifs that distinguish early and late stage plaques. Tonal-texture parameters could be linked to key histological features that characterize vulnerable plaque: the thickness and density of the fibrous cap, size of the atheroma, and the level of inflammation indicated through lipid deposition. Tonal and texture parameters from NLO images provide objective metrics that correspond to structural and biochemical changes that occur within the vessel wall in early and late stage atherosclerosis.

Label-free imaging of bulk arterial tissue is demonstrated using a multimodal nonlinear optical microscope based on a photonic crystal fiber and a single femtosecond oscillator operating at 800 nm. Colocalized imaging of extracellular elastin fibers, fibrillar collagen, and lipid-rich structures within aortic tissue obtained from atherosclerosis-prone myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits is demonstrated through two-photon excited fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering, respectively. These images are shown to differentiate healthy arterial wall, early atherosclerotic lesions, and advanced plaques. Clear pathological changes are observed in the extracellular matrix of the arterial wall and correlated with progression of atherosclerotic disease as represented by the age of the WHHLMI rabbits.

lex C. T. Ko,* Andrew Ridsdale, Michael S. D. Smith, eila B. Mostaço-Guidolin, Mark D. Hewko, drian F. Pegoraro, Elicia K. Kohlenberg, ernie Schattka, Masashi Shiomi, Albert Stolow, and ichael G. Sowa National Research Council Canada, Institute for iodiagnostics, 435 Ellice Avenue, Winnipeg, Manitoba, anada R3B 1Y6 National Research Council Canada, Steacie Institute for olecular Sciences, 100 Sussex Drive, Ottawa, ntario, Canada K1A 0R6 Kobe University School of Medicine, Institute of xperimental Animals, 7-5-1, Kusunoki-cho, Chuo-ku, obe 650-0017, Japan

A femtosecond CARS-based nonlinear optical microscope was used to simultaneously image extracellular structural proteins and lipid-rich structures within intact aortic tissue obtained from myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits (WHHLMI). Clear differences in the NLO microscopic images were observed between healthy arterial tissue and regions dominated by atherosclerotic lesions. In the current ex-vivo study, we present a single parameter based on intensity changes derived from multi-channel NLO image to classify plaque burden within the vessel. Using this parameter we were able to differentiate between healthy regions of the vessel and regions with plaque, as well as distinguish plaques relative to the age of the WHHLMI rabbit.