Characterization of Foodborne Pathogens in Biofilms: A Four-Dimensional Structural Dynamics Approach Using HCS-CLSM.

The functional properties of biofilms are intimately related to their spatial architecture. Structural data are therefore of prime importance to dissect the complex social and survival strategies of biofilms and ultimately to improve their control. Confocal laser scanning microscopy (CLSM) is the most widespread microscopic tool to decipher biofilm structure, enabling noninvasive three-dimensional investigation of their dynamics down to the single-cell scale. The emergence of fully automated high content screening (HCS) systems, associated with large-scale image analysis, has radically amplified the flow of available biofilm structural data. In this contribution, we present a HCS-CLSM protocol used to analyze biofilm four-dimensional structural dynamics at high throughput. Meta-analysis of the quantitative variables extracted from HCS-CLSM will contribute to a better biological understanding of biofilm traits.

Cryo-Electron Microscopy in the Study of Antiviral Innate Immunity.

Cryo-electron microscopy is a powerful methodology in structural biology and has been broadly used in high-resolution structure determination for challenging samples, which are not readily available for traditional techniques. In particular, the strength of super macro-complexes and the lack of a need for crystals for cryo-EM make this technique feasible for the structural study of complexes involved in antiviral innate immunity. This chapter presents detailed information and experimental procedures of Cryo-EM for determining the structures of the complexes using STING as an example. The procedures included a sample quality check, high-resolution data acquisition, and image processing for Cryo-EM 3D structure determination.

Comparing spatial distributions of ALA-PpIX and indocyanine green in a whole pig brain glioma model using 3D fluorescence cryotomography.

Significance: ALA-PpIX and second-window indocyanine green (ICG) have been studied widely for guiding the resection of high-grade gliomas. These agents have different mechanisms of action and uptake characteristics, which can affect their performance as surgical guidance agents. Elucidating these differences in animal models that approach the size and anatomy of the human brain would help guide the use of these agents. Herein, we report on the use of a new pig glioma model and fluorescence cryotomography to evaluate the 3D distributions of both agents throughout the whole brain. Aim: We aim to assess and compare the 3D spatial distributions of ALA-PpIX and second-window ICG in a glioma-bearing pig brain using fluorescence cryotomography. Approach: A glioma was induced in the brain of a transgenic Oncopig via adeno-associated virus delivery of Cre-recombinase plasmids. After tumor induction, the pro-drug 5-ALA and ICG were administered to the animal 3 and 24 h prior to brain harvest, respectively. The harvested brain was imaged using fluorescence cryotomography. The fluorescence distributions of both agents were evaluated in 3D in the whole brain using various spatial distribution and contrast performance metrics. Results: Significant differences in the spatial distributions of both agents were observed. Indocyanine green accumulated within the tumor core, whereas ALA-PpIX appeared more toward the tumor periphery. Both ALA-PpIX and second-window ICG provided elevated tumor-to-background contrast (13 and 23, respectively). Conclusions: This study is the first to demonstrate the use of a new glioma model and large-specimen fluorescence cryotomography to evaluate and compare imaging agent distribution at high resolution in 3D.

Reconstruction of 3D Chromosome Structure from Single-Cell Hi-C Data via Recurrence Plots.

We describe an approach for reconstructing three-dimensional (3D) structures from single-cell Hi-C data. This approach has been inspired by a method of recurrence plots and visualization tools for nonlinear time series data. Some examples are also presented.

Investigation of the furcation morphology of permanent mandibular first molars by using micro-computed tomography.

BACKGROUND: To investigate the anatomic features of the root furcation of permanent mandibular first molars. METHODS: A total of 50 extracted mandibular first molars (25 two-rooted and 25 three-rooted) were collected and scanned using micro-computed tomography. The digital models of teeth and root canal systems were reconstructed three-dimensionally. The tooth models were displayed in parallel projection mode from buccal and distal views. Screenshots were captured and subsequently analyzed using Image-Pro Plus 6.0 software after calibration. The furcation angle, root trunk length, maximum depth and level of distal root concaves of mesial roots, and length of enamel projections were measured, and the furcation types (classified into type V, type U and type W) were detected. Statistical analysis was performed using the Shapiro-Wilk's test, one-way analysis of variance, Student's t-test and Chi-square test. RESULTS: The mean furcation angle between the distobuccal (DB) and distolingual (DL) roots (in distal view) was the greatest (59.2°), whereas the furcation angle between the mesial and DL roots (in buccal view) was the smallest (25.4°) among the four furcation angles (all p < 0.05). Regarding the furcation types, bucco-lingual root trunk length, maximum depth and site of the distal root concavities, and enamel projection length, no significant differences were detected between the three- and two-rooted molar groups (all p > 0.05). The frequency of type V was the highest (54.0%), followed by type U (26.0%), and type W had the lowest occurrence rate (20.0%). The mean length of distal root trunk in the three-rooted mandibular molars was significantly greater than that of the buccal/lingual one (3.7 mm vs. 3.0 mm, p < 0.01). The maximum depth of the distal concavities of the mesial roots was on average 0.66 ± 0.19 mm, and the site was located at an average of 2.8 ± 1.3 mm below furcation. The mean length of buccal enamel projections was significantly longer than that of lingual ones (3.1 mm vs. 0.7 mm, p < 0.01). CONCLUSIONS: The furcation anatomy of the mandibular first molar is complex, and the presence of the DL root may further complicate its topography. A thorough understanding of these anatomic features is essential for successful periodontal treatment.

Intraoperative estimation of natural femoral anteversion from proximal femoral osseous orientation during total hip arthroplasty.

BACKGROUND: The purpose of this study was to elucidate the relationship between the orientation of the osseous structure of the proximal femur encountered during total hip arthroplasty (THA) and preoperative femoral anteversion (FA). METHODS: Three-dimensional models were constructed using full-length lower extremity computed tomography images from a total of 80 participants. Femoral neck cutting was performed at heights of 5, 10, and 15 mm relative to the lesser trochanter. Following neck cutting, the angles formed by the anterior outer cortex and posterior outer cortex with the posterior condylar line (PCL) were defined as the anterior cortical angle (ACA) and posterior cortical angle (PCA), respectively. Univariate linear regression analysis was conducted using the remaining measurements with FA as the dependent variable. RESULTS: The mean age of the participants was 60.98 ± 10.82 years (males, 60.50 ± 11.36 years; females, 61.45 ± 10.37 years) (p = 0.697). All cortical angles and FA were larger in women compared to those in men. When comparing measurements by age groups, no statistically significant differences were observed. Univariate linear regression analysis with FA as the dependent variable showed statistical significance for all cortical angles. The adjusted R2 values were 0.711 (ACA5), 0.677 (ACA10), 0.572 (ACA15), 0.493 (PCA5), 0.574 (PCA10), and 0.446 (PCA15). CONCLUSION: Natural FA can be inferred from the anterior cortical angle (ACA) from femoral neck cutting plane observed during the THA procedure without preoperative images. TRIAL REGISTRATION: Retrospectively registered.

AB089. Improving decision-making in traumatic brain injury patients via 3D volumetric analysis of epidural haematoma.

BACKGROUND: Epidural haematoma (EDH) is a common finding in many traumatic brain injury scenarios, which necessitates surgical evacuation if the volume equals 30 cm3. The Tada formula, despite its convenience, have been observed to inaccurately depict haemorrhage volume, which can lead to inappropriate decision-making. Objective: (I) to determine if there are statistical differences between EDH volumes as calculated using three-dimensional (3D) software versus Tada's formula; and (II) whether this difference leads to differences in treatment options. METHODS: Computed tomography (CT)-scan of 15 traumatic brain injury (TBI) patients with EDH in January-February 2024 were obtained, and the volumetric analysis was performed using the (I) 3D Slicer software; and the (II) Tada formula for each scan. In addition, characteristics such as patient demographics and region were noted. We performed a paired t-test to scrutinise whether there were any differences between the volumes obtained via the two methods. RESULTS: There was a significant difference (P≤0.05) between the EDH volumes as calculated via 3D software and the Tada formula. We also noted that some patients who should have been treated surgically were not operated on, and vice versa. The process of 3D segmentation only takes an average of 8.2 minutes per patient; which is comparable to using the Tada formula. The inaccuracy of the Tada formula could be attributed to the irregular volume of the bleeding foci, contrary to the prototypical biconvex shape. CONCLUSIONS: 3D segmentation should be routinely employed in EDH and other TBI-related haemorrhage cases when available, to aid in decision-making. Extensive development is needed to explore the utility of 3D software in emergency neurosurgery.

ANALYSIS OF THE USE OF COMPLEX DIGITAL TECHNOLOGIES IN THE DIAGNOSIS AND TREATMENT OF OCCLUSAL ANOMALIES.

BACKGROUND: Digital technologies have expanded in the field of dentistry, especially in the clinical and diagnostic aspects of occlusal abnormalities. Consequently, the purpose of this narrative review is to identify and synthesize data concerning the effects of these sophisticated digital technologies on improved diagnostic performance, treatment interventions, and patient outcomes. METHODS: Cochrane, Scopus, Web of Science, and PubMed were searched and, therefore, performed to find the pertinent digital technologies in dentistry from the published literature. The search was conducted in the period between 2000 and 2024. The criteria for inclusion of the studies targeted technologies that were Cone-Beam Computed Tomography (CBCT), intraoral scanners, 3D imaging, and Computer-Aided Design and Manufacturing (CAD/CAM). Some of the comparing between conventional and modern approaches were raised. RESULTS: Digital technologies have enhanced the diagnostic process due to extended visualization and precise evaluation of occlusal disturbances Conclusion: It has been seen that the application of information technologies in dentistry significantly improved the diagnostics and therapy of occlusion disturbances. While there are some invincible challenges posed by these advancements, the prospects are noteworthy when it comes to accuracy, efficiency, and patient benefits.

Three-dimensional numerical schemes for the segmentation of the psoas muscle in X-ray computed tomography images.

The analysis of the psoas muscle in morphological and functional imaging has proved to be an accurate approach to assess sarcopenia, i.e. a systemic loss of skeletal muscle mass and function that may be correlated to multifactorial etiological aspects. The inclusion of sarcopenia assessment into a radiological workflow would need the implementation of computational pipelines for image processing that guarantee segmentation reliability and a significant degree of automation. The present study utilizes three-dimensional numerical schemes for psoas segmentation in low-dose X-ray computed tomography images. Specifically, here we focused on the level set methodology and compared the performances of two standard approaches, a classical evolution model and a three-dimension geodesic model, with the performances of an original first-order modification of this latter one. The results of this analysis show that these gradient-based schemes guarantee reliability with respect to manual segmentation and that the first-order scheme requires a computational burden that is significantly smaller than the one needed by the second-order approach.

Spot Spine, a freely available ImageJ plugin for 3D detection and morphological analysis of dendritic spines.

Background: Dendritic spines are tiny protrusions found along the dendrites of neurons, and their number is a measure of the density of synaptic connections. Altered density and morphology is observed in several pathologies, and spine formation as well as morphological changes correlate with learning and memory. The detection of spines in microscopy images and the analysis of their morphology is therefore a prerequisite for many studies. We have developed a new open-source, freely available, plugin for ImageJ/FIJI, called Spot Spine, that allows detection and morphological measurements of spines in three dimensional images. Method: Local maxima are detected in spine heads, and the intensity distribution around the local maximum is computed to perform the segmentation of each spine head. Spine necks are then traced from the spine head to the dendrite. Several parameters can be set to optimize detection and segmentation, and manual correction gives further control over the result of the process. Results: The plugin allows the analysis of images of dendrites obtained with various labeling and imaging methods. Quantitative measurements are retrieved including spine head volume and surface, and neck length. Conclusion: The plugin and instructions for use are available at https://imagej.net/plugins/spot-spine.

Effect of interproximal enamel reduction on interradicular bone volume in clear aligner therapy: a three-dimensional cone-beam computed tomography study.

OBJECTIVES: To assess the effect of inter-proximal enamel reduction (IPR) on interradicular bone volume and incisal inclination in patients undergoing clear aligner therapy (CAT). MATERIALS AND METHODS: The study sample consisted of 60 cases which underwent orthodontic CAT, in a private clinic in Dammam, KSA. A total of 120 CBCT scans (60 pre-treatment and 60 post- treatment) were measured using the CS 3D Imaging software to examine bone volume (using height, width, and depth of the interproximal area) and incisal inclination. The corresponding ClinCheck models were collected to determine the amount and locations of interproximal reduction performed. Little's Irregularity Index values were measured using OrthoCAD software. Paired sample t-test was used to address the measurements of bone height, width, depth, bone volume, and inclination of upper and lower incisors before and after IPR. RESULTS: IPR did not affect the upper or lower bone volume except at LR3-2 and UL 2 - 1 where a significant difference between the bone volume with and without IPR was detected (p = 0.02 and p = 0.04 respectively). Upper and lower incisor inclination showed a statistically significant decrease after IPR. There was no correlation between IPR and bone volume difference between upper and lower teeth except at LR3-2 and UL 2 - 1. CONCLUSIONS: IPR had no significant effect on inter-radicular bone volume except at areas of lower right canine-lateral and at areas of upper left central-lateral. There was a positive correlation between the amount of IPR and incisal inclination. CLINICAL RELEVANCE: The current study findings suggest that while IPR has a minimal and localized effect on bone volume in certain areas, it plays a role in adjusting incisal inclination, highlighting its significance in the careful planning of orthodontic treatment using clear aligners.

Light-field tomographic fluorescence lifetime imaging microscopy.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful imaging technique that enables the visualization of biological samples at the molecular level by measuring the fluorescence decay rate of fluorescent probes. This provides critical information about molecular interactions, environmental changes, and localization within biological systems. However, creating high-resolution lifetime maps using conventional FLIM systems can be challenging, as it often requires extensive scanning that can significantly lengthen acquisition times. This issue is further compounded in three-dimensional (3D) imaging because it demands additional scanning along the depth axis. To tackle this challenge, we developed a computational imaging technique called light-field tomographic FLIM (LIFT-FLIM). Our approach allows for the acquisition of volumetric fluorescence lifetime images in a highly data-efficient manner, significantly reducing the number of scanning steps required compared to conventional point-scanning or line-scanning FLIM imagers. Moreover, LIFT-FLIM enables the measurement of high-dimensional data using low-dimensional detectors, which are typically low cost and feature a higher temporal bandwidth. We demonstrated LIFT-FLIM using a linear single-photon avalanche diode array on various biological systems, showcasing unparalleled single-photon detection sensitivity. Additionally, we expanded the functionality of our method to spectral FLIM and demonstrated its application in high-content multiplexed imaging of lung organoids. LIFT-FLIM has the potential to open up broad avenues in both basic and translational biomedical research.

Real-world application of a 3D deep learning model for detecting and localizing cerebral microbleeds.

BACKGROUND: Detection and localization of cerebral microbleeds (CMBs) is crucial for disease diagnosis and treatment planning. However, CMB detection is labor-intensive, time-consuming, and challenging owing to its visual similarity to mimics. This study aimed to validate the performance of a three-dimensional (3D) deep learning model that not only detects CMBs but also identifies their anatomic location in real-world settings. METHODS: A total of 21 patients with 116 CMBs and 12 without CMBs were visited in the neurosurgery outpatient department between January 2023 and October 2023. Three readers, including a board-certified neuroradiologist (reader 1), a resident in radiology (reader 2), and a neurosurgeon (reader 3) independently reviewed SWIs of 33 patients to detect CMBs and categorized their locations into lobar, deep, and infratentorial regions without any AI assistance. After a one-month washout period, the same datasets were redistributed randomly, and readers reviewed them again with the assistance of the 3D deep learning model. A comparison of the diagnostic performance between readers with and without AI assistance was performed. RESULTS: All readers with an AI assistant (reader 1:0.991 [0.930-0.999], reader 2:0.922 [0.881-0.905], and reader 3:0.966 [0.928-0.984]) tended to have higher sensitivity per lesion than readers only (reader 1:0.905 [0.849-0.942], reader 2:0.621 [0.541-0.694], and reader 3:0.871 [0.759-0.935], p = 0.132, 0.017, and 0.227, respectively). In particular, radiology residents (reader 2) showed a statistically significant increase in sensitivity per lesion when using AI. There was no statistically significant difference in the number of FPs per patient for all readers with AI assistant (reader 1: 0.394 [0.152-1.021], reader 2: 0.727 [0.334-1.582], reader 3: 0.182 [0.077-0.429]) and reader only (reader 1: 0.364 [0.159-0.831], reader 2: 0.576 [0.240-1.382], reader 3: 0.121 [0.038-0.383], p = 0.853, 0.251, and 0.157, respectively). Our model accurately categorized the anatomical location of all CMBs. CONCLUSIONS: Our model demonstrated promising potential for the detection and anatomical localization of CMBs, although further research with a larger and more diverse population is necessary to establish clinical utility in real-world settings.

Measurement of the Tilt Angle of the Optic Disc Using Spectral-Domain Optical Coherence Tomography and Related Factors in Myopia.

Purpose: This study presents a novel, three-dimensional method for measuring the tilt angle of the tilted optic disc (TOD) using spectral-domain optical coherence tomography (SD-OCT) and investigates the correlation between ocular-related parameters and TOD. Methods: We included the right eyes of 243 healthy young individuals, categorized by axial length. We measured the ovality index (OI) and dihedral angle (DA) using SD-OCT infrared ray fundus photographs and high-resolution cross-sectional images of the optic disc, respectively. The relationships between OI, DA, and ocular-related parameters were analyzed. Results: Eyes in the longer axial length group exhibited a lower OI and a higher DA, along with thinner nasal and inferonasal circumpapillary retinal nerve fiber layer (cpRNFL) and thicker temporal and superotemporal cpRNFL. There was a significant relationship between DA and cpRNFL thickness. The new method utilizing DA to measure the tilt angle of TOD demonstrated high repeatability. Conclusions: We propose a novel, three-dimensional, and quantitative method for evaluating the tilt degree of TOD. A higher degree of myopia indicated a greater tilt angle of the TOD, and a greater TOD suggested additional changes in cpRNFL thickness. These findings should be considered when interpreting increased susceptibility and early assessment of glaucoma in myopia. Translational Relevance: DA could serve as a superior indicator for describing TOD morphology during eyeball elongation and evaluating its impact on related parameters of the optic disc and peripapillary structures in the myopic population.

Detection of breast cancer in digital breast tomosynthesis with vision transformers.

Digital Breast Tomosynthesis (DBT) has revolutionized more traditional breast imaging through its three-dimensional (3D) visualization capability that significantly enhances lesion discernibility, reduces tissue overlap, and improves diagnostic precision as compared to conventional two-dimensional (2D) mammography. In this study, we propose an advanced Computer-Aided Detection (CAD) system that harnesses the power of vision transformers to augment DBT's diagnostic efficiency. This scheme uses a neural network to glean attributes from the 2D slices of DBT followed by post-processing that considers features from neighboring slices to categorize the entire 3D scan. By leveraging a transfer learning technique, we trained and validated our CAD framework on a unique dataset consisting of 3,831 DBT scans and subsequently tested it on 685 scans. Of the architectures tested, the Swin Transformer outperformed the ResNet101 and vanilla Vision Transformer. It achieved an impressive AUC score of 0.934 ± 0.026 at a resolution of 384 × 384. Increasing the image resolution from 224 to 384 not only maintained vital image attributes but also led to a marked improvement in performance (p-value = 0.0003). The Mean Teacher algorithm, a semi-supervised method using both labeled and unlabeled DBT slices, showed no significant improvement over the supervised approach. Comprehensive analyses across different lesion types, sizes, and patient ages revealed consistent performance. The integration of attention mechanisms yielded a visual narrative of the model's decision-making process that highlighted the prioritized regions during assessments. These findings should significantly propel the methodologies employed in DBT image analysis by setting a new benchmark for breast cancer diagnostic precision.

Revealing real-time 3D in vivo pathogen dynamics in plants by label-free optical coherence tomography.

Microscopic imaging for studying plant-pathogen interactions is limited by its reliance on invasive histological techniques, like clearing and staining, or, for in vivo imaging, on complicated generation of transgenic pathogens. We present real-time 3D in vivo visualization of pathogen dynamics with label-free optical coherence tomography. Based on intrinsic signal fluctuations as tissue contrast we image filamentous pathogens and a nematode in vivo in 3D in plant tissue. We analyze 3D images of lettuce downy mildew infection (Bremia lactucae) to obtain hyphal volume and length in three different lettuce genotypes with different resistance levels showing the ability for precise (micro) phenotyping and quantification of the infection level. In addition, we demonstrate in vivo longitudinal imaging of the growth of individual pathogen (sub)structures with functional contrast on the pathogen micro-activity revealing pathogen vitality thereby opening a window on the underlying molecular processes.

Neuroanatomical photogrammetric models using smartphones: a comparison of apps.

OBJECTIVES: A deep knowledge of the surgical anatomy of the target area is mandatory for a successful operative procedure. For this purpose, over the years, many teaching and learning methods have been described, from the most ancient cadaveric dissection to the most recent virtual reality, each with their respective pros and cons. Photogrammetry, an emergent technique, allows for the creation of three-dimensional (3D) models and reconstructions. Thanks to the spreading of photogrammetry nowadays it is possible to generate these models using professional software or even smartphone apps. This study aims to compare the neuroanatomical photogrammetric models generated by the two most utilized smartphone applications in this domain, Metascan and 3D-Scanner, through quantitative analysis. METHODS: Two human head specimens (four sides) were examined. Anatomical dissection was segmented into five stages to systematically expose well-defined structures. After each stage, a photogrammetric model was generated using two prominent smartphone applications. These models were then subjected to both quantitative and qualitative analysis, with a specific focus on comparing the mesh density as a measure of model resolution and accuracy. Appropriate consent was obtained for the publication of the cadaver's image. RESULTS: The quantitative analysis revealed that the models generated by Metascan app consistently demonstrated superior mesh density compared to those from 3D-Scanner, indicating a higher level of detail and potential for precise anatomical representation. CONCLUSION: Enabling depth perception, capturing high-quality images, offering flexibility in viewpoints: photogrammetry provides researchers with unprecedented opportunities to explore and understand the intricate and magnificent structure of the brain. However, it is of paramount importance to develop and apply rigorous quality control systems to ensure data integrity and reliability of findings in neurological research. This study has demonstrated the superiority of Metascan in processing photogrammetric models for neuroanatomical studies.

An innovative evaluation method for clinical comparative analysis of occlusal contact regions obtained via intraoral scanning and conventional impression procedures: a clinical trial.

OBJECTIVES: To compare the occlusal contact regions (OCRs) obtained through an intraoral scanning system and conventional impression procedures via an innovative evaluation method. MATERIALS AND METHODS: Fifteen participants with complete dentitions and stable centric occlusion were included. Three groups were created based on the technique used to obtain the OCRs of quadrant posterior teeth at the maximal intercuspal position: 100 µm articulating paper (Control), an intraoral scanner (Test 1, T1) and conventional impression procedure (Test 2, T2). OCRs of control group were digitized by the intraoral scanner, while all conventional impressions were cast and digitized by an extraoral scanner. The virtual occlusal records of the 2 test groups were obtained by buccal bite registration. The OCRs within 100 µm in the 3 groups were three-dimensionally superimposed based on the tooth surfaces and the area of OCRs (SC, ST1, ST2) was calculated. The area of overlapping OCRs (SO) between the test groups and the control group was calculated. In the two test groups, the consistency rate of OCRs (SO/SC) and the positive rate of OCRs (SO/ST) were calculated and compared. For occlusal tightness evaluation, the mean occlusal clearances (OC) as well as minimum OC between the upper and lower models were calculated and compared. RESULTS: The consistency rate of OCRs was 0.73 ± 0.17 for T1 group and 0.23 ± 0.13 for T2 group (p < 0.001). The positive rate of OCRs was 0.67 ± 0.15 for T1 group and 0.56 ± 0.23 for T2 group (p = 0.143). The mean OC was 51.32 ± 16.04 µm for T1 group and 68.20 ± 18.15 µm for T2 group (p = 0.024). The minimum OC was - 61.74 ± 35.38 µm for T1 group and 4.09 ± 27.15 µm for T2 group (p < 0.001). CONCLUSIONS: For obtaining occlusal records in the quadrant posterior region, the tested intraoral scanning system was more reliable for recording occlusal contact regions and showed higher occlusal tightness compared with conventional impression procedures. CLINICAL RELEVANCE: (1) The evaluation method can assist clinicians in making more objective analysis and comparisons among different sources of virtual occlusal records. (2) Occlusal tightness is a key and indispensable indicator in the evaluation of virtual occlusal records, and it can be quantified by measuring the occlusal clearance utilizing the current evaluation method.

Enhancing surgical occlusion setting in orthognathic surgery planning using mixed reality technology: a comparative study.

OBJECTIVES: Orthognathic surgery necessitates precise occlusal alignment during surgical planning, traditionally achieved through manual alignment of physical dental models as the recognized gold standard. This study aims to evaluate the efficacy of mixed reality technology in enhancing surgical occlusion setting compared to traditional physical alignment and an established virtual method, addressing the research question: Can mixed reality technology improve the accuracy and efficiency of occlusion setting in orthognathic surgery planning? MATERIALS & METHODS: This experimental study compared the surgical occlusion settings of 30 orthognathic cases using three methods: a new virtual method with mixed reality technology, the traditional gold standard of physical alignment, and an established virtual occlusion method using the IPS Case Designer (KLS Martin SE & Co. KG, Tuttlingen, Germany). RESULTS: Results indicated that surgical occlusions set with mixed reality technology were comparable to the conventional method in terms of maxillary movement and occlusal relationship. Differences observed were within the inter-observer variability of the gold standard. Both virtual methods tended to position the maxilla more anteriorly, resulting in fewer occlusal contacts. However, virtual occlusion demonstrated clinical applicability, achieving an average of 11 occlusal contacts with a bilaterally symmetrical distribution along the dental arch. CONCLUSIONS: The mixed reality environment provides an intuitive and flexible experience for setting surgical occlusion, eliminating the need for costly 3D-printed physical models or the automatic calculations required by other virtual occlusion methods, thereby offering maximum freedom. CLINICAL RELEVANCE: As a novel form of virtual occlusion, it presents a comprehensive tool that contributes to a timely and cost-effective full digital workflow of orthognathic surgery planning.