## Materials and Methods
### In Vitro Motion Experiment  
This study was approved by China Medical University and Hospital Research Ethics Committee (No: DMR101-IRB1-139). The knee specimen used in the current in vitro experiment was obtained from a male donor who received a below-hip amputation for disorders other than the knee joint and had provided written informed consent. Access to information identifying the donor was not provided to the authors. To evaluate the performance of the proposed MIBFT in measuring tibiofemoral kinematics with interleaved biplane fluoroscopy, we conducted a simulated knee-motion experiment on the knee specimen described as follows. Lengths of the femur and tibia of the fresh-frozen knee  
Figure 3: The 3D/2D image registration procedure that numerically searches for the optimal pose parameters of the bone such that its forward projected digitally reconstructed radiographs best match the real fluoroscopic image and the image interpolated from the adjacent image frames. specimen were approximately 260 and 290 mm, respectively. A plastic connector was affixed onto the proximal end of the femur and distal end of the tibia with bone cement (Figure 4A). Four marker clusters, each attached with four infrared retroreflective markers with a 7 mm radius, were rigidly implanted into the femur and its connector. Three other clusters were implanted into the tibia and its connector. The knee specimen with the clusters and connectors was scanned using a clinical CT scanner (Optima CT660, GE Healthcare, Chicago, IL, USA) to acquire CT images of the whole construct (peak voltage = 120 kVp, tube current = 180 mA, pixel size = 0.709 mm \(\times\) 0.709 mm, slice thickness = 0.625 mm, matrix size = \(512\times 512\times 945\)). A clinical biplane fluoroscopy system (Allura Xper FD20/20, Philips Medical Systems, Amsterdam, the Netherlands) was configured with source-to-detector distances of 1195 and 1050 mm for the two X-ray units and was operated with tube voltages ranging from 50 to 59 kVp, a tube current of 5 mA and a frame rate of 30 frames/s per X-ray unit, which allowed us to obtain \(512\times 512\) 8-bit interleaved fluoroscopic images at an effective frame rate of 60 frames/s. Prior to fluoroscopic image acquisition, the specimen was mounted on a custom-made motion testing frame through the connector. While the femoral connector was rigidly mounted to the frame, the femur was kept immobile, and the tibia was allowed to manually rotate about the knee by the operator, and vice versa. Following the cadence from a metronome, the operator extended and flexed the knee by moving the end of the freely moving segment at five rotation speeds (0.25 cycle/s, 0.375 cycle/s, 0.5 cycle/s, 0.625 cycle/s, and 0.75 cycle/s), during which the interleaved biplane fluoroscopic images were acquired. These examinations were termed femoral and tibial isolated flexion/extension (FE). The connector was then detached from the frame, and the specimen was manually bent and extended by the operator to acquire fluoroscopic images of the tibofemoral FE at four prescribed speeds (0.375 cycle/s, 0.5 cycle/s, 0.625 cycle/s, and 0.75 cycle/s). Overall, three trials per motion speed, each with at least one complete motion cycle, were collected for the femoral isolated FE, tibial isolated FE, and tibofemoral FE scenarios. During the motion examinations, the 3D trajectories of the markers on the cluster frames were reconstructed using  
Figure 4: **(A)** A cadaveric experiment was conducted on a knee specimen to provide the interleaved biplane fluoroscopic images for model-based interleaved biplane fluoroscopy image tracking (MIBFT) analysis and to record the 3D trajectories of the bone-anchor markers via a motion capture system (MoCap). **(B)** The marker data obtained from the MoCap were also imported into the virtually reconstructed biplane imaging environment, which were then used to determine the reference pose parameters of the femur and tibia.