Abstract <p>Markerless tumor tracking in x-ray fluoroscopic images is an important technique for achieving precise dose delivery for moving lung tumors during radiation therapy. However, accurate tumor tracking is challenging due to the poor visibility of the target tumor overlapped by other organs such as rib bones. Dual-energy (DE) x-ray fluoroscopy can enhance tracking accuracy with improved tumor visibility by suppressing bones. However, DE x-ray imaging requires special hardware, limiting its clinical use. This study presents a deep learning-based DE subtraction (DES) synthesis method to avoid hardware limitations and enhance tracking accuracy. The proposed method employs a residual U-Net model trained on a simulated DES dataset from a digital phantom to synthesize DES from single-energy (SE) fluoroscopy. Experimental results using a digital phantom showed quantitative evaluation results of synthesis quality. Also, experimental results using clinical SE fluoroscopic images of ten lung cancer patients showed improved tumor tracking accuracy using synthesized DES images, reducing errors from 1.80 to 1.68 mm on average. The tracking success rate within a 25% movement range increased from 50.2% (SE) to 54.9% (DES). These findings indicate the feasibility of deep learning-based DES synthesis for markerless tumor tracking, offering a potential alternative to hardware-dependent DE imaging.</p> Graphical abstract <p>The U-Net model was first trained on simulated single-energy and dual-energy subtraction image pairs, then applied to synthesize DES from clinical X-ray images for improving tumor tracking</p>