## 2 Materials and Methods  
### Clinical Workflow  
The clinical procedure for OPT at the CNAO consists of two phases: a treatment simulation performed in the CT room with the patient immobilized in the supine position (Figure 2b) and an irradiation phase that takes place in the therapy bunker with the patient in a sitting position (Figure 3). The ETS is used in both phases, and the stream of frames acquired in real time by the stereo cameras is processed through a proprietary custom software aimed at monitoring any involuntary eye motion. The ETS provides also an aid in maintaining the gaze stability by means of a source of light (a red LED) enclosed in the rigid chassis. As the eye bulb is reflected by the mirror and seen by the cameras, the patient can see the reflection of the LED. As a consequence, the relative position of the ETS in reference to the isocenter can be used to uniquely define the gaze direction. The ETS position is refined during setup preparation, on a patient-specific basis. The adopted strategy foresees the minimization of 3D displacements between the ETS position in space and a nominal model. This operation is performed via a point-based rigid registration of surrogates, represented in this case by spherical markers placed in a predefined configuration on the ETS. During clinical routine, 3D coordinates of markers are calculated with optical-tracking systems. For safety reasons we adopt two different sets of markers: made of aluminum and thus radiopaque during CT acquisitions; made of plastic during treatment. Figure 2: Elements for ETS alignment in the CT room: the OTS-CT, in blue (**a**), and setup in the CT bunker (**b**). Elements for ETS alignment in a dedicated room: Smart-DX motion analysis system equipped with three cameras (**c**) and setup optimized in the laboratory (**d**). In order to highlight the impact that the explored novel procedure could have in the clinical workflow of OPT at CNAO, we report the essential steps of the currently adopted procedure. It is worth recalling that, in order to localize the malignant lesion inside the eye, a set of radiopaque fiducial clips are sutured around the lesion during a preliminary surgical intervention and used as tumor localization surrogates for treatment planning and patient setup. At CNAO, the treatment plan is elaborated by means of a dedicated software (Eclipse, ver. 13.5.01, Varian medical systems, Palo Alto, CA, USA), which outputs the optimal geometrical solution to focus the dose on the target, while sparing radiosensitive healthy structures within the eye. The main geometrical parameters optimized by the treatment planning system are the gaze direction angles (expressed in polar coordinates) required to expose the tumor mass to the delivered horizontal proton beam. Patient cooperation is required to maintain the planned gaze direction during irradiation, and proper verification is needed to ensure setup stability at all phases of the irradiation session. In particular, the treatment simulation consists of three main steps:  
1. Planning CT scan (CT0): The patient is fitted with the immobilization thermoplastic mask and undergoes a first CT scan of the diseased eye while keeping the gaze straight ahead (Figure 4). Proper sight direction is ensured by a corresponding positioning of the ETS and ocular motions are detected through the video stream coming from the two stereo-cameras embedded in the ETS. The manual contouring of the iris and pupil on the ETS PC monitor serves as reference for the qualitative detection of ocular motion. 2. Gaze direction and model generation: On the CT0, the treatment plan is elaborated, and the deviated optimal gaze direction provided by the treatment planning software is translated into a corresponding ETS position and orientation expressed in terms of reference coordinates of the radiopaque, retroreflective markers attached to the ETS case [18].