vergence stimulus, shown in figure 1. The stimulus was a green vertical line, 2 cm in height by 2 mm in width, and was presented on a black background. The two stimuli (green vertical lines) from each computer screen were projected onto the two partially reflecting mirrors and into the line of sight of the subject. Prior to the experiment, the stimuli from the computer screens were adjusted with the partially reflecting mirrors to calibrate the visual stimulus with real targets located at measured distances from the subject’s midline. An inter-pupillary distance of 6 cm was assumed. During the experiment, only the visual stimulus displayed on the computer screens was seen by the subject. The subject’s head was restrained using a custom chin rest to eliminate head movement and avoid any vestibular influences. No other visual stimuli were presented to the subject except for the visual stimuli presented on the computer screens. The stimuli screens were placed 40 cm away from the subject, hence the accommo- dation stimulus was held constant at 2.5D. A previous study showed that accommodative-vergence does not influence the initial peak velocity measurement [25]. Hence, vergence peak velocities observed in this present study will be assumed to be associated with the disparity-vergence system. Eye Tracking Instrumentation and Data Analysis Eye movements were recorded using an infrared (l = 950 nm) system manufactured by Skalar Iris (model 6500, Netherlands). All of the eye movements were within the linear range of the system (625u). The left eye and right eye responses were recorded, calibrated and saved separately for offline data analysis. Eye movement digitization was preformed with a 12-bit digital acquisition (DAQ) hardware card (National Instruments 6024 E series, Austin, TX, USA). Visual stimuli and data digitization were controlled by a custom LabVIEWTM program (National Instru- ment, Austin, TX, USA). The eye movement signals were digitized at 200 Hz. Calibration for the 4u disparity-vergence responses was composed of two points which were the initial and final vergence positions of the convergence step stimuli. The two-point calibration was viewed binocularly and was the initial and final combined vergence demand of the step stimuli. This calibration method was validated in our previous study [26]. The system has a high degree of linearity, within 3% between 625 degrees horizontally [27]. Hence, a two-point calibration was adequate for this present study. Changes in Phoria and Convergence Peak Velocity A custom MATLABTM program (Waltham, MA, USA) was used for all data analysis. Left and right eye movement data were converted into degrees using the individual calibration data discussed above. Disparity-vergence was calculated by subtracting the right eye movement from the left eye movement. Any responses with blinks were excluded in the data analysis. Convergence peak velocity was computed using a two-point central difference algorithm [28]. Convergence responses are plotted as positive throughout the entire paper. Phoria Measurements The subjects binocularly viewed a pair of vertical lines that stimulated 4.22u of rotation which corresponded to a target 40 cm away from the subject’s midline, similar to the distance used clinically. The right eye was used to measure near dissociated phoria. A binocular target was presented for 2.5 seconds. Then, the right eye stimulus was extinguished and the eye position decayed to the steady state phoria level which was recorded for 15 seconds. This method of phoria measurement using our eye movement recording system was validated in our previous study [29]. A four-point calibration was utilized to assess the linearity of the eye movement recording system over the range of possible eye movement. A four-point calibration was used to ensure that the eye movement responses were within the calibration range since it was unknown prior to the study the extent to which each subject’s phoria would be changed after the sustained fixation. The calibration points were observed monocularly with the right eye. The first calibration stimulus was 2u into the left visual field from the midline. The second calibration stimulus was on midline. The third and fourth points were 4u and 9u into the right visual field which equated to a potential phoria range of 3.5D esophoria to 15.8D exophoria. The right eye decay to phoria signal was converted into prism diopters because those are the units used clinically (One prism diopter = 100tan (h)). Experimental Protocol During each session, baseline phoria was initially recorded. Baseline phoria was measured to determine whether the near or far sustained fixation adapted the phoria compared to baseline. The subjects fixated on a binocular target at 16u (near sustained fixation session, figure 2A) or 1u (far sustained fixation session, figure 2B) for three minutes. Subjects then performed a 4u convergence step starting at an initial vergence position of 12u and Figure 1. Haploscope experimental setup which stimulates retinal disparity while keeping accommodation constant. All visual stimuli were 40 cm away along midline for a constant 2.5D accommodative stimulus. doi:10.1371/journal.pone.0020883.g001 PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20883