Workflows for the Photopic Negative Response and the Baby Vision Test

Summary

This article presents standardized workflows for the photopic negative response (PhNR) and the Baby Vision Test, two underutilized but clinically valuable electrophysiological modalities, to reduce procedural variability and enhance their diagnostic utility in glaucoma and pediatric visual disorders.

Abstract

Visual electrophysiological tests are noninvasive, objective tools widely used in clinical ophthalmology. However, two promising modalities, the photopic negative response (PhNR), which assesses inner retinal and retinal ganglion cell function, and the dynamic target-based Baby Vision Test, remain underutilized in clinical practice despite their ability to provide valuable objective functional data. Limited adoption is primarily due to procedural variability, numerous confounding factors, and challenges in result interpretation. To address these barriers, clear and standardized operational protocols are essential for maximizing their diagnostic potential. Here, a standardized 6E workflow is proposed, encompassing Environment, Equipment, Examinee, Electrode, Examination, and Exit, designed to improve the clinical feasibility and implementation of these tests. In a retrospective analysis, implementation of the 6E protocol significantly increased the proportion of infants who successfully completed testing. Specifically, the PhNR test success rate increased from 61.54% to 86.45% (p < 0.05), while the average duration of the Baby Vision Test decreased from 17.44 ± 9.89 min to 13.28 ± 9.24 min (p < 0.05). These findings demonstrate that the 6E workflow enhances the clinical utility and efficiency of PhNR and Baby Vision testing, supporting their broader application in the diagnosis and management of glaucoma and pediatric visual disorders.

Introduction

Visual electrophysiology provides an objective, quantitative means of assessing visual pathway function from the retina to the cortex^1,2. Conventional tests such as full‑field electroretinography (ERG) and visual evoked potentials (VEPs) are widely used in clinical ophthalmology^3,4. However, several specialized techniques with proven clinical value remain underutilized in routine practice.

Two such techniques are the photopic negative response (PhNR), a negative wave that follows the b-wave of the light-adapted electroretinogram (ERG) and reflects retinal ganglion cell function^5,6, and the Baby Vision Test, a visual assessment modality that uses a dynamic target-based testing approach. The baby vision test uses a horizontally drifting grating (with luminance matched to the background) while recording eye movements. Spatial resolution is increased progressively, and visual acuity (VA) is estimated from the highest resolution that elicits stable tracking⁷. Although both techniques have demonstrated utility in research settings^7,8, their widespread clinical adoption remains limited.

Several factors contribute to this gap. Electrophysiological recordings are susceptible to numerous confounds, including electromagnetic interference, grounding, electrode type and placement, stimulation parameters, patient factors (age, pupil, refraction, anesthesia), and environmental variables^9,10,11,12. Procedural variability and lack of consistent reference data further undermine clinical confidence¹³.

To address these challenges, detailed, standardized workflows for PhNR and Baby Vision examinations are presented. These workflows are built upon a structured, checklist‑based 6E framework (Environment, Equipment, Examinee, Electrode, Examination, and Exit) that explicitly specifies environmental conditions, standardizes each testing phase from patient preparation to electrode placement, and translates technical parameters into practical, step‑by‑step actions. By reducing operator‑dependent variability and minimizing omitted steps, the 6E approach directly improves intra‑ and inter‑subject reproducibility compared to conventional, less protocol‑driven methods. The protocol is designed for routine clinical settings such as pediatric ophthalmology and electrophysiology laboratories. The aim is to facilitate broader clinical adoption of these valuable tools.

Protocol

The procedures described here focus on the photopic negative response (PhNR) and the Baby Vision Test. All procedures were approved by the Institutional Review Board (IRB) of the Eye Hospital, Wenzhou Medical University (IRB approval no. H2025-071-K59 for the retrospective analysis). The requirement for informed consent was waived because of the retrospective nature of the study. This protocol is based on the 6E examination framework, which comprises Environment, Equipment, Examinee, Electrode, Examination, and Exit (Figure 1).

1. Workflow for the photopic negative response (PhNR)

1. Environmental requirements
   ​NOTE: The environmental specifications described below are based on a combination of ISCEV recommendations, equipment manufacturer operating specifications, international electrical safety standards, published laboratory environmental recommendations, and pediatric clinical practice^3,4. Where ISCEV does not prescribe numerical values, practical operating ranges were selected to provide stable recording conditions while maintaining patient comfort, particularly for infants and young children.
   1. Lighting conditions
      1. Equip the examination room to provide both conventional ambient illumination (200 lux in this study) and complete darkness as required by the testing protocol.
      2. Maintain consistent ambient lighting across all recording sessions to facilitate high-quality recordings and improve interlaboratory comparability.
      3. Use directly switched lighting fixtures without dimmers to minimize mains-borne electrical interference.
         ​NOTE: Consistent ambient lighting is essential for collecting reference data and obtaining reliable recordings⁴.
   2. Electromagnetic shielding and electrical installation
      1. Locate the examination room away from sources of strong electromagnetic fields, including radiology suites, elevators, large air-conditioning units, MRI scanners, physiotherapy equipment, refrigerators, and computer server rooms.
      2. Install a dedicated low-impedance grounding electrode with a measured ground resistance of ≤5 Ω.
      3. Standardize the grounding installation and ensure that it remains independent of other electrical circuits.
      4. Supply the electrophysiological equipment through a dedicated power line connected to the main distribution panel and equipped with a voltage stabilizer.
         NOTE: Visual electrophysiological signals are recorded at the microvolt (µV) level and are highly susceptible to electromagnetic interference.
      5. Avoid sharing electrical circuits with large appliances.
      6. Arrange all recording cables neatly and keep them separated from one another.
         ​NOTE: Do not bundle amplifier cables with other cables because capacitive coupling may increase electrical noise.
   3. Temperature and humidity
      1. Maintain room temperature between 22 °C and 26 °C.
         NOTE: Temperature variations may affect cellular response kinetics and response amplitudes¹⁴.
      2. Maintain relative humidity between 30% and 65%.
         ​NOTE: Humidity variations may affect electrode stability and impedance. Excessive moisture can promote electrode corrosion, whereas excessive dryness can increase electrical noise¹⁵.
      3. Use a dedicated heating, ventilation, and air-conditioning system to maintain environmental stability.
   4. Acoustic control
      1. Maintain background noise below 30 dB(A) using acoustic insulation measures, such as double-glazed windows, soundproof doors, and rubber flooring.
      2. Avoid sudden noises that may evoke startle responses or myogenic artifacts during recording.
      3. Minimize sources of mechanical vibration and acoustic interference within the examination room.
2. Equipment preparation
   1. Perform routine calibration and verification of all stimulus and recording instruments according to ISCEV recommendations³.
   2. Verify that the ERG system is properly connected to the visual stimulator.
   3. Inspect all cable connections and confirm that they are intact and functioning correctly.
      NOTE: Thorough equipment preparation is essential because device malfunction may reduce examination success rates, particularly in young children with limited testing tolerance.
   4. Disinfect the headrest and chinrest using alcohol-soaked cotton swabs before testing.
   5. Turn on the computer and wait until the desktop is fully loaded.
   6. Switch the stimulator rocker switch from O to I.
   7. Launch the acquisition software (Supplementary Figure 1A).
      NOTE: Follow the correct power-on sequence to ensure stable system operation.
   8. Select the ISCEV extended PhNR protocol from the examination menu (Supplementary Figure 1B).
      ​NOTE: Create a new protocol if the predefined protocol is unavailable. Use the following settings: blue background, 450 nm at 10 cd/m²; red flash, 630 nm at 2.0 cd·s/m²; stimulus frequency, 1 Hz; 50 artifact-free sweeps averaged (minimum 8–10 sweeps); amplifier bandpass, 0.3–300 Hz; automatic artifact rejection threshold, ±500 µV^16,17,18. Maintain identical acquisition settings throughout the study and across all participants to ensure data comparability.
   9. Position and focus the camera correctly before beginning the examination.
3. Examinee preparation
   1. Explain the procedure to the parent or guardian.
   2. Inform the parent or guardian that the examination is noninvasive and painless.
   3. Confirm that the child is awake and calm.
   4. Avoid testing children who are drowsy, dozing, or actively crying¹⁹.
      NOTE: Feed the child, change the diaper, and allow adequate rest before testing whenever possible.
   5. Use the flying-hold position for infants younger than 3 months with poor head control.
   6. Seat older infants on a parent's lap or position them standing when appropriate (Figure 2).
      NOTE: Stabilize the infant gently during testing. Avoid rigid head fixation. Pause the recording if excessive movement or crying occurs and resume once the infant is calm.
   7. Clean the periocular skin using a cotton swab.
   8. Confirm baseline pupil status.
   9. Instill one drop of 0.5% tropicamide every 3–5 min for a total of three applications.
   10. Confirm adequate pupil dilation after 30 min.
       NOTE: Inform parents that photophobia and blurred near vision may occur for 4–6 h following dilation.
   11. Allow the patient to adapt to photopic conditions for at least 10 min before recording.
4. Electrode placement
   1. Inspect all skin electrodes and confirm that they are intact, uncontaminated, and free of deterioration (Figure 3).
      NOTE: Skin electrodes are used in this protocol because they are well tolerated by infants and young children and facilitate examination in patients who cannot comfortably undergo corneal electrode placement.
   2. Apply conductive gel to the cleaned periocular skin.
   3. Fill the skin electrodes with conductive paste.
   4. Secure the electrodes using medical tape.
   5. Place the active electrodes on the lower eyelids.
   6. Place the reference electrodes at the external canthi.
   7. Place the ground electrode on the forehead (Table 1, Figure 4).
   8. Measure electrode impedance.
   9. Confirm that impedance is below 10 kΩ and preferably ≤5 kΩ.
      ​NOTE: A green quality bar indicates acceptable impedance, whereas a red quality bar indicates abnormal impedance (Supplementary Figure 1C).
   10. Repeat steps 1.4.2–1.4.7 if impedance exceeds the acceptable range.
5. Examination execution
   1. Click on the Patient ID/Eye icon and enter the patient's information (Supplementary Figure 1D).
   2. Select binocular testing.
   3. Position the child comfortably at the examination station.
   4. Adjust the table height and chinrest position.
   5. Recheck electrode impedance.
   6. Click on Exam to begin acquisition (Supplementary Figure 1D).
   7. Monitor the waveform and artifact-rejection indicator continuously.
   8. Instruct the patient to fixate on the central fixation target.
   9. Encourage the patient to minimize blinking during acquisition.
   10. Use verbal cues to maintain attention when cooperation is limited.
       NOTE: The system automatically rejects sweeps containing artifacts.
   11. Continue recording until a reliable waveform is obtained.
       ​NOTE: Define a reliable waveform as one containing at least 8–10 artifact-free sweeps, a signal-to-noise ratio (SNR) ≥3, and no visible movement or electrical artifacts. These criteria are based on published statistical analyses of electrophysiological signal significance and ISCEV extended PhNR recommendations while accounting for the higher noise levels commonly encountered during pediatric recordings^16,18,20.
   12. Stop acquisition by pressing S or by clicking on Results.
6. Exit examination and reporting
   1. Remove all electrodes.
      NOTE: Corneal electrodes may be used in cooperative patients when no contraindications are present.
   2. Clean the skin using a cotton swab.
   3. Disinfect the headrest and chinrest using alcohol-soaked cotton swabs.
   4. Display the averaged waveform.
   5. Identify the a-wave, b-wave, and PhNR trough.
   6. Measure the PhNR amplitude from baseline to the trough of the negative wave following the b-wave.
   7. Measure peak time from stimulus onset to the PhNR trough (Figure 5).
   8. Compare the measured values with established reference values.
   9. Classify a PhNR amplitude reduction greater than 2 standard deviations below the normal mean as abnormal.
      NOTE: Establish device-specific normative data whenever possible. Total preparation and testing time is approximately 25–35 min. Whenever possible, establish laboratory-specific reference intervals using appropriately selected healthy participants. According to current recommendations, new reference intervals should ideally be derived from at least 120 individuals per clinically relevant subgroup, whereas transferred or verified reference intervals may require fewer participants after accepted validation procedures¹³.

2. Workflow for the baby vision test

1. Environmental requirements
   ​NOTE: Environmental requirements for the Baby Vision Test are identical to those described for the PhNR examination (Section 1.1), including lighting consistency, electromagnetic shielding, grounding, temperature and humidity control, and acoustic management.
   1. Darken the room as needed to minimize distractions and facilitate visual attention.
2. Equipment preparation
   1. Perform routine equipment calibration according to the manufacturer's instructions and laboratory procedures.
   2. Verify that all cables are connected properly and functioning normally.
   3. Disinfect the headrest and chinrest using alcohol-soaked cotton swabs.
   4. Turn on the computer and launch the acquisition software.
   5. Select Baby Vision from the examination menu.
   6. Select the appropriate testing distance according to the child's age (Supplementary Figure 2A).
   7. Use a testing distance of 33–40 cm for infants younger than 3 months.
      NOTE: Shorter testing distances are used for younger infants to evaluate lower visual acuity levels.
   8. Set the stimulus velocity to 7°/s for infants younger than 3 months (Supplementary Figure 2B).
   9. Use a testing distance of 33–40 cm for infants aged 3–6 months.
   10. Set the stimulus velocity to 30°/s for infants aged 3–6 months (Supplementary Figure 2C).
   11. Use a testing distance of 100 cm for children older than 6 months.
   12. Set the stimulus velocity to 10°/s for children older than 6 months (Supplementary Figure 2D).
       ​NOTE: Testing parameters are adjusted according to the developmental stage of visual function^21,22,23. Faster stimulus velocities may improve attention and tracking responses in infants aged 3–6 months.
   13. Position and focus the camera before beginning the examination.
3. Examinee preparation
   1. Explain the examination procedure to the parent or guardian.
   2. Inform the parent or guardian that the examination is noninvasive and painless.
   3. Confirm that the child is awake and alert.
      NOTE: Feed the child, change the diaper, and allow adequate rest before testing whenever possible.
   4. Use the flying-hold position for infants younger than 3 months who have poor head control. 
   5. Seat older children on a parent's lap or position them standing when appropriate (Figure 2).
   6. Clean the forehead using a cotton swab.
   7. Confirm that the pupils have not been pharmacologically dilated.
      ​NOTE: Do not perform the Baby Vision Test after pupil dilation because mydriasis may affect fixation behavior and visual performance.
4. Electrode placement: applying the reflective dot
   1. Inspect the reflective dot and confirm that it is intact and uncontaminated.
   2. Clean the forehead before applying the reflective dot.
   3. Clean the skin before applying the reflective marker to improve adhesion and ensure accurate positioning.
      ​NOTE: Conductive gel may be used as a convenient skin-cleaning medium, but is not required; alternative skin cleansers are acceptable.
   4. Place the reflective dot on the forehead equidistant from both eyes.
   5. Position the reflective dot approximately 10 mm above the horizontal eye level (Figure 6).
   6. Confirm that the reflective dot is firmly attached before proceeding.
5. Examination execution
   1. Click on the Patient ID/Eye icon and enter the child's information.
   2. Select the eye to be tested. Test the right eye first, followed by the left eye.
   3. Cover the fellow eye with an occluder during monocular testing.
   4. Ensure that the occluder does not cover the reflective dot.
      ​NOTE: Proceed with binocular testing if monocular testing cannot be completed.
   5. Click the appropriate visual acuity (VA) test icon according to the child's age group.
   6. Position the child in front of the testing device.
   7. Adjust the examination table height and chinrest position until the eyes are centered within the camera frame.
   8. Observe the quality bar displayed on the monitor.
   9. Adjust the child's position until the quality bar is green.
      NOTE: A green quality bar indicates that the eye-tracking system has correctly identified the reflective dot and pupil(s) (Supplementary Figure 2E).
   10. Acquisition of tracking responses
       1. Press ENTER or click on Exam to begin acquisition when the quality bar is satisfactory.
       2. Present the moving grating stimulus and observe the tracking response.
          NOTE: The Gabor patch moves horizontally, and active tracking is indicated by a green stimulus curve.
       3. Record whether the child successfully tracks the presented stimulus.
       4. Classify a stimulus as not seen if the child fails to track it in two or more of three presentations.
       5. Increase the spatial frequency by one step after two or more successful tracking responses in three presentations.
       6. Decrease the spatial frequency by one step after two or more unsuccessful tracking responses in three presentations.
          ​NOTE: Visual acuity is determined using a 2-up, 1-down staircase procedure.
       7. Continue testing until at least two staircase reversals are obtained or the maximum spatial frequency is reached.
       8. Determine the visual acuity threshold as the highest spatial frequency at which the child consistently demonstrates successful tracking.
       9. Record the final visual acuity value.
       10. Stop acquisition by pressing S or by clicking on Results.
   11. Repeat the examination in the fellow eye during monocular testing.
   12. Apply a new reflective dot if the original dot is no longer securely attached.
6. Exit examination and reporting
   1. Remove the reflective dot.
   2. Disinfect the headrest and chinrest using alcohol-soaked cotton swabs.
   3. Review the recorded tracking curves.
      NOTE: The gray curve represents stimulus position, the blue curve represents eye movement, and the black curve represents head movement (Figure 7).
   4. Verify that the recorded data meet the predefined quality criteria.
   5. Generate a report containing the recorded tracking curves and the final visual acuity value.
   6. Document patient identification, examination date, examiner identification, pupil status, alertness, and relevant clinical history.
   7. Document the examination mode and acquisition parameters.
   8. Document artifact management procedures and quality-control findings.
   9. Document the final visual acuity threshold and supporting tracking data.
   10. Save all examination data and reports according to institutional procedures.
       NOTE: Reporting should follow the Standardized Reporting Checklist for the PhNR and Baby Vision Test (Supplementary Table 1).

3. Test sequence considerations

1. Perform the Baby Vision Test before the PhNR examination when both tests are scheduled during the same visit.
   NOTE: The Baby Vision Test requires natural pupil status, whereas the PhNR examination requires pharmacologic pupil dilation.
2. Schedule multiple electrophysiological examinations in an order that maximizes patient cooperation and minimizes fatigue.

4. Application of the 6E framework

1. Apply the 6E framework to other visual electrophysiological examinations when appropriate.
   NOTE: Although the software instructions described here are system-specific, the 6E framework can be adapted to any ISCEV-compliant platform.

Results

The clinical utility of the 6E workflow was evaluated by comparing examination outcomes before and after its implementation. Specifically, PhNR data were analyzed during the one-year period before and the one-year period after adoption of the 6E protocol (Figure 8), as well as Baby Vision Test data collected during the two-year periods before and after implementation (Figure 9).

Following implementation of the 6E protocol, the number ...

Discussion

While the photopic negative response (PhNR) and automated eye-tracking–based grating acuity assessments offer substantial promise for the objective evaluation of inner retinal function and infant visual acuity (VA), with the PhNR requiring neither refractive correction nor fixation monitoring⁵ and eye-tracking methods providing reliable acuity measurements without verbal responses⁷, both techniques remain underutilized in many clinical settings.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Compound tropicamide eye drops	Kingyork Group Hebei Univision Pharmaceutical,Tianjin, China	8327304	Fixed combination ophthalmic solution containing 0.5% tropicamide and 0.5% phenylephrine hydrochloride. Used for pupillary dilation and cycloplegia.
Durapore	3M Deutschland GmbH,Neuss,Germany	108099995	High strength, non stretch silk like cloth medical tape.
GT20 Conductive Paste	GREENTEK, Wuhan, China	20142072064	Water soluble conductive paste for non adhesive electrodes in electrophysiological examination
GT5 Conductive Gel	GREENTEK, Wuhan, China	20152072150	Combined skin prep and conductive gel for electrophysiological recordings
MonpackOne instrument	Metrovision, Perenchies, France	20162160840,K211643	Integrated electrodiagnostic system for visual electrophysiology. Supports full field flash ERG, pattern ERG, multifocal ERG, VEP, multifocal VEP, and sensory EOG et al. Used in hospitals and clinics by trained ophthalmologists and medical professionals.
Vision Monitor Software	Metrovision, Perenchies, France	Mon2018F,K211643	Integrated software platform for visual electrophysiology. Controls stimulus generation (flash, pattern, multifocal), acquires bioelectrical signals via up to 5 channels, and processes raw data using spatial filtering and artifact rejection algorithms. Produces digitized ERG, VEP, EOG waveforms et al.