Edited by: Andrew J. Zele, Queensland University of Technology, Australia
Reviewed by: Sei-ichi Tsujimura, Kagoshima University, Japan; Dingcai Cao, University of Illinois at Chicago, United States
This article was submitted to Neuro-Ophthalmology, a section of the journal Frontiers in Neurology
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The human pupillary light response is driven by all classes of photoreceptors in the human eye—the three classes of cones, the rods, and the intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin. These photoreceptor classes have distinct but overlapping spectral tuning, and even a monochromatic light with a wavelength matched to the peak spectral sensitivity of a given photoreceptor will stimulate all photoreceptors. The method of silent substitution uses pairs of lights (“metamers”) to selectively stimulate a given class of photoreceptors while keeping the activation of all others constant. In this primer, we describe the method of silent substitution and provide an overview of studies that have used it to examine inputs to the human pupillary light response.
At the input level, the size of the pupil is controlled by the activity of the different photoreceptors in the human eye (
Photoreception in the human retina is based on the signals produced by the three types of cones—the long[L]-wavelength-sensitive cones, the medium[M]-wavelength-sensitive cones, and the short[S]-wavelength-sensitive cones—, the rods, and the intrinsically photosensitive retinal ganglion cells (ipRGCs), which contain the photopigment melanopsin (
An important desideratum for examining how the different photoreceptors contribute to the human pupillary light response is that stimuli produce responses specific to a given photoreceptor class. One consequence of the extensive spectral overlap of the photoreceptors is that most light sources activate all photoreceptors, and therefore, the responses elicited are largely nonspecific. For example, monochromatic light with a peak spectral output of 490 nm will activate melanopsin maximally relative to the other photoreceptors, but it will also lead to substantial activation of rods and the cones (Figure
One property of photoreceptors is the
Because photoreceptors weight input light by their spectral sensitivity function, in the case of two photoreceptors, it is possible to find two lights and scale them such that the excitation of one of the photoreceptors remains constant in this wavelength exchange, while the other one “sees” a difference. This is shown in Figure
In the method of silent substitution, pairs of light are found that have the property that they stimulate the targeted photoreceptor class (or classes) whilst not changing the excitation of the other photoreceptors, the silenced ones. The method has a long history for determining the properties of the mechanisms of human color vision (
To introduce the method of silent substitution we begin with an example from human color vision. Human color vision is trichromatic under daylight conditions, i.e., when rods do not participate: A color-normal observer can match the color appearance of any light using a combination of three primary lights (
In general, to stimulate one class of photoreceptor classes out of
For the case of four photoreceptor classes in the human retina (three classes of cones and melanopsin), four lights are necessary to match the activation of cones and stimulate melanopsin. When including the rods, five lights are necessary to match the activation of cones and rods and stimulate melanopsin.
It is possible to have more primary lights than photoreceptors under consideration, i.e.,
The term contrast refers to a specific quantity, which is the fractional difference of activation of a photopigment around a background:
Intuitively, when the light-adapted background activates a given photoreceptor by some amount, e.g., 100 (arbitrary units), and the modulation activates it by a higher amount, e.g., 120 (arbitrary units), the contrast in that case would be 0.2 or 20%. Contrast can be specified either as fractions or as percentages.
We now describe an example case for the method of silent substitution corresponding to the stimuli used in Spitschan, Jain, Brainard and Aguirre (
Background spectrum: In the first instance, we begin with a background spectrum of known spectral power distribution (Figure
Increasing melanopsin activation: Pragmatically, we can increase the amount of light seen by melanopsin by simply increasing the amount of light emitted near the melanopsin peak. This is shown in Figure
Silencing S cones: To zero, or silence, the S cones, we decrease the amount of short-wavelength light, to which the S cones are most sensitive (Figure
Silencing L and M cones: To silence the L and M cones, a similar trick is applied: Light near the peak spectral sensitivity of L and M cones is decreased to reduce the overall absolute activation of L and M cones (Figure
Inverting the melanopsin activation: The modulation spectrum shown in Figure
We provide a quantitative example along with code in Appendices
The method of silent substitution has enjoyed use in empirical work well before the discovery of melanopsin and the ipRGCs. We point the reader to Estévez and Spekreijse (
We provide an overview of extant studies examining specifically melanopsin photoreception using the method of silent substitution in Table
Studies examining human pupil responses with silent substitution.
Tsujimura et al. ( |
4 | 470, 500, 525, 615 nm ± 20–36 nm | Diffusing screen in front of integrating sphere | 20° field size | Melanopsin [−53%] |
301 cd/m2 to 642 cd/m2 to 982 cd/m2 | 6 | 10 min stimuli |
Cones: CIEPO2006 |
Viénot et al. ( |
5 | 473 ± 25 nm |
Light booth with white paint | Ganzfeld | Melanopsin-only (cone and rod silent) [3.4%] |
35 cd/m2 | 10 | Measurement after 1 min of continuous exposure | Cones: CIEPO2006 |
Tsijumura and Tokuda, ( |
4 | 468, 524, 599, and 633 nm (test) |
Diffusing screen in front of integrating sphere | Annulus id 5 od 18° |
8% | 612 cd/m2 background1,109 cd/m2 test field | 6 | Sinusoidal & square wave stimuli | Cones: CIEPO2006, 10° |
Spitschan et al. ( |
128 | n/a | Viewing of surface through lens | 27.5° circular, central 5° blocked | S, (L+M), melanopsin, (L+M+melanopsin) [50%] | 382–1,033 cd/m2 | 16 | Sinusoidal, 0.01 – 2 Hz | 10° Stockman–Sharpe/CIE cone fundamentals, melanopsin estimated by shifting Stockman-Sharpe nomogram to λmax = 480 nm, corrected for prereceptoral filtering (same as cones, optical density 0.3). |
Barrionuevo et al. ( |
4 | 442, 516, 594, and 634 nm (one set) |
Ganzfeld | 54° field | Mixed joint modulations, no melanopsin-isolaing modulation | 0.002–100 cd/m2 | 3 (authors) | Sinusoidal, 0.5–8 Hz | Smith–Pokorny cone fundamentals |
Cao et al. ( |
5 | 456, 488, 540, 592, 633 | Maxwellian view | 30° circular, central 10.5° blocked | Experiment 1: S, M, L, Rod, Melanopsin [16%] |
Experiment 1: 200 Photopic Td |
3 | Sinusoidal, 1 Hz | Smith–Pokorny cone fundamentals applied for the CIE 1964 10° Standard Observer |
Barrionuevo and Cao, ( |
5 | 456, 488, 540, 592, 633 | Maxwellian view | 30° circular, central 10.5° blocked | Experiment 1: S, M, L, Rods, Melanopsin, (L+M+S) [17%], red-green [4% M,−4% L] |
2–20,000 Photopic Td | 3 (2 authors) | Sinusoidal, 1 Hz | Smith–Pokorny cone fundamentals applied for the CIE 1964 10° Standard Observer |
Spitschan et al. ( |
56 | n/a | Viewing of surface through lens | 64° circular, central 5° blocked | 25–400% | 100–200 cd/m2 | 4 | Tapered pulses (3 s, 14–16 s ISI) | CIE 2006 parametric model |
Woelders et al. ( |
5 | 465, 500, 515, 595 | Diffusing screen in front of LEDs | 24.68° horizontal, 12.13° vertical | S, M, L, Melanopsin [23%] | Background or average of 8.5 melanopic lux | 16 | Square-wave (0.25–4 Hz) | α-opic lux (Lucas et al.): Govardovski nomograms, λmax from Dartnall, optical densities 0.3, 0.38, 0.38 (S, M, L) |
Murray et al. ( |
4 | 460, 524, 590, 635 | Ganzfeld | Central 7° of surface covered with disk of no reflective black material. | L, M, (L+M+S) [11% Weber] | 17 cd/m2 | 5 | Square-wave (1 s increment/ |
Stockman Sharpe cone fundamentals |
Zele et al. ( |
5 | 456, 488, 540, 592, 633 | Maxwellian view | 30° circular, central 10.5° blocked | Color:[7%, 22% or 24% Weber] |
2,000 photopic Td (detection thresholds and pupil) |
4 (2 authors) | Pupil: 1 Hz sinusoidal | Smith–Pokorny cone fundamentals applied for the CIE 1964 108 Standard Observer CIE 1951 scotopic luminosity function Enezi et al. melanopsin function |
As described above (
In the case where the primary lights are discrete (such as LEDs), the peak emission wavelengths are subject to design considerations when building the apparatus. Both the choice of peak wavelengths and primary widths affects the contrast available for the silent substitution modulations. The contrast available is also called the
Typical viewing geometries include Ganzfeld viewing conditions (in which the stimulus is a homogenous field in an integrating sphere) or Maxwellian view (in which an image is focused on the entrance pupil of the observer). These again depend on the type of design used when building the stimulation system.
As can be seen in the table, the field sizes used in the field vary somewhat, and will again depend on constraints set by the optical apparatus used to deliver the stimuli, as well as theoretical considerations such as the distribution of the photoreceptor types across the retina.
Depending on the spectra of the primary lights, different amounts of contrast are available to stimulate melanopsin. Typically, the highest contrast can be achieved when LEDs are chosen of which the distribution of peak wavelengths is as broad as possible.
The choice of background light level is again somewhat arbitrary in many situations, though experimenters typically strive to be well in photopic conditions, where rods are assumed to be saturated, and can therefore be ignored (but see
The extent to which a given melanopsin-stimulating modulation silences the cones depends on the spectral sensitivities assumed. Various spectral sensitivities are available (
There are various sources of uncertainty when using silent substitution stimuli. We highlight a few of these here.
The human retina is inhomogeneous. One obvious feature of the retina making it inhomogeneous is the spatial location of the macular pigment around the fovea, with a drop-off toward the periphery. A consequence of macular pigment is that all light seen by the fovea is filtered through the pigment, thereby shifting the effective peak spectral sensitivity of the foveal cones vs. the peripheral cones. In addition, there are also differences in how much photopigment is expressed in foveal vs. peripheral cones—the optical densities are different. Another source of retinal inhomogeneity is that cones that are in the partial shadow of retinal blood vessels—penumbral cones—have a different spectral sensitivity than the open-field cones (
There are individual differences in the spectral sensitivities of the cones and this biological variability will affect the degree to which the cones are truly silenced in a melanopsin-directed modulation. Inter-observer differences have been a concern in the accurate specification of cone signals well before the discovery of melanopsin (
The method of silent substitution assumes that melanopsin the spectral sensitivity of melanopsin can be described by a single function. There is ample evidence that melanopsin is a bistable (
Under daylight conditions, rods are typically thought to be saturated (
The human eye is an imperfect optical system. In cases where the stimulus is a spatially extended light source and there is light outside the primary stimulation area (both centrally, if the macular region is blocked, and in the far periphery), there will be undesired stimulation of potentially unadapted photoreceptors (such as the rods). This can be addressed by adding a light outside the primary stimulation area that light-adapts the photoreceptors outside of the primary stimulation area.
The light source used may not be stable over time and change spectral output between operations, or throughout the sessions. These drifts in device output need to be either calibrated, or at least characterized.
We have noted in the introduction that the photoreceptors contributing to pupillary control differ not only in their spectral sensitivity (as is exploited in the method of silent substitution) but also in their temporal properties, their operating range and their distribution across the retina. These properties might also be exploited to selectively stimulate melanopsin. For example, the retinal location corresponding to the blind spot does not contain rods and cones, but light might stimulate melanopsin in the axons of ipRGCs. Delivering a stimulus only in the blind spot would therefore ensure that only melanopsin would be activated (
The method of silent substitution is a powerful technique to stimulate a specific photoreceptor class or specific photoreceptor classes in the living human retina while leaving other classes un-stimulated. The method has been used successfully to examine the photoreceptor contributions to the human pupillary light responses. The method is not failsafe as several factors need to be considered (retinal inhomogeneities, individual differences, rod intrusion, scatter, and device uncertainty), but these can be addressed experimentally or in simulation. We hope that the method of silent substitution will gain traction to tease apart the contributions of different photoreceptors to human vision and to elucidate their role in the non-invasive assessment of the human visual system using pupillometry.
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Prof. Hannah Smithson for comments on the manuscript.
The Supplementary Material for this article can be found online at: