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The human eye operates over an enormous variety of light levels ranging from bright sunlight to starlight, with light intensity in bright sunlight being up to 10,000,000,000 times greater than in starlight thus equating to a 10log unit difference. Visual system's ability to cope and adjust to such levels of darkness and light is known as adaptation. For example, when we move from a relatively well-lighted or bright area to a relatively dark area, a brief amount of time is taken before the eye can perceive any detail or existence of any objects that may be present in relatively dark area. This is called 'dark adaptation'. It is achieved through the eye recovering its sensitivity to the dim light following the exposure to bright light. The first cells to adapt are the cones followed by rods which continue to adapt for up to 4 hours.
As the retina is exposed to high illuminance, all the photoreceptor pigments become bleached and are unable to absorb any photons whilst being in this state. Once placed into low illuminance, the photopigments revert back to their unbleached state, known as regeneration state, and displays an increase in sensitivity. One way of quantifying this change in sensitivity is by determining the changes in absolute threshold, that is, the minimum luminance of a test object required to produce visual sensation. However, there are several factors affecting the threshold luminance including colour, size, presentation time, Vitamin A deficiency etc.
The purpose of this experiment was to measure the time taken for the retina to fully dark adapt following exposure to high illuminance for both red and green test stimulus. This was done by recording subject's absolute threshold over time span of 20minutes following exposure to high illuminated Goldmann-Weekers Adaptometer and a computer with pre-set program designed to stimulate investigated conditions. The adaptometer allowed the subject control the luminance of the flicker test field, thus helping determine absolute threshold for different stimulus.
Before the start of the experiment, it was important that the subject familiarised with the adaptometer and the examiner with the computer program. Next the test field luminance was set to its maximum by rotating clockwise until it reached the stop. Run Experiment was selected from the menu displayed on the computer screen and details of the subject along with the colour of the test field were entered so that the graph produced by the computer would incorporate all this information.
The subject was then made aware of the bright light about to be displayed in front of them along with the time span of the whole experiment. Once the subject had been positioned, any key was pressed to automatically switch on the adaptation light. The subject was then encouraged to gaze around the interior of the brightly- lit globe for five minutes to attain a sufficient level of light adaptation. This is known as pre-adaptation and is designed to bleach most of the photopigment in the photoreceptors and ensure that both rods and cones are stimulated to obtain a bi-phasic curve at the end of experiment.
After 5minutes the adaptation light was switched off by the program and the subject was encouraged to fixate on the fixation point (above the test field) for the remainder of the experiment. Presentation of the test stimulus (10deg below the fixation point), on the peripheral visual field allowed isolation of the rods from the cones since the peripheral retina has higher density of rods. Also, the subject focusing on the fixation point ensured that adjusting of the luminance of the test field was based upon the peripheral vision and not central vision.
As soon as the test field became visible, the subject was asked to decrease the luminance so that it can only just be seen (threshold). The subject was then asked to maintain the luminance of the test field as close to the threshold as possible by continually altering the luminance up and down for the next 20 minutes. The subject was also told that it was normal to see the test field randomly disappear and reappear due to the change from cone phase to rod phase. The colour appearance of the test field near threshold during the cone and rod phases of dark adaptation was noted in order to demonstrate the change.
The colour of the test field was then changed from green light to red light (initially started with green light) and the experiment was repeated with the dark adaptation function recorded again. The colour of the test field was changed in order to compare the spectral sensitivity of the rods and the cones for different wavelengths.
Results and Discussion:
The graph above shows the difference in log luminance threshold over time. Lower log threshold values meant that the subject had to decrease the luminance of the test stimulus in order to maintain threshold and thus indicating greater intensity. As the graph shows there was a general decrease in the luminance threshold for both red and green lights as time progressed. This was due to more and more photopigments becoming unbleached as time passed by and thus resulting in increased sensitivity as retina dark adapted.
With reference to the red light curve, over a period of 20minutes, the threshold improved by about 2 log units, i.e. after 20 minutes, the subject became more sensitive to the red test stimuli and with reference to the green light curve, over a period of 20 minutes, the threshold improved by about 3 log units.
Both the curves to some extent show two regions of recovery, dominated by the cones and rods respectively. The initial phase, where the curve plateaus for first time (cone plateau), for both red and green test stimuli is credited to foveal adaptation, involving the cones since they are most abundant there, and is completed within 8 minutes for red and 5minutes for green, during which there is an increase in the sensitivity of the visual system by about 1 to 1.5log units, thus low threshold. Thereafter, the abrupt change in slope of both curves is referred to as rod-cone break. It's the point at which rods become more sensitive as near cessation of cone vision occurs. Prior to the point, the cones detect the stimuli as they are much faster at dark adapting then rods, and after this point the rods detect the stimuli. The final phase of the curve for both light stimuli's represents rod adaptation as the curve once again plateaus (rod plateau) to about 20 minutes, further sensitivity change of about 1 to 2 log units.
The cone plateau represents lowest photopic threshold for a stimulus whilst the rod plateau represents lowest scotopic threshold for the same stimulus. The difference between the two plateaus corresponds to the photochromatic interval. For the red test stimuli, it is relatively small as compared to the photochromatic interval of the green. This is due to scotopic system being more sensitive than photopic system at all wavelengths except in the long wavelength (red) region of the spectrum where rods and cones both are equally sensitive.
One of the core factors determining the dark adaption function is the bleaching of the photopigment present in the outer segments of cones and rods. Rhodopsin is the photopigment in rods which consists of opsin and retinal. Retinal in rhodopsin is the light sensitive part and exists in 2 forms, a cis form and a trans form. When in dark, retinal is in the cis form but on absorption of light, it switches to the trans form and changes its colour from purple to transparent, i.e. becomes bleached, hence making the rods non-functional. This means that whilst being in this state they are unable to absorb photons of light, resulting in becoming less sensitive, thus explaining the higher luminance threshold initially obtained after being light adapted, for red test field i.e. -0.3cdm-2 and green test field i.e. -0.4 cdm-2.
Thereafter, with cone mechanism for dark adaptation under way first, the decrease in the luminance threshold for both red and green test field was due to the regeneration of the photopigment in cones. Bleached photopigment molecules spontaneously revert back to the unbleached state when placed under darkness, with cone photopigments taking just under 10 minutes to regenerate after bleaching whereas rods being much slower and taking about 30 minutes for all it's photopigment to regenerate. Hence the half life of regeneration, which is an exponential function that shows the time it takes for half the photopigment molecules to revert back to their unbleached state following bleaching, for the cone photopigments, i.e. approximately 1.5 minutes, is evident from the graph as from about 1 minute onwards the luminance threshold seems to be decreasing since the sensitivity of the visual system is brought back. The initiation of the slower rod dark adaptation, i.e. regeneration of rhodopsin molecules, is shown by the further dip in the slope for both red and green at 8minutes and 5 minutes respectively, thus the decreasing luminance threshold as the visual system was becoming increasingly sensitive to light stimulus.
A different wavelength stimulus is another factor that affects the dark adaptation function. As previously mentioned, when the illumination is reduced to a low level, the visual mechanism shifts from photopic vision to scotopic vision as the eye slowly dark adapts. As a result, the sensation produced by longer wave stimuli decreases rapidly as compared to that produced by shorter wave stimuli and this shift in sensitivity of eye towards the blue end of spectrum at low illumination levels is known as the purkinje shift. Moreover, taking into account the relative luminous efficiency, both the rods and the cones have similar sensitivities to longer wavelength light, i.e. red, due to a small photochromatic interval. On the other hand however, with a large photochromatic interval and therefore a prominent rod-cone breakpoint, the rods are much more sensitive to a medium wavelength of light, i.e. green, than the cones, with rods peak absorption and sensitivity being at 505nm as compared to cones at 555nm once the visual system is dark adapted. This explains the dip in the green curve as compared to red curve since the rods were more sensitive to green light stimuli hence giving low luminance threshold readings whereas red light stimuli giving comparatively higher luminance threshold readings. The rods poor absorption of red light, thus insensitivity to it explains the difficulty experienced by the visual system to identify red objects under scotopic vision.
The rod's ultimate sensitivity makes them naturally more sensitive to the green light stimuli than the cones and is evident from the graph. In context to absolute threshold, a flash of light in the peripheral field would be detected by a dark adapted subject when about 50-150 photons are incident on the cornea. However, many of these photons are lost even before they reach the retina and are not absorbed by any photoreceptors, therefore making it unlikely that any rod receives more than 1 photon. Hence in a dark adapted subject, for perception to occur, only 1 photon is needed to be absorbed by the rod, and thus explaining rod's ultimate sensitivity.
The ultimate sensitivity of the rods can be explained by their display of spatial and temporal summation. Wiring of the rods is such that there is a lot of convergence in their pathway and therefore allowing them to poses spatial and temporal summation properties. Many rods (with ratio of rods to ganglion cell being up to 500:1) synapse onto a single bipolar cell which then synapses onto a single ganglion cell. Photopigments in the photoreceptors are naturally unstable, with retinal in rhodopsin isomerising even if no photons are absorbed. However, the retinal cell ganglions have a failsafe mechanism which allows firing of a potential provided an input is received from at least 10 isomersisations. Since many rods converge onto same ganglion cell, there is a greater chance of 10 isomerisation input occurring than there is for cones in the fovea as there is only 1:1 ratio of cone to ganglion cell, with no convergence in the foveal cone pathway. This gives the rods its greater sensitivity in comparison to the cones, however with a drawback of poorer spatial resolution of the rods i.e. decreased visual acuity.
Temporal summation is another mechanism used by the rods under the scotopic conditions to increase their sensitivity. Temporal summation is when rods combine their response arriving from numerous photons over a period of time and thus like spatial summation, increasing the chances of the retinal ganglion cell receiving the input from 10 isomerisations necessary in order for potential to be fired. However, just as spatial summation results in decline in spatial resolution, similarly temporal summation causes reduction in temporal resolution, which explains why rods have a lower flicker fusion frequency than cones.
A factor affecting the sensitivity of the cones is the position of the stimulus on the retina and can be explained by stiles-crawford effect. For light to be maximally effective at bleaching photopigment, it must strike cone perpendicular to its surface rather than obliquely. Moreover a light stimulus entering through the centre of the pupil is more likely to be absorbed by a photoreceptor than the light entering through periphery. Taking the two mentioned points above into consideration, the cones display a considerable directional sensitivity with peak sensitivity being only when light is entering through the centre of the pupil and hitting the cones perpendicularly. On the other hand, the rods display no directional sensitivity and therefore the angle at which a photon strikes a rod is relatively insignificant since the rods are designed to operate under the conditions, where already limited photons are available, i.e. scotopic.
There are a number of diseases affecting the rods before the cones and vice versa. The dark adaptation function is often clinically measured in order to reveal the relative rate of decline of the sensitivity of the rods and the cones caused by diseases.
One such disease is retinitis pigmentosa, which is degeneration of the rods in the early stages with cone degeneration at latter stage. A patient suffering from it would initially report difficulty seeing at night and lose their peripheral visual field, ending up with 'tunnel vision'.
The graph on the left shows how dark adaptation function would change over time of a subject suffering from retinitis pigmentosa. The cone phase would appear relatively normal but a change in rod phase would be recorded as well as delayed rod-cone break, indicating decrease in the rod's ultimate sensitivity. With progression of the disease, the rod-cone break would delay further as well as rods losing their sensitivity. Eventually with complete degeneration of the rods, the subject would just be left with the cone component of the dark adaptation function and will have real difficulties viewing under low light levels.
The graph on the left shows how the dark adaptation function would look for a subject that is asked to fixate on small central test field.
Like the graph above, the cone phase would be normal with only change in the rod phase. This is because under these conditions, only foveal/macula area would be functioning and since there are no or very few rods here, a rod phase would not be recorded for the dark adaptation function. With cones having highest density at the centre of the fovea (160,000 mm2), they would be successful in capturing the photons of light and producing a cone phase. Moreover when a small test stimulus is used, a single branch is recorded since only cones are found at the fovea. With a larger test spot however, both rods and cones would be stimulated resulting in rod-cone break to be recorded.
The graph on the left shows how the dark adaptation function would look for a patient with condition of rod monochromotism. This is where the retina would not contain any functional cones and therefore report having poor colour vision, photophobia and at times associated nystagmus and myopia.
Thus as expected, the rod phase would be normal but the cone phase will be affected as the curve displays rods taking much longer to adapt as compared to cones since it takes less time for the cones to achieve maximum sensitivity.
The graph here is for a subject being shown red test field. The dark adaption function would display rods being less sensitive to red light than the cones. This is due to the low probability of rhodopsin molecule absorbing photon of longer wavelength, i.e. red. It's only after a considerable amount of rhodopsin having regenerated that the scotopic system will become more sensitive than the photopic system.
To conclude, the results showed that the cone threshold for red light stimuli was reached at around 8minutes and for green at 5minutes. The rod threshold for both green and red light stimuli could not be recorded as it is expected that it would take up to 30minutes for all rods to be regenerated but the experiment was set to run for only 20minutes. Moreover it was expected that the rods would become more sensitive as compared to the cones than the observed results, increasing by about 3 log units more to around -5log units. If the experiment would have been carried for up to 300minutes, it is possible that the anticipated rods threshold would have been achieved.
Factors that could have affected this experiment include results being affected by the mentally tiring and tedious nature of the procedure which would have altered subject's response. To minimise the errors, the experiment could have been conducted on variety of subjects and an average could have been calculated. This would have helped reduce influence on results caused by particular subject personality factors.
To further improve the experiment, more factors affecting dark adaptation such as effects of field size or duration of stimulus presentation, could have been investigated.