Module: HNSS

Photochemistry of Vision

Alissa Laurel N. Calderon,

MD, FPAFP, MMHA
Objective:
At the end of the session, the students
should be able to:
1. To review the parts of the eye
2. To understand the roles of
photoreceptors in vision.
3. To understand the role of Vitamin A in
vision.
4. The understand what is
phototransduction.
5 Major Sensory Systems
• Vision – the detection of light
• Olfaction – the detection of small
molecules in the air (sense of smell)
• Taste or Gustation – the detection of
selected organic compounds and ions by
the tongue
• Hearing – the detection of sound (or
pressure wave in the air)
• Touch – the detection of changes in
pressure, temperature and other factors by
the skin
Each of these primary sensory systems contains
specialized sensory neurons that transmit nerve
impulses to the CNS

In the CNS theses signals are processed

and combined with other information to
yield a perception that may trigger a
change in behavior.
By these means, our senses allow us to
detect changes in our environments and
adjust our behavior appropriately
VISION
Pigment epithelium

Neuronal layers
The Retina – inner layer of the
eye
• Contains photoreceptor cells (rods and cones) and
associated interneurones and sensory neurones

light

to optic ganglion bipolar rod cone pigmented

nerve cells neurones cells cells retina
• Two types of photoreceptor cells
• rods and cones
• Rods are in the periphery
• more sensitive to light; they are not involved
in distinguishing color
• function in night vision; and then only in black
and white
• @ 125 M
• Cones are at the center of retina
• Cones are responsible for bright light and
color vision
• @6M
Rods vs Cones
• Differ in their sensitivities to light of different intensity and
wavelength and their retinal distributions
• Rods are highly sensitive to low (mesopic) levels of
illumination, and under complete darkness (scotopic)
and full dark adaptation can detect a single photon of
light.
• Cones require high levels of photon capture for
activation, and therefore work best under bright
(photopic) light conditions.
Cones Rods
• Cone-shaped • Rod-shaped
• Pigment: Iodopsin • Pigment: Rhodopsin
• Function: highly discriminatory • Function: peripheral vision and
central vision and color vision scotopic vision (vision of low
• Low sensitivity illumination)
• Mesopic-Photopic light range • High sensitivity (single photon)
• Low convergence • Scotopic-Mesopic light range
• High density in fovea • High convergence
• Trichromatic: 3 cone • Low spatial resolution
opsins
Wavelength • Low density in fovea

Sensitivities
Short: 419nm
(blue)
Medium: 531nm
(green)
Photochemistry of Vision
• Photopic Vision
• Day light vision due to cones
• Color vision
• Brightness above 1mA
• Scotopic Vision
• Dim light vision due to rods
• Below 0.001 mA
• Mesotopic Vision
• Full moonlight vision
• Both rods and cones
Electromagnetic Spectrum
• Photons are classified according to their wavelength
• Longest wavelength: radio and television waves
• Shortest wavelength : gamma rays
• Middle of the spectrum : visible light
Rods and Cones
• Retinal photoreceptors contain pigments that
preferentially absorb photons with wavelengths 400-700
nm
• Shortest wavelength : blue and green
• Longer wavelength : yellow, orange and red
Visual Pigments
• Four visual pigments:
• Rhodopsin : present in rods
• 3 cone pigments
• Erythrolabe (R cones) : red , 570 nm
• Cyanolabe (B cones): blue, 440 nm
• Chlorolabe (G cones): green, 540 nm
 The neural circuits in the retina
Vision---rod/cones of a primate
-The incoming light reaches the
photoreceptor cells (rods and cones) only
after passing through several thin,
transparent layers of other neurons.

-The pigment epithelium absorbs the light

that is not absorbed by the photoreceptor
cells and thus minimizes reflections of
stray light.

The ganglion cells communicate to the

thalamus by sending action potentials
down their axons.

However, the photoreceptor cells and

other neurons communicate by graded
synaptic potentials that are conducted
electronically.
Phototransduction
• The primary function of the photoreceptors is the conversion
of light energy (photons) to electrical signals.
• The initial step in phototransduction is the absorption of a
photon of light by the photopigments within the disk
membranes of their outer segments
• in rods, the photopigment is rhodopsin,
which absorbs light
over a broad range of wavelengths.
• Cones contain one of three cone opsins, which have
peak
sensitivities at wavelengths corresponding to red,
green, and blue.
• All other perceived colors are represented by
differences
in the absorption balance of these three pigments.
The Rod Cell

Scanning electron micrographs of retinal rod cells

Schematic representation
of a rod cell Photoperception
100,000,000 rod cells 1000 disks, 16nm thick
in human retina

Rod cell (1x40µm)

Biochemistry. L. Stryer
 RODS

• @ 125M
• Detect the degree of light entering the eye
• Contain the pigment rhodopsin or visual purple, which
is generated within the cells
• Only work in low light as at high illumination the
reduced level of this photosensitive pigments leads to
a very low sensitivity.
• The speed at which rhodopsin adjusts to darkness
depends on a sufficient supply of Vitamin A in the
body
• Humans cannot make rhodopsin instead they use an
external source, β-carotene, which is found in food
• Rod vision is acute but coarse
• Rods do not provide a sharp image for several reasons:
• Adjacent rods are connected by gap junctions and so
share their changes in membrane potential
• Several nearby rods often share a single circuit to one
ganglion cell
• A single rod can send signals to several different
ganglion cells
• If only a single rod is stimulated, the brain has no way
of determining exactly where on the retina it was
• Rods are sensitive to light
• A single photon absorbed by a small cluster of
adjacent rods is sufficient to send a signal to brain
• Rods provide us a grainy, colorless image, they
permit us to detect light that is over a billion times
dimmer than what we see on a bright sunny day
• Human peripheral vision is Rod based..
• We cannot see color at the edges of our vision
ROD VISION
• Rhodopsin: visual purple; light absorbing pigment of the
rods
• Scotopsin (opsin): protein part
• Retinene1 : 11-cis retinal, derivative of Vitamin A
• Serves as chromophore
• Main receptor for photons which enter the eye
• Metarhodopsin II – activated rhodopsin; brings about
electrical changes in rods

• Photoreceptor molecule in the rods: rhodopsin

consists of opsin linked to 11-cis retinal
(polyene- with 6 alternating double and single bonds)
Illustration of
Rhodopsin (blue)
with
11-cis retinal (red)
Vision Process for Monochrome Vision (Rod
Cells)
• three important steps:
1. isomerization of retinal
2. protein conformational changes following retinal
isomerization
3. signal transduction cascade to generate a nerve
impulse.
1. Isomerization of Retinal
• first step in the monochrome vision process, after light
hits the rod cell, is for the chromophore 11-cis-retinal to
isomerize to all-trans-retinal
 11-cis-retinal 11-trans-retinal

➢ When light hits the retinal, the energy is used to break

the double bond contained in the molecule
➢ Light causes a photoisomerization of 11-cis retinal into
an intermediate form that after going through a series
of spontaneous changes, finally converted to all-trans
retinal
➢ All-trans retinal dissociates from opsin and then is
converted to 11-trans retinol (Vitamin A)
• When the chromophore absorbs a photon it isomerizes
to the all-trans configuration without (at first) any
accompanying change in the structure of the protein
• Rhodopsin containing the all-trans isomer of retinal is
known as bathorhodopsin.
• However, the trans isomer does not fit well into
the protein, due to its rigid, elongated shape.
• While it is contained in the protein, the all-trans
chromophore adopts a twisted conformation, which
is energetically unfavorable.
• Therefore, a series of changes occurs to expel
the chromophore from the protein.
 2. Protein Conformational Changes Following
Retinal Isomerization
• the isomerization of retinal has an important effect on
special proteins in the rod cell:
• the isomerization event actually causes the proteins
to change their shape.
• This shape change ultimately leads to the
generation of a nerve impulse.
• For vision to occur, the important intermediate is
metarhodopsin II.
• This intermediate activates the enzyme transducin,
which starts the impressive signal transduction
cascade resulting in the production of a nerve
impulse to the brain
• The all-trans-retinal is important for the multiple-step
regeneration of 11-cis-retinal.
• The regenerated 11-cis-retinal is re-incorporated into
rhodopsin.
Schematic diagram of the cyclic GMP cascade of vision
3. Signal Transduction Cascade to Generate a
Nerve Impulse
• After the metarhodopsin II is formed, there are
approximately four more steps in the vision process
1. activation of the enzymes transducin and
phosphodiesterase,
2. hydrolysis of cyclic GMP,
3. closing of Na+ channels, and
4. propagation of an electrical impulse to the brain
Signal Transduction Cascade to
Generate a Nerve Impulse
• The starting point for this process is the production
of metarhodopsin II
• This initiates the following cascade of events: First,
metarhodopsin II complexes with the enzyme
transducin and activates it. Transducin in turn activates
another enzyme, phosphodiesterase.
Phosphodiesterase catalyzes the hydrolysis of cyclic
GMP,
• Phosphodiesterase catalyzes the hydrolysis of cyclic
GMP, adding a water molecule (red) to GMP and
breaking the bond between the phosphate group (blue)
and carbon.
• Cyclic GMP is required to open Na+ channels in
the plasma membrane.
• In the dark, cyclic GMP is abundant and these
channels stay open.
• Sodium cations enter freely into the rod cell,
because the cell typically has a lower potential
(is more negative) than the external
environment, thus attracting the positively-
charged ions.
• when cyclic GMP is hydrolyzed (gains an H2O and
breaks a bond) by the now-activated phosphodiesterase,
it is no longer available to keep the Na+ channels open.
• Sodium cations can no longer enter the cell freely, and
so the cell's potential suddenly becomes even lower
relative to the external environment.
• A large charge difference across the membrane is built
up; this is known as hyperpolarization.
• The large potential difference travels as an electrical
impulse down the rod cell to the synaptic terminal, and is
then transferred to an adjoining nerve cell.
• The nerve cell carries this impulse all the way to the
brain.
• The brain then determines where the nerve impulse
originated, and interprets the image,
• https://www.youtube.com/watch?v=58OOO0zk6mY
• https://www.youtube.com/watch?v=aSND0v18cks
Rhodopsin Bleaching and Regeneration
• When the eye is exposed to light, the 11-cis-retinal
component of rhodopsin is converted to all-trans-retinal,
resulting in a fundamental change in the configuration of
the rhodopsin molecule.
• The change in configuration initiates a phototransduction
cascade within the rod, whereby light is converted into
an electrical signal that is then transmitted along the
optic nerve to the visual cortex in the brain.
• The change in configuration also causes opsin to
dissociate from retinal, resulting in bleaching.
• Bleaching limits the degree to which the rods are
stimulated, decreasing their sensitivity to bright light and
allowing cone cells (the other type of photoreceptor in
the retina) to mediate vision in bright environments.
Bleaching of rhodopsin
• When exposed to light, the color of rhodopsin changes from
red to yellow by a process known as bleaching
• Bleaching occurs in a few milliseconds and many unstable
intermediates are formed during this process

• Rhodopsin Prelumirhodopsin Lumirhodopsin

All-trans-retinal Metarhodopsin II Metarhodopsin I

+ Opsin
• All-trans-retinal that is released during bleaching is either
stored or changed back to 11-cis-retinal and transported
back to the rods.
• The latter process, which is known as recycling,
allows for the regeneration of rhodopsin.
• Rhodopsin regeneration takes place in darkness and is
central to dark adaptation, when rhodopsin levels,
depleted from bleaching in a brightly lit environment,
gradually increase, enabling rod cells to become
increasingly sensitive to dim light.
• The all-trans-retinal is isomerized to 11-cis-retinal by
retinal isomerase (retinal epithelium)
• This combines with opsin to regenerate rhodopsin and
compete the visual cycle
• Most of the all-trans retinal is transported to liver and
converted to all-trans retinol by alcohol dehydrogenase
Summary of Steps of Phototransduction
• The photosensitive element is rhodopsin, which is
composed of opsin, a protein, belonging to the
superfamily of G-protein-coupled receptors and retinal
(an aldehyde of Vitamin A)
a. Light on the retina converts 11-cis retinal to all-trans
retinal, a process called photoisomerization.
• A series of intermediates is then formed, one of which
is Metarhodopsin II
• Vitamin A is necessary for the regeneration of 11-cis
retinal
b. Metarhodopsin II activates a G-
protein called transducin, which
in turn activates a
phosphodiesterase
c. Phosphodiesterase catalyzes the
conversion of cyclic guansine
monophosphate (cGMP) to 5’-
GMP and cGMP levels decrease
d. Decrease levels of cGMP cause
closure of Na+ channels,
decreased inward Na+ current,
resulting into hyperpolarization of
the cell membrane
• Increasing light intensity
increases the degree of
hyperpolarization
• It is an excitatory response
transmitted through the
neuron network to the
visual cortex of brain
Rhodopsin-Retinal Visual Cycle
• Regeneration of rhodopsin: all-
trans retinol is enzymatically
isomerized to 11-cis retinol
and then enzymatically
oxidized to 11-cis retinal.
• The 11-cis retinal then
spontaneously combines with
opsin, reforming rhodopsin
1. The conversion of the retinol
to retinal is dependent on the
pigment epithelium
2. Deficiency of vitamin A
prevents the regeneration of
rhodopsin
• Lack of vitamin A leads to night
blindness
Regulation of Retinal Sensitivity
• Dark Adaptation: person exposed to light for many hours
is suddenly exposed to darkness
• Rhodopsin stores are depleted and vision is impaired
• After a few minutes rhodopsin is resynthesized and
vision is improved
• Usually completed within 1 hour
• Both cones and rods take part
• Cones adapt earlier than rods
• Light Adaptation: reverse of the above
• Sensitivity of eye can change by 1 million times
• Registration of image requires both light and dark
spots
In the Dark…

• In the dark the channel is open → Na+ flow in can

cause rod cells to depolarise.
• Therefore in total darkness, the membrane of a rod cell is
polarised
• Therefore rod cells release neurotransmitter in the
dark
• However the synapse with bipolar cells is an
inhibitory synapse i.e. the neurotransmitter stops
impulse
In the Light…

As cis retinal is converted to trans retinal, the

Na+ channels begin to close
i
less neurotransmitter is produced. If the
threshold is reached, the bipolar cell will be
depolarised
i
forms an impulse which is then passed to the
ganglion cells and then to the brain
Rods and Cones
Rods Cones
Outer segment is rod Outer segment is cons
shaped shaped
109 cells per eye, 106 cells per eye, found
distributed throughout the mainly in the fovea, so can
retina, so used for only detect images in
peripheral vision. centre of retina.
Good sensitivity Poor sensitivity
Only 1 type → 3 types (R, G & B) →
monochromatic vision colour vision
Many rods connected to Each cone is connected to
one bipolar cell → poor one bipolar cell → good
acuity = poor resolution acuity = good resolution
Mechanisms of Dark & Light Adaptations
1. Availability of light sensitive pigments
2. Changes in pupillary size
3. Neural adaptation
Night Blindness (Nyctalopia)
• Impaired vision at night or in dim light situations
• Rhodopsin deficiency affecting rods
• Rods degenerate
• Most common cause – prolonged vitamin A deficiency
Mechanism for Vitamin A Improvements for
Night Vision
• As it relates to eyesight, vitamin A (also called all-trans-
retinol) has been shown to be useful in helping with night
vision
• Vitamin A is a precursor of rhodopsin, the photopigment
found in rods within the retina of our eye that helps us to
see at night. .
Cones
• Around 3 M
• Concentrated in the center of the retina (macula)
• Contain the pigment iodopsin
• Sensitive to light levels but retain their function up to high
illumination via use of pigment
• Detection of color is the function of 3 types of cone cells
present within the retina
Cone Vision
• Cones operate only in relatively bright light
• Provide us with sharpest images and enable us to see
color
• Most of the cones in each retina are confined to a small
region just opposite the lens called the fovea
• Sharpest and colorful images are limited to a small
area of view
 Color Vision
• When we look at something the image falls on the
fovea and we see it in color and sharp detail.

• Objects in the periphery of our field of view are not

seen in colour, or detail.

• The fovea has high density of cones.

• Each cone has a synapse with one bipolar cell and

one ganglion → each cone sends impulses to the
brain about its own small area of the retina → high
visual acuity
Color Vision

• 3 different cone cells. Each have a different form of

opsin (they have the same retinal)

• 3 forms of rhodopsin are sensitive to different parts of

the spectrum
• 10% red cones
• 45% blue cones
• 45% blue cones
 Color Vision
• Colored light will stimulate these 3 cells differently - by
comparing the nerve impulses from the 3 kinds of cones
the brain can detect any colour
• Red light → stimulates R cones
• Yellow light → stimulates R and G cones equally
• Cyan light → stimulates B and G cones equally
• White light → stimulates all 3 cones equally

• Called the trichromatic theory of color vision

Summary: Wald’s Visual Cycle
1. Rhodopsin, which is the chromoprotein that is present in the outer
segment of the rods, is bleached on exposure to light.
2. When light falls on retina, the rhodopsin passes rapidly through
several stages of very short half – lives.
3. It is finally converted to metarhodopsin – II that splits in to all –
trans retinene1 and opsin.
4. This all – trans retinene1 is then reduced to vitamin – A1 by alcohol
dehydrogenase (otherwise called retinene reductase) using NADH
(note that usually NADPH serves as coenzyme for reductase
enzymes but, here is one of the exceptions). It is then stored in the
pigment epithelium.
5. Similarly, 11 – cis retinene1 can be converted to 11 – cis vitamin –
A1, by alcohol dehydrogenase. These two are reversible reactions.
6. The photopigment should be regenerated after being bleached so
as to get ready for the next round of response.
Summary (Con’t)
7. Therefore in the dark, retinene isomerase and also vitamin –
A1 isomerase, converts all – trans retinene1 and all – trans vitamin
– A1, to 11 – cis retinene1 and 11 – cis vitamin – A1, respectively.
These two are also reversible reactions. This reaction consumes
energy and takes place in light of short wavelength.
8. Similar changes takes place in cones where instead of rhodopsin
we have iodopsin which splits up to photopsin and all – trans
retinene1.
9. The trans form of vitamin – A (retinol), which is abundant in the liver
and blood, is the most active isomer in all physiological roles of the
vitamin, except vision.
10. Bleaching and regeneration of photosensitive pigments are
independent of nerves but takes place only as long as the rods
remain in contact with the stratum pigmenti.
11. Bleaching itself or products formed during bleaching stimulate the
rods to generate visual impulses, i.e., light energy is converted to
nerve impulse.
https://www.youtube.com/watch?v=HG5BfsaoiE0
• https://www.youtube.com/watch?v=TeFm1ca2ozo
References:
• Nelson, et al. Lehninger: Principles of Biochemistry.
”Sensory Transducton in Vision, Olfaction and
Transduction.” Chapter 12.7