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Department of Ophthalmology

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Yiannis Koutalos - Research Description

Research Interests

Eye, Retina, Photoreceptors

Phototransduction and Visual Cycle

Fluorescence Imaging


Research Interests

I am interested in the coordination and integration of physicochemical processes into cell function.  As a model system we use photoreceptor cells.  Photoreceptors are the cells that convert light to an electrical signal through a network of biochemical reactions that is called phototransduction.  The primary detector that absorbs the incoming photons and begins phototransduction is the visual pigment protein contained in the photoreceptor cells.  The process of light detection destroys the visual pigment necessitating its regeneration.  This regeneration is accomplished through a series of reactions that is called the Visual Cycle.  We use fluorescence imaging techniques to study the reactions of phototransduction and Visual Cycle in relation to cell metabolism, oxidative stress and aging.

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Eye, Retina and Photoreceptors

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The photoreceptor cells convert incoming photons to an electrical signal through a process called phototransduction.  Photoreceptors are part of the retina, a thin nerve tissue in the back of the eye.  Light enters through the cornea and is focused by the lens on the retina.  The photoreceptors are located in the back of the retina.


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Vertical section through a human retina (from J. Dowling.  The Retina, Harvard University Press, Cambridge, MA. 1987).
Photoreceptor cells are located in the back of the eye, adjacent to the pigment epithelium; light reaches them after it crosses the other neuronal layers of the retina.

Salamander rod and cone
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Brightfield images of living rod and cone photoreceptors isolated from a salamander retina.  Phototransduction takes place in the outer segment, while the ellipsoid is densely packed with mitochondria.  Rods are responsible for dim light vision, cones for bright light vision.

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Phototransduction and Visual Cycle

Photoreceptor light excitation

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Phototransduction.  Phototransduction takes place in the outer segments of the rod and cone photoreceptor cells, beginning with the absorption of incident light by visual pigment molecules.  In the absence of light, Na+ (and Ca2+) ions enter the photoreceptor outer segment, so that the cell is partially depolarized and maintains a high release of neurotransmitter from the synaptic terminal.  Light absorption begins a series of reactions that stop the Na+ influx, resulting in hyperpolarization and a reduction in transmitter release.


Visual CycleVisual Cycle.  Detection of light destroys the visual pigment, necessitating its regeneration.  The visual pigment, rhodopsin in the case of rod photoreceptors, is composed of a chromophore, 11-cis retinal, covalently linked to a protein, opsin . Absorption of a photon isomerizes the chromophore to all-trans and results in the production of an active intermediate that initiates the reactions leading to the change in the membrane potential of the photoreceptor cell .  The photoisomerization of the chromophore also results in the destruction of the visual pigment.  The series of reactions that remake the visual pigment comprise the visual cycle, and, in the case of the rods, take place within the photoreceptor outer segment and the adjacent pigment epithelial cells.  Within the rod outer segment, all-trans retinal is released from the photoactivated pigment and reduced to all-trans retinol by retinol dehydrogenase.  All-trans retinol is then transported to the pigment epithelial cells, where it is refashioned into 11-cis retinal.  All-trans retinal is toxic!

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Fluorescence Imaging

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Experimental setup.  For fluorescence imaging experiments, the sample of cells or tissues is placed in a chamber on a microscope stage.  Excitation light from an intense light source is focused on the sample by an objective lens.  The emitted fluorescence is collected by the objective lens and imaged with a CCD camera.


Frog and mouse photoreceptors

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Model systems.  Bright field images of isolated living frog and mouse rod photoreceptors and of a living mouse retinal slice.  These are the preparations we use for fluorescence imaging of photoreceptor function. [mouse slice image from Chen et al.,Biophys J 88:2278-2287, 2005].


All-trans retinol emits sufficient fluorescence to allow its imaging in living photoreceptors.  The formation of all-trans retinol in photoreceptor outer segments reflects the clearance of the toxic byproduct all-trans retinal.  Mitochondrial NADH can also be measured from its fluorescence and provides a way for measuring metabolic activity.


Color of retinol fluorescence

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All-trans retinol and NADH fluorescence in an intact frog rod photoreceptor.  The fluorescence image is shown in true color.  NADH fluorescence is blue-shifted compared to that of all-trans retinol.  [adapted from Wu et al., Biochemistry 46:8669-8679, 2007].

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Real time measurement of all-trans retinol formation in the outer segment of an isolated frog rod.  The cell is initially dark-adapted.  Upon exposure to light the chromophore of the visual pigment is isomerized to all-trans retinal.  All-trans retinol is generated through the reduction of the all-trans retinal released from the photoactivated pigment.


The fluorescence of a molecule can be sensitive to the binding of ions, to membrane potential, to oxidation, or other factors of biological interest.  Exogenous fluorophores sensitive to the appropriate factor can be incorporated into cells and used to measure ionic concentrations (Ca2+, Mg2+, H+, and others), mitochondrial membrane potential, oxidative stress, etc., etc.

Fluorophores can also be used as markers/tracers of different cellular compartments or organelles.

Rod and cone Mitotrackers

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Measurement of mitochondrial membrane potential.  Living rod and cone photoreceptor cells isolated from a salamander retina.  The cells have been stained with MitoTracker Red, a fluorescent probe that accumulates in the membrane of respiring mitochondria.


Free Mg2+ measurement

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Measurement of free Mg2+ concentration.  Living rod and cone photoreceptor cells isolated from a salamander retina were loaded with the Mg2+-sensitive probe FURAPTRA.  The fluorescence of the probe from the outer segment was converted to free Mg2+ concentration. [from Chen et al., J Physiol 553:125-135, 2003].

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