Name: preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging
Uploaded: 2011-06-22T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging at JoVE.com

Supported by NEI grant EY014850.

1.	Preparation of Sylgard-covered dishes, experimental chambers, and razor blades
<ol>
<li>35 mm Falcon Petri dishes coated with Sylgard elastomer are needed for the proper chopping of an isolated retina to obtain single photoreceptor cells. The elastomer is prepared according to the supplier's instructions and a small amount is poured into each dish to cover its bottom with a layer. Replace the dish covers after coating and store them. In a few days' time the elastomer hardens and the dishes are ready.</li>
<li>Iso...

<ol>
	<li>Burns, M. E., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Counting+Photons:+Trials+and+Trends+in+Vertebrate+Visual+Transduction.">Beyond Counting Photons: Trials and Trends in Vertebrate Visual Transduction.</a> Neuron. 48, 387-401 (2005).</li><li>Ebrey, T., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+Photoreceptors.">Vertebrate Photoreceptors.</a> Prog Retin Eye Res. 20, 49-94 (2001).</li><li>Fain, G. L., Matthews, H. R., Cornwall, M. C., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+in+Vertebrate+Photoreceptors.">Adaptation in Vertebrate Photoreceptors.</a> Physiol Rev. 81, 117-151 (2001).</li><li>Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+Protein-Coupled+Receptor+Rhodopsin.">G Protein-Coupled Receptor Rhodopsin.</a> Annu Rev Biochem. 75, 743-767 (2006).</li><li>Saari, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+Visual+Pigment+Regeneration:+The+Friedenwald+Lecture.">Biochemistry of Visual Pigment Regeneration: The Friedenwald Lecture.</a> Invest Ophthalmol Vis Sci. 41, 337-348 (2000).</li><li>Lamb, T. D., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dark+Adaptation+and+the+Retinoid+Cycle+of+Vision.">Dark Adaptation and the Retinoid Cycle of Vision.</a> Prog Retin Eye Res. 23, 307-380 (2004).</li><li>Imanishi, Y., Lodowski, K. H., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Photon+Microscopy:+Shedding+Light+on+the+Chemistry+of+Vision.">Two-Photon Microscopy: Shedding Light on the Chemistry of Vision.</a> Biochemistry. 46, 9674-9684 (2007).</li><li>Sampath, A. P., Matthews, H. R., Cornwall, M. C., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleached+Pigment+Produces+a+Maintained+Decrease+in+Outer+Segment+Ca2++in+Salamander+Rods.">Bleached Pigment Produces a Maintained Decrease in Outer Segment Ca2+ in Salamander Rods.</a> J Gen Physiol. 111, 53-64 (1998).</li><li>Sampath, A. P., Matthews, H. R., Cornwall, M. C., Bandarchi, J., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Dependent+Changes+in+Outer+Segment+Free-Ca2++Concentration+in+Salamander+Cone+Photoreceptors.">Light-Dependent Changes in Outer Segment Free-Ca2+ Concentration in Salamander Cone Photoreceptors.</a> J Gen Physiol. 113, 267-277 (1999).</li><li>Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Cytoplasmic+Calcium+Concentration+in+the+Rods+of+Wild-Type+and+Transducin+Knock-out+Mice.">Measurement of Cytoplasmic Calcium Concentration in the Rods of Wild-Type and Transducin Knock-out Mice.</a> J Physiol. 542, 843-854 (2002).</li><li>Leung, Y. T., Fain, G. L., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Measurement+of+Current+and+Calcium+in+the+Ultraviolet-Sensitive+Cones+of+Zebrafish.">Simultaneous Measurement of Current and Calcium in the Ultraviolet-Sensitive Cones of Zebrafish.</a> J Physiol. 579, 15-27 (2007).</li><li>Matthews, H. R., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Spot+Confocal+Technique+to+Measure+Cytoplasmic+Calcium+Concentration+in+Photoreceptors.">Laser Spot Confocal Technique to Measure Cytoplasmic Calcium Concentration in Photoreceptors.</a> Methods Enzymol. 316, 146-163 (2000).</li><li>Szikra, T., Cusato, K., Thoreson, W. B., Barabas, P., Bartoletti, T. M., Krizaj, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+Calcium+Stores+Regulates+Calcium+Influx+and+Signal+Transmission+in+Rod+Photoreceptors.">Depletion of Calcium Stores Regulates Calcium Influx and Signal Transmission in Rod Photoreceptors.</a> J Physiol. 586, 4859-4875 (2008).</li><li>Krizaj, D., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+Calcium+Extrusion+Mechanisms+in+the+Outer+and+Inner+Segments+of+Photoreceptors.">Compartmentalization of Calcium Extrusion Mechanisms in the Outer and Inner Segments of Photoreceptors.</a> Neuron. 21, 249-256 (1998).</li><li>Chen, C., Nakatani, K., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+Magnesium+Concentration+in+Salamander+Photoreceptor+Outer+Segments.">Free Magnesium Concentration in Salamander Photoreceptor Outer Segments.</a> J Physiol. 553, 125-135 (2003).</li><li>Chen, C., Jiang, Y., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Behavior+of+Rod+Photoreceptor+Disks.">Dynamic Behavior of Rod Photoreceptor Disks.</a> Biophys J. 83, 1403-1412 (2002).</li><li>Tsina, E., Chen, C., Koutalos, Y., Ala-Laurila, P., Tsacopoulos, M., Wiggert, B., Crouch, R. K., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+Microfluorometric+Studies+of+Reduction+and+Clearance+of+Retinal+in+Bleached+Rod+Photoreceptors.">Physiological and Microfluorometric Studies of Reduction and Clearance of Retinal in Bleached Rod Photoreceptors.</a> J Gen Physiol. 124, 429-443 (2004).</li><li>Ala-Laurila, P., Kolesnikov, A. V., Crouch, R. K., Tsina, E., Shukolyukov, S. A., Govardovskii, V. I., Koutalos, Y., Wiggert, B., Estevez, M. E., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Cycle:+Dependence+of+Retinol+Production+and+Removal+on+Photoproduct+Decay+and+Cell+Morphology.">Visual Cycle: Dependence of Retinol Production and Removal on Photoproduct Decay and Cell Morphology.</a> J Gen Physiol. 128, 153-169 (2006).</li><li>Koutalos, Y., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluorometric+Measurement+of+the+Formation+of+All-Trans-Retinol+in+the+Outer+Segments+of+Single+Isolated+Vertebrate+Photoreceptors.">Microfluorometric Measurement of the Formation of All-Trans-Retinol in the Outer Segments of Single Isolated Vertebrate Photoreceptors.</a> Methods Mol Biol. 652, 129-147 (2010).</li><li>Wu, Q., Blakeley, L. R., Cornwall, M. C., Crouch, R. K., Wiggert, B. N., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interphotoreceptor+Retinoid-Binding+Protein+Is+the+Physiologically+Relevant+Carrier+That+Removes+Retinol+from+Rod+Photoreceptor+Outer+Segments.">Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments.</a> Biochemistry. 46, 8669-8679 (2007).</li><li>Kolesnikov, A. V., Ala-Laurila, P., Shukolyukov, S. A., Crouch, R. K., Wiggert, B., Estevez, M. E., Govardovskii, V. I., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Cycle+and+Its+Metabolic+Support+in+Gecko+Photoreceptors.">Visual Cycle and Its Metabolic Support in Gecko Photoreceptors.</a> Vision Res. 47, 363-374 (2007).</li><li>Chen, C., Blakeley, L. R., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+All-Trans+Retinol+after+Visual+Pigment+Bleaching+in+Mouse+Photoreceptors.">Formation of All-Trans Retinol after Visual Pigment Bleaching in Mouse Photoreceptors.</a> Invest Ophthalmol Vis Sci. 50, 3589-3595 (2009).</li></ol>

If healthy isolated cells are not obtained, the problem lies either with the isolation or health of the retina or with its chopping. Typically, after removing the front of the eye and the vitreous, the retina readily lifts off the pigment epithelium. If it does not, try to peel it off starting from the periphery of the eyecup. If it is still difficult to separate, a likely possibility is that the animal has not been dark-adapted for an adequate period of time, or the red light is too bright. Ensure proper dark-adapta...

<table border="1">
 <tbody>
 <tr>
 <th>Name</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
 </tr>
 <tr>
 <td>Dark room (100-150 ft2)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Red lights19</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Online stores</td>
 </tr>

 <tr>
 <td>Infrared light sources and
infrared image viewers
</td>
 <td>FJW Optical Systems, Inc.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 <td>Dissecting microscope19</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Outfitted with infrared viewers</td>
 </tr>

 <tr>
 <td>Epifluorescence microscope enclosed in a light-tight cage19</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 <td>Dissecting tools
(scissors, forceps, blade holder) 
</td>
 <td>Roboz or
Fine Science Tools
</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 <td>Sylgard elastomer</td>
 <td>Essex (Charlotte, NC)</td>
 <td>Sylgard 184 elastomer kit</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 <td>Poly-L-ornithine (0.01%)</td>
 <td>Sigma-Aldrich</td>
 <td>P4957</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 <td>Poly-L-lysine (0.1%)</td>
 <td>Sigma-Aldrich</td>
 <td>P8920</td>
 <td>Dilute to 0.01%</td>
 </tr>

 <tr>
 <td>Experimental chambers</td>
 <td>Warner Instruments (Hamden, CT)</td>
 <td>D3512P</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 <td>Petri dishes, plastic pipettes</td>
 <td>Fisher Scientific</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr> 
 </tbody>
</table>

preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that 
initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The 
recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca2+ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca2+ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.
Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the 
photoisomerization of its 11-cis chromophore to all-trans5-7. This regeneration begins with the release of all-trans retinal by 
the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all-
trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is 
then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).
Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca2+-sensitive fluorescent dyes can be used to examine in detail the interplay between outer 
segment Ca2+ changes and response to light8-12 as well as the role of inner segment Ca2+ stores in Ca2+ homeostasis13,14. 
Fluorescent dyes can also be used for measuring Mg2+ concentration15, pH, and as tracers of aqueous and membrane compartments16. 
Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single 
photoreceptor cells17-19.

Watch this Scientific Journal Video about Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging at JoVE.com

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

Research

JoVE Journal

Neuroscience

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...