Name: Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy (Video) | JoVE Journal
Uploaded: 2009-06-09T00:00:00.000+00:00
Duration: 11 min 54 s
Description: Learn to image single synaptic vesicle exocytosis in goldfish retinal bipolar cells using TIRF microscopy. This method enables real-time visualization of neurotransmitter release mechanisms.

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5 minutes

This work was supported by NIH Grant EY 14990.

Part 1: Dissection and Bipolar Cell Isolation
<ol>
<li>Prepare solutions listed in Table 2; The pH of ringers’ (external) solutions should be adjusted to 7.4 with NaOH and the pH of the internal solution should be adjusted to 7.2 with CsOH. Protect internal solution from light with aluminum foil and keep it at 4 °C until use;</li>
<li>Dark-adapt a goldfish for at least 30 minutes prior to dissection;</li>
<li>While the animal dark-adapts, prepare 5 mL of the hyaluronidase (type V hyaluronidase,1100 units/mL in low Ca2+ ringer’s; Sigma, St. Louis, MO) and 10 mL of the L-cysteine (0.5 mg/mL in low Ca2+ ringer’s) solutions and weigh the papain (lyophilized powder, 40 units/mL; Sigma, St. Louis, MO) for 5 mL of the digestion solution;</li>
<li>Euthanize the goldfish by quick decapitation with surgical scissors and destroy the brain and spinal cord with a #11 scalpel blade;</li>
<li>Remove the eyes by destroying the extra-ocular muscles with the help of a #7 curved Dumont forceps and cutting the optic nerve with iris scissors;</li>
<li>Place one eye bulb on a piece of filter paper and puncture the scleral limbus with the tip of a #11 scalpel blade;</li>
<li>Introduce the blade of a pair of vannas scissors inside the punctured whole and cut the whole anterior segment away;</li>
<li>Place a small piece of filter paper on top of the remaining optic cup and exert some pressure in order to have the paper soak with vitreous humor;</li>
<li>Lift the filter paper with the retina attached to it and cut the optic nerve with a par of vannas scissors;</li>
<li>Place the filter paper containing the retina in a 35 mm plastic culture dish with hyaluronidase solution and peel off the retina from the filter paper with the help of #7 Dumont tweezers;</li>
<li>Cut the retina into 4-6 pieces with half of an industrial carbon steel single-edged blade and let it sit in the hyaluronidase solution for 20 minutes;</li>
<li>While waiting for hyaluronidase to take effect, add 5 mL of the L-cysteine solution to the papain and let it sit until the liquid becomes transparent (approximately 5-10 minutes);</li>
<li>Wash the pieces of retina 3x in low Ca2+ Ringer’s and let them sit in the papain solution for 30-35 minutes;</li>
<li>Wash the pieces of retina 3x in low Ca2+ Ringer’s and store them until use at 4 °C in a 35 mm plastic culture dish containing low Ca2+ Ringer’s;</li>
<li>To dissociate the cells, put a piece of retina in a microcentrifuge tube containing 500 mL of low Ca2+ Ringer’s and slowly triturate the retina by pipetting it up and down with a glass dissociation pipette, carefully not to produce any air bubbles. Dissociation pipettes are fabricated by heating up the tip of a glass Pasteur pipette with a Bunsen burner and slightly bending it with the help of anatomical forceps;</li>
<li>Plate the isolated cells by adding a drop of the retinal suspension to a home-made recording chamber previously filled with 2 mL of low Ca2+ ringer’s. The chamber consists of the bottom half of a 35-mm plastic culture dish with a circular whole in the middle and a circular coverslip of 1.78 refractive index glass (PlanOptik, Germany) glued to the bottom with a silicon elastomer (Sylgard 184; Dow Corning, Midland, MI).</li>
</ol>
Part 2: Bipolar Cell Loading and Wash Out
TIRF imaging of synaptic vesicles is best carried out using an objective-type TIRFM microscope with a very high NA objective and a sensitive camera. For our experiments, we choose to use a 1.65 NA objective (Apo x100 O HR, N.A. 1.65, Olympus, Japan) with an EMCCD (Cascade 512B, Roper Scientific, Tucson, AZ). The use of the very high NA objective necessitates the use of high refractive glass coverslips and immersion fluid (di-iodomethane with sulfur). Under our conditions, excitation light is limited to an exponentially decaying field with a length constant of approximately 50 nm.
<ol>
<li>Add a drop of high refractive index liquid (M series, refractive index = 1.7800, Cargille Labs, Cedar Grove, NJ) to the microscope objective;</li>
<li>Place the recording chamber carefully on top of the microscope objective and carefully mount ground electrode and superfusion exit pipe to chamber;</li>
<li>Let chamber sit on microscope for 10-20 minutes to allow cells to sink and adhere to the bottom;</li>
<li>In the meantime, prepare 5 mL of a 1mM trolox® ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; Sigma, St. Louis, MO) solution in high K+ ringer’s. Sonicate until dissolved;</li>
<li>Prepare 15 mL of ADVASEP-7 washing solution: 1 mM ADVASEP-7 (Sigma, St. Louis, MO) in low Ca2+ ringer’s. Note that ADVASEP-7 use is optional and can be omitted if desired;</li>
<li>Purge the superfusion lines and add ADVASEP-7 washing solution, low Ca2+ ringer’s and control ringer’s to the superfusion system; </li>
<li>Pull loading pipettes from thin-walled borosilicate glass (Kwik-Fil® TW150-3; WPI, Sarasota, FL). Puffer pipette resistances are in the 1.5-2.5 MΩ range;</li>
<li>Prepare the FM1-43® (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide, “special packaging”; Invitrogen, Carlsbad, CA) solutions. First, make a 1 mM stock by adding 160 μL distilled water to one vial (100 mg) of FM1-43®. This stock can be kept at 4 °C for up to one week. Then, add 5 μL of FM1-43® to 1 mL high K+ ringer’s + 1mM trolox®. Protect solution from light with aluminum foil and keep it at 4 °C until use; </li>
<li>Turn the microscope bright field light on and search for intact bipolar cells. Slightly tap the microscope to make sure that the neurons are firmly attached to the bottom of the chamber;</li>
<li>Position the superfusion pen close to the cell of interest and continuously perfuse the preparation with low Ca2+ ringer’s;</li>
<li>Turn off the room lights and add a red long pass filter (i.e. RG630; Schott, Germany) to the optical path to minimize excitation of the FM dye;</li>
<li>Fill a loading pipette with 10 μL of the FM dye solution, mount the pipette in the micromanipulator and lower the pipette onto the preparation without overpressure until it is at the same focal plane as the bipolar cell you want to load. Make sure you have at least two electrode holders: the one used for the FM dye cannot be used for patch clamping, or else it may contaminate the intracellular solution;</li>
<li>Position the puffer opening at a distance of around 10 μm from the axon terminal, turn the superfusion system off and puff the dye solution for 10 seconds by turning the pipette overpressure on;</li>
<li>Turn the overpressure off and, without moving the pipette, wait for 30 seconds;</li>
<li>Turn the superfusion on and bathe the chamber in ADVASEP-7 solution for 5 minutes. In the meantime, remove the puffer pipette from the bath;</li>
<li>After 5 minutes, switch to low Ca2+ ringer’s and perfuse the chamber for 25-30 minutes to allow removal of excess dye.</li>
</ol>
Part 3: Patch Clamping and TIRFM Imaging
<ol>
<li>While the preparation is washing out, pull patch pipettes from thick-walled borosilicate glass (B150-86-10; Sutter Instrument Company, Novato, CA). Patch pipette resistances are in the 8-10 MΩ range;</li>
<li>After wash out is complete, place the axon terminal in the center of the field of view;</li>
<li>Fill a patch pipette with 7μL of internal solution, tap the pipette to get rid of air bubbles, coat the pipette with molten dental wax (Sticky Wax; Kerr Corporation, Orange, CA) and mount it in the micromanipulator;</li>
<li>Turn the pipette overpressure on and lower the pipette slowly onto the preparation. Check pipette resistance in the amplifier and correct pipette offsets and capacitance with the respective amplifier controls;</li>
<li>Switch the superfusion to control ringer’s. To create a gigaseal between the pipette and the cell, slightly touch the tip of the electrode against the cell body and turn the pipette overpressure off while gently applying negative pressure to the electrode;</li>
<li>While sealed, choose “whole cell” mode in the amplifier and set the cell holding potential to -60 mV;</li>
<li>Choose a region of interest with the imaging software that encompasses the whole axon terminal, turn the bright field light off and, by briefly exposing (30 ms) the terminal to the 488 nm laser, find the right focal plane for TIRF imaging;</li>
<li>Break into the cell by using the “zap” command of the amplifier while applying slightly negative pressure to the pipette;</li>
<li>Correct for cell capacitance and series resistance, and then apply the voltage protocol of interest while imaging the movement of synaptic vesicles. It is at this stage important to have the voltage protocol synchronized to the camera frame rate. In our case, each frame is 30 ms long, so the voltage changes occur in multiples of this value (i.e., every 300 ms or 10 frames);</li>
<li>Wait at least 40 seconds between trials to allow for recovery;</li>
<li>To check the position of synaptic ribbons, take pictures while turning the 561 nm laser on.</li>
</ol>
<img src="/files/ftp_upload/1305/fig1.jpg" alt="Optical microscopy setup diagram; laser excitation, fluorescence emission, dichroic filter." data-altupdatedat="1787630408000" data-alt="figure 1"> Figure 1: The experimental setup. A 488 nm laser (blue) is focused to the periphery of the back focal plane of the objective and suffers total internal reflection when it reaches the glass-aqueous medium interface. The electromagnetic field generated by the reflected beam excites the fluorophore loaded into the synaptic vesicles closest to the bottom of the glass chamber, which then fluoresce (green). The fluorescent light is then guided to the observer’s eye (depicted) or to a CCD camera. The membrane potential of the imaged cells is controlled simultaneously by patch-clamping them. This approach allows the study of the relationship between incoming signals (the membrane voltage) and the neuronal output (exocytosis).
<img src="/files/ftp_upload/1305/fig2.jpg" alt="Isolated bipolar cell axon terminal microscopy; fluorescence, FM 1-43, Rpep-Rhodamine labeling." data-altupdatedat="1787630408000" data-alt="figure 2"> Figure 2: Typical results. Left: bright field image of an isolated goldfish bipolar cell. Upper right: TIRF image of the synaptic vesicles in the bipolar cell axon terminal loaded with FM 1-43® and imaged with the 488 nm laser (FM dye). Bottom right: image of the same terminal after patching the cell and imaging the axon terminal with the 561 nm laser. Synaptic ribbons are labeled by the rhodamine-based RIBEYE-binding peptide (Rpep_rhod).
Table 1: Specific Reagents and Equipment.
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td> Name</td>
<td>Type</td>
<td>Manufacturer</td>
<td>Catalog</td>
<td>Comment</td>
</tr>
<tr>
<td>-</td>
<td>Air Table</td>
<td>Newport Corporation</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>IX70</td>
<td>Inverted Microscope</td>
<td>Olympus</td>
<td>-</td>
<td>Equipped with a tungsten lamp for bright field and a lateral opening port for TIRF</td>
</tr>
<tr>
<td>TH4-100</td>
<td>Lamp Power Source</td>
<td>Olympus</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>FF498_581-Di01</td>
<td>Dichroic Filter</td>
<td>Semrock</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>NF01-405_488_568</td>
<td>Emission Filter</td>
<td>Semrock</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>Apo x100 O HR</td>
<td>Objective</td>
<td>Olympus</td>
<td>-</td>
<td>N.A. 1.65</td>
</tr>
<tr>
<td>RG630</td>
<td>Red Glass Filter</td>
<td>Schott</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>-</td>
<td>488 nm Laser</td>
<td>Coherent</td>
<td>-</td>
<td>Use at minimum power</td>
</tr>
<tr>
<td>-</td>
<td>Shutter</td>
<td>Uniblitz</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>VMM-D1</td>
<td>Shutter Driver</td>
<td>Uniblitz</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>-</td>
<td>561 nm Laser</td>
<td>Melles Griot</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>-</td>
<td>Shutter</td>
<td>Uniblitz</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>VMM-D3</td>
<td>Shutter Driver</td>
<td>Uniblitz</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>Perfusion Pressure Kit</td>
<td>Superfusion System</td>
<td>Automate Scientific</td>
<td>09-04</td>
<td>-</td>
</tr>
<tr>
<td>Perfusion Pen</td>
<td>Superfusion System</td>
<td>Automate Scientific</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>Valvelink 8</td>
<td>Superfusion System Controller</td>
<td>Automate Scientific</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>Cascade 512B</td>
<td>EM CCD Camera</td>
<td>Roper Scientific</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>Metamorph 7.1</td>
<td>Imaging Software</td>
<td>Molecular Devices</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>EPC-9</td>
<td>Patch Clamp Amplifier</td>
<td>HEKA Elektronik</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>Pulse</td>
<td>Amplifier Software</td>
<td>HEKA Elektronik</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>MP-285</td>
<td>Micromanipulator</td>
<td>Sutter Instrument</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>-</td>
<td>Electrode Holder</td>
<td>HEKA Elektronik</td>
<td>-</td>
<td>For 1.5 mm O.D. glass, 2 units</td>
</tr>
<tr>
<td>Kwik-Fil® TW150-3</td>
<td>Borosilicate Capillary Glass</td>
<td>WPI</td>
<td>-</td>
<td>Without filament</td>
</tr>
<tr>
<td>B150-86-10</td>
<td>Borosilicate Capillary Glass</td>
<td>Sutter Instrument</td>
<td>-</td>
<td>With filament</td>
</tr>
<tr>
<td>P-97</td>
<td>Microelectrode Puller</td>
<td>Sutter Instrument</td>
<td>-</td>
<td>Equipped with 3x3 box filament and environmental chamber</td>
</tr>
<tr>
<td>-</td>
<td>Pressure Vacuum Air Pump</td>
<td>Thomas Scientific</td>
<td>7893B05</td>
<td>Creates vacuum to remove liquids from chamber and overpressure for pipettes</td>
</tr>
<tr>
<td>MatLab R2008a</td>
<td>Analysis Software</td>
<td>MathWorks</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>353001</td>
<td>35 mm Plastic Culture Dishes</td>
<td>Falcon</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>-</td>
<td>High Refractive Index Glass</td>
<td>PlanOptik</td>
<td>-</td>
<td>Refractive index488 nm = 1.78</td>
</tr>
<tr>
<td>Series M</td>
<td>High Refractive Index Liquid</td>
<td>Cargille Labs</td>
<td>-</td>
<td>Refractive index = 1.78</td>
</tr>
<tr>
<td>Sylgard 184</td>
<td>Silicon Elastomer Kit</td>
<td>Dow Corning</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>Glutathione</td>
<td>Tripeptide</td>
<td>EMD Chemicals</td>
<td> </td>
<td>Free radical scavenger</td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>Enzyme</td>
<td>Sigma</td>
<td>H6254</td>
<td>Type V</td>
</tr>
<tr>
<td>L-cysteine</td>
<td>Amino Acid</td>
<td>Fluka</td>
<td>30090</td>
<td>Activates papain</td>
</tr>
<tr>
<td>Papain</td>
<td>Enzyme</td>
<td>Fluka</td>
<td>76220</td>
<td>From Carica papaya</td>
</tr>
<tr>
<td>Trolox®</td>
<td>Soluble Vitamin E</td>
<td>Sigma</td>
<td>56510</td>
<td>Free radical scavenger</td>
</tr>
<tr>
<td>ADVASEP-7</td>
<td>Sulfonated B-Cyclodextrin Derivative</td>
<td>Sigma</td>
<td>A3723</td>
<td>Reduces FM 1-43® background fluorescence</td>
</tr>
<tr>
<td>FM 1-43®</td>
<td>Fluorescent Dye</td>
<td>Invitrogen</td>
<td>T35356</td>
<td>“Special packaging”</td>
</tr>
<tr>
<td>Sticky Wax</td>
<td>Pipette Coating Agent</td>
<td>Kerr Corporation</td>
<td>-</td>
<td>Decreases pipette capacitance</td>
</tr>
</tbody>
</table>
Table 2: Physiological Solutions Used in This Study.
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td> Substance</td>
<td>Low Ca2+ Ringer’s</td>
<td>Control Ringer’s</td>
<td>High K+ Ringer’s</td>
<td>Internal Solution</td>
</tr>
<tr>
<td>NaCl</td>
<td>120 mM</td>
<td>120 mM</td>
<td>97.5 mM</td>
<td>-</td>
</tr>
<tr>
<td>KCl</td>
<td>2.5 mM</td>
<td>2.5 mM</td>
<td>25 mM</td>
<td>-</td>
</tr>
<tr>
<td>MgCl2</td>
<td>1 mM</td>
<td>1 mM</td>
<td>1 mM</td>
<td>4 mM</td>
</tr>
<tr>
<td>CaCl2</td>
<td>0.5 mM</td>
<td>2.5 mM</td>
<td>2.5 mM</td>
<td>-</td>
</tr>
<tr>
<td>HEPES</td>
<td>10 mM</td>
<td>10 mM</td>
<td>10 mM</td>
<td>10 mM</td>
</tr>
<tr>
<td>EGTA</td>
<td>0.75 mM</td>
<td>-</td>
<td>-</td>
<td>0.5 mM</td>
</tr>
<tr>
<td>Glucose</td>
<td>10 mM</td>
<td>10 mM</td>
<td>-</td>
<td>-</td>
</tr>
<tr>
<td>Glutathione</td>
<td>2 mM</td>
<td>2 mM</td>
<td>-</td>
<td>1 mM</td>
</tr>
<tr>
<td>CH3CsO3S*</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>100 mM</td>
</tr>
<tr>
<td>TEACl</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>10 mM</td>
</tr>
<tr>
<td>ATP-Mg</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>10 mM</td>
</tr>
<tr>
<td>GTP-Li</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>1 mM</td>
</tr>
<tr>
<td>Rpep-rhod**</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>5 mM</td>
</tr>
<tr>
<td>Volume</td>
<td>200 mL</td>
<td>100 mL</td>
<td>5 μL</td>
<td>100 μL</td>
</tr>
</tbody>
</table>
* Cesium methanesulfonate. ** RIBEYE-binding peptide: rhodamine+EQTVPVDLSVARDR-cooh (mw 1997.75).

<ol><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Total+internal+reflection+fluorescence+microscopy+in+cell+biology%5BTitle%5D)+AND+%22Traffic%22%5BJournal%5D)">Total internal reflection fluorescence microscopy in cell biology.</a> Traffic. 2, 764-774 (2001).</li>
<li>Zenisek, D., Steyer, J. A., Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transport%2C+capture+and+exocytosis+of+single+synaptic+vesicles+at+active+zones%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Transport, capture and exocytosis of single synaptic vesicles at active zones.</a> Nature. 406, 849-854 (2000).</li>
<li>Zenisek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vesicle+association+and+exocytosis+at+ribbon+and+extraribbon+sites+in+retinal+bipolar+cell+presynaptic+terminals%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">Vesicle association and exocytosis at ribbon and extraribbon sites in retinal bipolar cell presynaptic terminals.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 4922-4927 (2008).</li>
<li>Zenisek, D., Horst, N. K., Merrifield, C., Sterling, P., Matthews, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+synaptic+ribbons+in+the+living+cell%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Visualizing synaptic ribbons in the living cell.</a> J. Neurosci. 24, 9752-9759 (2004).</li>
<li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives%5BTitle%5D)+AND+%22J.+Biomed.+Opt%22%5BJournal%5D)">Selective imaging of surface fluorescence with very high aperture microscope objectives.</a> J. Biomed. Opt. 6, 6-13 (2001).</li>
<li>Rouze, N. C., Schwartz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continous+and+transient+vesicle+cycling+at+a+ribbon+synapse%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Continous and transient vesicle cycling at a ribbon synapse.</a> J. Neurosci. 18, 8614-8624 (1998).</li>
</ol>

The advantages of objective-type TIRF microscopy are that 1) it provides excellent optical sectioning by restricting excitation light to a narrow region within the focal plane of the objective, thereby minimizing out-of-focus light; 2) since light drops exponentially with distance, movement in a vertical direction can be monitored as a change in fluorescence intensity; 3) efficient light collection through the high numerical aperture objective1,5.
The main drawback of the technique...

imaging exocytosis in retinal bipolar cells with tirf microscopy

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

This procedure starts with the dissection of the neural retina of adult goldfish, which is the nervous tissue at the back of the eye. Retinas are placed in an enzymatic solution for 35 minutes, rinsed and ated with a fire polished glass pipette to dissociate cells. Cells are then plated on high refractive glass cover slips and mounted into plastic tissue culture dishes.Isolated bipolar cells are identified by their distinct morphology to load terminals. We puff on them for 10 seconds with a solution containing FM 1 43 and wait 30 seconds before washing the dye away. After a 30 minute wash cells are patch, clamped and imaged with TIRF microscopy.Hi, my name is Christina Jovi from the ZEC Lab in the Department of Cellular and Molecular Physiology at Yale University School of Medicine. Today we'll show you a procedure to study exocytosis in living retinal bipolar cells using turf microscopy. We use this procedure in the lab to study mechanisms of synaptic release.So let's get started. To begin dark adapt a goldfish for at least 30 minutes prior to dissection. Following decapitation and eye removal, place one eye bulb on a piece of filter paper and use the tip of a number 11 scalpel blade to puncture the scleral limbus Inside the punctured hole.Use a pair of bonna scissors to cut the whole anterior segment away. Next place a small piece of filter paper on top of the remaining optic cup and exert some pressure causing it. To soak up the vitreous humor, lift the filter paper with the retina attached and using a pair of VNA scissors cut the optic nerve.Place the filter paper containing the retina in a 35 millimeter plastic cultured dish containing high alur solution and use number seven dumont tweezers to peel the retina off the filter paper. Next, using half of an industrial carbon steel single edged blade, cut the retina into four to six pieces and let them sit in the hyaluronidase solution for 20 minutes while waiting For the hyaluronidase to take effect, add five milliliters of L cysteine solution to the papain and let it sit for approximately five to 10 minutes until the liquid becomes transparent. Wash the pieces of retina three times in low calcium ringer solution and let them sit in the papain solution for 30 to 35 minutes.After 35 minutes incubation in the papain solution. Wash the retinal pieces three times. Transfer the pieces to a 35 millimeter plastic cultured dish containing low calcium ringers, and store the dish at 12 to 14 degrees Celsius until you are ready to begin dissociation of the cells to dissociate the cells.First, fabricate a dissociation pipette by using a bun and burner to heat up the tip of a glass PEs or pipette and then use anatomical forceps to slightly bend it. Put a piece of retina in a micro centrifuge tube containing low calcium ringer solution and slowly iterate the retina by carefully pipetting it up and down with the fabricated dissociation pipette. Being careful not to produce any air bubbles plate the isolated cells by adding a drop of the retinal suspension to a homemade recording chamber filled with low calcium ringer solution.The chamber consists of the bottom half of the 35 millimeter plastic culture dish with a circular hole in the middle and a circular cover slip made of 1.78 refractive index glass glued to the bottom. With dissection and bipolar cell isolation complete, we are ready to begin bipolar cell loading and washout out. In this experiment, it is best to use an objective type total internal reflectance fluorescence microscope with a very high numerical aperture objective and a sensitive camera.In this video, we will be using a 1.65 numerical aperture objective and an electron multiplying charge coupled device. To begin, add a drop of high refractive index liquid to the microscope.Objective. Place the recording chamber on top of the microscope.Objective.Carefully mount the ground electrode and superfusion exit pipe to the chamber. Let the chamber sit on the microscope for 10 to 20 minutes, allowing the cells to sink and adhere to the bottom. Next, purge the superfusion lines and add washing solution.Low calcium ringer solution and control ringer solution to the superfusion system. Turn the microscope brightfield light on and search for intact bipolar cells. Slightly tap the microscope to make sure that the neurons are firmly attached to the bottom of the chamber.Position the superfusion pen close to an intact cell and continuously perfuse the preparation with low calcium ringer solution. Now turn the lights off in the room and add a red long pass Filter to the optical path to minimize excitation of the fluorescent dye. Fill a loading pipette with 10 microliters of fluorescent dye solution.Mount it in the micro manipulator and then lower it onto the preparation without using the over pressure option until it is at the same focal plane as the selected bilar cell. Position the puffer opening at a distance of around 10 micrometers from the axon terminal. Turn the superfusion system off and puff the dye solution for 10 seconds by turning the pipette over pressure on.Now turn the over pressure off and without moving the pipette. Wait 30 seconds, turn the superfusion system on and bathe the chamber in washing solution for five minutes. In the meantime, remove the puffer pipette from the bath.After five minutes, switch the SUPERFUSION system to low calcium ringer solution and perfuse it for 25 to 30 minutes, allowing for the removal of excess dye With bipolar cell loading and washout complete. We are ready to begin patch clamping and T-I-R-F-M imaging. After the washout is complete, change electrode holders to avoid contamination of your patch pipette with dye.Place the axon terminal in the center of the field of view. Fill a patch pipette with seven microliters of internal solution. Tap it to get rid of air bubbles.Coat it with molten dental wax and then mount it in the micro manipulator. Turn the pipette over pressure on and slowly lower the pipette onto the preparation. Check the pipette resistance and make any needed corrections.Next, with the superfusion system set to control ringer solution, create a giga seal between the pipette and the cell by slightly touching the tip of the electrode against the cell body. With the pipette over pressure off, gently apply negative pressure to the electrode while sealed. Select whole cell mode in the amplifier and set the cell holding potential to negative 60 millivolts.Now use imaging software to select a region of interest that encompasses the whole axon terminal. Turn the bright field light off and by briefly exposing the terminal to the 488 nanometer laser. Find the right focal plane for TIRF imaging.Next break into the cell by using the zap command of the amplifier while applying slightly negative pressure to the pipette correct for cell capacitance and series resistance. And then apply the voltage protocol while imaging the movement of synaptic vesicles. Wait at least 40 seconds between trials to allow for recovery.First, we choose a bipolar cell in brightfield microscopy. We then zoom in and load its axon terminal with the fluorescent dye. After a 30 minute wash, the loaded terminal can be imaged inter microscopy along with the movement of fluorescent glutamatergic vesicles.Here we introduce a rib eye binding peptide through the patch pipette, which allows us to see the position of synaptic ribbons when imaged with a 561 nanometer laser. We then depolarize the cells and image vesicle movement with the 488 nanometer laser. We have just shown you how to dissociate plate and load retinal bipolar cells with a fluorescent dye as well as to patch, clamp these neurons and simultaneously image them with turf microscopy.When doing this procedure, it's important not to expose the cells too much to the laser. So that's it. Thanks for watching and good luck with your experiments.

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Research

JoVE Journal

Biology

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Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

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Cell Imaging Using Total Internal Reflection Fluorescence Microscopy

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TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

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Imaging FITC-dextran as a Reporter for Regulated Exocytosis

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Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

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Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

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Learn to image single synaptic vesicle exocytosis in goldfish retinal bipolar cells using TIRF microscopy. This method enables real-time visualization of neurotransmitter release mechanisms.

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

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