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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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 bl

imaging-exocytosis-in-retinal-bipolar-cells-with-tirf-microscopy

Research

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Biology

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

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

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Source: Daniele, F., et. al TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis. J. Vis. Exp. (2015).This video demonstrates the imaging of human neuroblastoma cells using a total internal reflection fluorescence (TIRF) microscope to visualize fluorophore-tagged synaptic vesicles. The cells are first focused in epifluorescence mode. Adjustments are then made to achieve total internal reflection,...

Cell Imaging Using Total Internal Reflection Fluorescence Microscopy

Source: Winkle, C. C., et al. Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis. J. Vis. Exp. (2016)This video demonstrates total internal reflection fluorescence (TIRF) microscopy for visualizing exocytic events in cortical neurons. A pH-sensitive green fluorescent protein (GFP) marker fluoresces upon vesicle fusion, with selective TIRF excitation capturing membrane-proximal exocytosis in time-lapse imaging.

Total Internal Reflection Fluorescence Microscopy for Visualization of Exocytic Events 

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

Imaging Plasma Membrane Deformations With pTIRFM

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

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

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

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

This paper demonstrates a method for visualizing and quantifying microglia motility and contact with postsynaptic puncta in the retina using spinning disk confocal microscopy.

Live Imaging and Characterization of Microglia Dynamics and Interactions with Synapses in Diseased Murine Retina

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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