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.
Part 1: Dissection and Bipolar Cell Isolation
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.
Part 3: Patch Clamping and TIRFM Imaging
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).
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.
Name |
Type | Manufacturer | Catalog | Comment |
– | Air Table | Newport Corporation | – | – |
IX70 | Inverted Microscope | Olympus | – | Equipped with a tungsten lamp for bright field and a lateral opening port for TIRF |
TH4-100 | Lamp Power Source | Olympus | – | – |
FF498_581-Di01 | Dichroic Filter | Semrock | – | – |
NF01-405_488_568 | Emission Filter | Semrock | – | – |
Apo x100 O HR | Objective | Olympus | – | N.A. 1.65 |
RG630 | Red Glass Filter | Schott | – | – |
– | 488 nm Laser | Coherent | – | Use at minimum power |
– | Shutter | Uniblitz | – | – |
VMM-D1 | Shutter Driver | Uniblitz | – | – |
– | 561 nm Laser | Melles Griot | – | – |
– | Shutter | Uniblitz | – | – |
VMM-D3 | Shutter Driver | Uniblitz | – | – |
Perfusion Pressure Kit | Superfusion System | Automate Scientific | 09-04 | – |
Perfusion Pen | Superfusion System | Automate Scientific | – | – |
Valvelink 8 | Superfusion System Controller | Automate Scientific | – | – |
Cascade 512B | EM CCD Camera | Roper Scientific | – | – |
Metamorph 7.1 | Imaging Software | Molecular Devices | – | – |
EPC-9 | Patch Clamp Amplifier | HEKA Elektronik | – | – |
Pulse | Amplifier Software | HEKA Elektronik | – | – |
MP-285 | Micromanipulator | Sutter Instrument | – | – |
– | Electrode Holder | HEKA Elektronik | – | For 1.5 mm O.D. glass, 2 units |
Kwik-Fil® TW150-3 | Borosilicate Capillary Glass | WPI | – | Without filament |
B150-86-10 | Borosilicate Capillary Glass | Sutter Instrument | – | With filament |
P-97 | Microelectrode Puller | Sutter Instrument | – | Equipped with 3×3 box filament and environmental chamber |
– | Pressure Vacuum Air Pump | Thomas Scientific | 7893B05 | Creates vacuum to remove liquids from chamber and overpressure for pipettes |
MatLab R2008a | Analysis Software | MathWorks | – | – |
353001 | 35 mm Plastic Culture Dishes | Falcon | – | – |
– | High Refractive Index Glass | PlanOptik | – | Refractive index488 nm = 1.78 |
Series M | High Refractive Index Liquid | Cargille Labs | – | Refractive index = 1.78 |
Sylgard 184 | Silicon Elastomer Kit | Dow Corning | – | – |
Glutathione | Tripeptide | EMD Chemicals | Free radical scavenger | |
Hyaluronidase | Enzyme | Sigma | H6254 | Type V |
L-cysteine | Amino Acid | Fluka | 30090 | Activates papain |
Papain | Enzyme | Fluka | 76220 | From Carica papaya |
Trolox® | Soluble Vitamin E | Sigma | 56510 | Free radical scavenger |
ADVASEP-7 | Sulfonated B-Cyclodextrin Derivative |
Sigma | A3723 | Reduces FM 1-43® background fluorescence |
FM 1-43® | Fluorescent Dye | Invitrogen | T35356 | “Special packaging” |
Sticky Wax | Pipette Coating Agent | Kerr Corporation | – | Decreases pipette capacitance |
Table 2: Physiological Solutions Used in This Study.
Substance |
Low Ca2+ Ringer’s | Control Ringer’s | High K+ Ringer’s | Internal Solution |
NaCl | 120 mM | 120 mM | 97.5 mM | – |
KCl | 2.5 mM | 2.5 mM | 25 mM | – |
MgCl2 | 1 mM | 1 mM | 1 mM | 4 mM |
CaCl2 | 0.5 mM | 2.5 mM | 2.5 mM | – |
HEPES | 10 mM | 10 mM | 10 mM | 10 mM |
EGTA | 0.75 mM | – | – | 0.5 mM |
Glucose | 10 mM | 10 mM | – | – |
Glutathione | 2 mM | 2 mM | – | 1 mM |
CH3CsO3S* | – | – | – | 100 mM |
TEACl | – | – | – | 10 mM |
ATP-Mg | – | – | – | 10 mM |
GTP-Li | – | – | – | 1 mM |
Rpep-rhod** | – | – | – | 5 mM |
Volume | 200 mL | 100 mL | 5 μL | 100 μL |
* Cesium methanesulfonate.
** RIBEYE-binding peptide: rhodamine+EQTVPVDLSVARDR-cooh (mw 1997.75).
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 is that it is limited to imaging events happening within 100& nm of the cell surface, which is roughly equivalent to an ultrathin section in electron microscopy. Therefore, visualization of these events depends critically on the cells being firmly adhered to the glass, on the presence of synaptic ribbons close to the patch of membrane adhered to the glass and on the successful loading of vesicles. Our protocol enables the loading of only 1-2% of the total number of vesicles within the bipolar cell synaptic terminal2,6. With that said, it is clear that there are much more events happening at the cell surface than the ones we are able to image.
This work was supported by NIH Grant EY 14990.