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1. Imaging Exocytic Events via Total Internal Reflection Fluorescence (TIRF) Microscopy
Note: This protocol requires specialized microscopy equipment, including an environmental chamber to maintain temperature, humidity, and carbon dioxide (CO2), an inverted total internal reflection fluorescence (TIRF) microscope equipped with an epifluorescent illumination, a high magnification/ high numerical aperture (NA) TIRF objective, an automated XYZ stage, and a sensitive charge-coupled device (CCD) detector. This protocol uses a fully automated inverted microscope equipped with a 100x 1.49 NA TIRF objective, a solid state 491 nm laser, and an electron multiplying CCD (EM-CCD). All equipment is controlled by imaging and laser control software. Prior to the beginning of the imaging protocol, power on the environmental chamber, stage, lamp, computer, and camera.
- Select the objective within imaging software. Once the objective is in place, fasten the objective heater around the collar.
- Confirm that the objective is entirely lowered before securing the stage incubator in the stage slot. Pour distilled water evenly into the stage incubator inlets to prevent spills.
- Turn on the incubation system. Open the valve to the CO2 tank and confirm the pressure is appropriate for the system per the manufacturer's instructions. Allow the chamber some time to reach 5% CO2 and 37 °C. Prior to adding the imaging dish, place an empty dish in the incubator to avoid water condensation on the objective.
- Power on the laser source.
- Open the stage incubator and add immersion oil to the lens. Place the sample in the incubator and stabilize it with stability arms or a dish weight. Raise the objective until the oil makes contact with the bottom of the sample. Switch to transmitted light illumination and find the neuronal focal plane through the oculars.
- Start the laser software and connect it to the laser control software. Set illumination to widefield and select the objective (100x 1.49 NA TIRF is recommended). Set the sample's refractive index (cells ~1.38). Adjust the laser intensity by unchecking "TTL" (Transistor-Transistor Logic) for the 491 nm laser. Adjust the slider to 100, then bring it back down to a value between 20% and 40%. Recheck "TTL."
- Focus on the sample again in transmitted light illumination. Go to imaging software, select 491 nm laser illumination, and open the shutter. Fine-adjust the focal point of the laser on the ceiling and center it to the center of the closed-field diaphragm with the condenser removed. Place the condenser upside down on the optical bench so as not to scratch the lens.
- Replace the condenser, go to TIRF software, and set penetration depth (PD) to 110 nm. Then, switch from widefield illumination to TIRF illumination mode for imaging.
- Find vesicle-associated membrane protein 2 (VAMP2)-phluorin-expressing cells through the oculars using widefield epifluorescence with the epifluorescent light source.
- Adjust imaging parameters (exposure time, gain, and laser power) to maximize signal-to-noise ratio and dynamic range using the minimal exposure time and laser intensity to reduce photobleaching and phototoxicity (for example, exposure between 50 - 100 msec, with a 15 - 30 gain at 30% maximum laser power).
- Set continuous autofocus per cell. Acquire a timelapse image set with acquisition occurring every 0.5 sec for 5 min. For the netrin-1 stimulated condition, add 500 ng/ml netrin-1 to the dish of cells in a laminar flow hood and return the dish to the incubator for 1 hr before imaging.