Bioluminescence Imaging for Studying Calcium Transients in Drosophila Brain Structures

プロトコル

1. Imaging

1. Setup
   1. Start the recording system by turning on the microscope, computer, camera, and drainage system and setting the room environmental controls to 25 °C.
   2. Open the Measurement and Automation Explorer program on the computer. Click on “Devices and Interfaces”, then double click on “NI Motion Devices,” and finally right click on “PCI-7334” and select “initialize device”.
   3. Open Photon Imager. Create a new folder and name the first recording file. The camera must reach -80 °C before the system will function.
   4. Set up the perfusion system: Add KCl (potassium chloride), nicotine, and Ringer’s solution to the reservoirs and adjust so each solution discharges at 2 ml/min. Wash with Ringer’s solution prior to starting the experiment.
2. Prepare sample
   1. Place the imaging mount block dish on the mount under a microscope at 5X magnification and insert the perfusion tube into the channel through the puncture.
   2. Set the microscope to fluorescent mode, then center and bring mushroom bodies (MBs) into focus. Adjust to 20X, re-center, and focus.
   3. Position the drainage apparatus over the drainage pool, adjust the height of the drainage shunt so that the tip is just above the pool, and run Ringer’s solution for 30 sec to ensure adequate drainage.
   4. Pull down the shield to seal off the apparatus from light. Using the automated system in the photon imager, make fine adjustments to the focus and take a reference fluorescent image of the MBs.
3. Recording
   1. Adjust photon speed to the desired recording speed (from 50 msec up to 1 sec or more). Select photon mode and open the shutter. Record genotype, sex, age, and sample number in log entries. Adjust the position of ROI (Region of interest) boxes over calyx and cell bodies (CCB) and medial lobes by clicking on the ROI boxes and dragging while holding control.
   2. Record baseline for about 5 to 10 min (as desired).
   3. Apply nicotine for 1 min. Note start/stop in the log. Wash with Ringer’s solution for 5 min.
   4. Wait 5 min for recovery and prepare KCl (potassium chloride) log entry and timer.
   5. Apply KCl for 1 min. Note start/stop in the log. Wash with Ringer’s solution for 1 min.
   6. Switch to fluorescent mode, take the final fluorescent image, and turn off the Ringer’s solution.
   7. Press “Stop.” A dialog box will ask if you want to shut off the system. Select “No” to continue recording.
   8. Open the shield, move the drainage system, switch the lens objective to 5X, and remove the perfusion tube. Remove the sample/recording dish from the stage, and clean off the 20X lens by rinsing and wiping the lens twice with water.
   9. Press the white play button at the top of the program window to initiate the next recording.

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