Prepare the imaging buffer. The pre-made saline will be labeled for 2P Ca+2 imaging. Put a small amount (usually <5mL) in a fresh falcon tube. Bubble in CO2 by using the appropriate cap (labeled Ca+2) and turning the nozzle to let the air through. Close when finished (30 sec - 1 min).  

Anesthetize flies by chilling on ice. When no longer moving, place in the imaging holder using blunt forceps. Push down on the thorax and position the head gently in place. The fly’s head should poke out of the concave part of the holder. This is where the saline will eventually go. 
 
Use UV glue to glue the fly’s head and thorax into position on the concave side. UV glue can be found in a black and yellow bottle in the top drawer across from Shahar’s rig, to the left of the light microscope. Dollop a small dot on a piece of paper  and use the toothpick with a small wire hoop on the end to bring to the fly. Cure the glue with the UV light gun for 5 seconds. Don’t look at the UV light - wear yellow goggles while UV light is on.
 
Turn the imaging holder over. Grab a bit of wax and melt it on the hot tool. Once melted, coat the proboscis and legs in wax, trying to use only one stroke of the tool. The fly shouldn’t be waxed to the holder, the extremities just need to be waxed together to minimize movement.  
  
 
Put enough saline to cover the fly into the concavity in the holder. It doesn’t need to be filled all the way, just so that there will be a buffer between the brain and the objective lens. 
 
Using a surgical needle (take a new one every 4-5 flies), make an incision starting at the back of the fly’s head where the cuticle is slightly darker. The fly should be facing away from you. This bit of cuticle will be tougher than most. Take great care when cutting here in order to make a clean cut. Trace along the edge of the eyes to continue cutting the cuticle towards the front of the face, exposing the brain.  
 
Turn the fly around so that it is on a diagonal pointing to your left. Using the needle and the edge of the holder as a guide, carefully cut along the front of the cuticle until it releases from the fly’s face. 
 
Rotate the fly to face the other diagonal (or any other comfortable position). Take your time to carefully remove the fat, extra pieces of cuticle, and hair from the sample. 
 
You may notice that the brain is still moving. You can (mostly) get rid of this movement by clipping the muscles of the proboscis very carefully with fine forceps. Be careful not to go too deep and clip the antennal nerve!
 
Walk your sample over to the 2P and get imaging!