Olympus Confocal Microscope

1.    Turn on microscope/lasers/computer 2.    Prepare slide (see next page for illustration)

a. Put four dots of vacuum grease in a square

b. Put a dot of glycerol in the center of the four dots

c.     Pick larvae into water to rinse off food

d. Dry larva off with Kimwipe

e. Put coverslip on and squish slightly (larva should be vertical on the slide)

f. Move the coverslip left and right to roll the larva so two veins are visible, and roll so that the vein on the right is just about to the middle

g. Move the coverslip up and down to stretch the larva

3.    Pick the objective (20X, 40X, 60X, etc.)

4.    Put some immersion oil on the objective and place the slide upside down

5.    Bring the objective up until the immersion oil spreads slightly (about pencil eraser size)

6.    Press button to change to fine focus!

7.    Check settings on FV1000 control panel

a. Laser power should ideally be between 10-15% (for brighter samples, lower is fine ~5%)

b. HV should be set between 750-900 (600-900 range is okay)

c. Pinhole size: 110um d. Pixels 1024x1024

e. Generally, want 0.5-1s/frame

f. Generally, image 5 min (600 frames for 0.5s/f or 300 frames for 1s/f)

g. For Z stack step size should be 0.3-0.5um

8.    Turn on epifluorescence or BF (trans) and focus on larva

9.    Find the head and move down to segment A2/3/4 (A3 the best) by focusing out and passing over three sets of denticles (or, “dentricles” if you are Paige)

For live imaging: image A3 and A4 (one 5 min movie per segment). For morphology, image A2/3/4.

a. Alternatively: if on 40X or 20X focus on neurons and differentiate segments by gaps in the arbor. The 3rd cluster of neurons is in the A1 segment

10.  When in the correct segment, focus on neuron and arbor

11.  Move the field of view so that you can see the entire arbor. 12.  Press “XY repeat” and image will appear on computer screen rather than through binoculars. Adjust if necessary

13.  To take a Z stack, focus out until the neurons are no longer visible and press “set”, the focus in (the opposite way) until the neurons are no longer visible and press the other “set” (one is start and one is end of the Z stack)

14.  Pick stack size

15.  Stop the “XY Repeat” and choose “depth” under “XY LZ1” button

16.  Let run and press complete when done. 17.  Save all images and put onto mcb server

Crude rendering of slide-making process by Paige

Close up of denticles. Larval body segments Will not look so dark

When all neurons are lit up with GFP, the lowest neuron in the cluster is Class 1.

In class 1-GFP specific larva (Gal4^221, UAS-MCD8-GFP), image the

lower neuron as it is the most morphologically consistent between segments and larvae.