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Important notes:

- Don’t go to live mode without checking the Exposure and gain to protect the optics and your
sample.
- Always double check the focusing mode to avoid damage to the objective.
- Always check the laser power to protect the optics.
- Avoid putting tension on the objective at all costs.
1) Uncover Microscope and turn the APC (power) on.
2) Put a drop of oil on your objective.
3) Open IQ3 then Fusion.
4) Rotate the laser’s key to turn it on.
5) In IQ3, select Fusion with Micropoint.
6) The black disk should be on A.
7) Put on the mirror slide and grab it in contact with the objective. (Find focus)
8) Find the mirror slide focus, by turning on the Eye mode and white LED light and adjust focus
from the knob.
9) After finding the focus, turn PFS on, and start using this knob to adjust the focus.
10) In Fusion, select your desired protocol.
11) In Fusion, select your desired channel.
12) Turn down exposure and gain to start viewing without harming the optics.
13) Snap  Live mode, repeat as necessary to optimize your desired gain and exposure.
14) PFS: on Conti
15) Enable or disable Automap to preference.
16) In IQ3, go to Micropoint and input your settings.
17) Hit Record, and start rotating the laser’s knob to make it pulse and test ablation on a mirror slide
by moving the X-Y joystick and adjusting the laser’s power accordingly, till you are barely
ablating the slide.
18) Increase brightness and adjust focus to small ablated holes.
19) From the laser knob with the green arrow, play around with it till you mazimize holes.
20) Make sure your image is as focused and sharp as it can be.
21) Start Acquiring
22) IQ3  Options  Edit  drag the windows  start Live
23) Adjust Black corners, such that there are no black edges inside the red box.
24) IQ3  Wizards  Frappa Calibration  EMCCD  Next
25) Adjust micropoint Energy (Power) as needed.
26) Bleach Sample  Add calibration point, repeat 16 times.
27) Lower the objective and replace the mirror slide with your slide, bring them in contact, find
focus, make sure that it reads PFS: Off
28) Switch to L100 instead of Eye
29) Start Ablating
30) Export your files with the metadata.
31) Shut down all softwares.
32) Turn off PC, APC and Laser.
33) Clean objective in a sweeping motion.
34) Cover the microscope.