Startup the setup (D1.210)
Procedure for startup
Before you can start any measurement there are a few steps that need to be done.
- Make sure the interlock in D1.210 is switched ON (otherwise the lasers will not work)
- Go to the setup (left curtain)
- Immobilize HJ on the slide with the base mix (if you have not done yet)
- switch TEC ON on the temp control of the laser, let the temperature stabilize. Be careful not to turn the knob (which changes the set temperature)
- Leave laser off till the sample is in position (default settings laser controller: display P_LD, mode const P, LD POL AG)
- when (later on) you do switch on the laser, set the power to 7 mW. (feel free to use the power meter to check the output level, which will be <7 mW)
- Turn on the computer, open Andor Solis, cool camera till -80 C (left click left bottom)
- Turn off the ceiling lights (near the door, left button) and remove the cap in front of the camera. Place back all shielding to prevent overexposure (and damage) of the EMCCD)
- Set the camera settings to have EMgain disabled, and Pre-Amp Gain 1x, as shown below.
Tetraspeck beads are bright (big plastic beads with lots of fluorescent labels and do not need gain). For now acquire with 'take videos', the leftmost acquisition symbol. Make sure to save on image for mapping later on.
- Place the Tetraspeck sample: first oil on top of the objective, then the sample (in an aluminium covered box, the cover is to prevent bleaching)
- To get the sample in focus, turn on the LED, move the sample to the sticky tape and focus on the tape.
Get a feeling for how far to move the objective up and down (turn wheel below sample plate) to go from bottom surface (to be imaged in tirf) and top surface.
It is approximately a quarter turn
Use the shape of the oil between objective and sample to discriminate the direction of (de)focus.
- If in focus, move the sample so you don't see tape anymore.
- Switch of LED, switch on laser (first temperature to 25 C, if temp stabilized , put on laser goggles and switch on laser power at 4 mW).
- Remove goggles if the power is & stays below 5 mW.
- Continue to the acquisition