STORM protocol
- Switch on (note on the wall):
Laser (switch on the back) and laser lines (rectangles on the front).
Lumencor.
Microscope (switch to the right).
Andor.
Widefield illumination.
Filter Wheels.
Computer.
Stage. - Place suitable stage.
- Place a drop of oil on the objective (make sure it’s the right objective).
- Place chamber on stage.
- Click on NIS and choose N-STORM Andor.
- Right-click, acquisition control, N-STORM launch pad.
- Click on the lasers. Choose laser lines.
- Press autofocus button. It will start flashing. Start rolling the wheel at your direction to move up the objective. Once it gets closer it will stop flashing. Pay attention to your imaging buffer refractive index.
- Check that STORM lens is inside. Lift red tape to move the laser beam with knob.
- Check whether beam is collimated. Unlock screw. Move screw right and left to collimate and lock it.
- Make sure that the detection pathway is set to the Andor camera (says TIRF on microscope).
- Press live view. Press auto scale. Set reporter laser power to ~ 0.5 while looking for focus. Use joystick to look for cells. Can rotate handle to switch from coarse to fine and extra fine. Since autofocus is active use “perfect focus offset” (button for fine and coarse). Use ~100 ms exposure time. Play with TIRF angle for better resolution.
- Once you found a cell, stop live view.
- Choose ROI, Define ROI. Take a snapshot.
- Switch buffer to STORM buffer. Repeat step 14 and other steps if necessary.
- Turn up reporter laser power to 100. Change exposure time to ~10-20 ms (play with exposure time, not laser power).
- Define save pathway.
- Hit run.
- Transfer files.
- Analysis: 4th (most right) button in the launch pad.
Open file.
ID settings (wheel).
Choose a frame.
Allocate a low-intensity spot. Scroll-in with mouse roller to look at the peak height.
Press red button (circle). Press test to see if you chose a good threshold.
Click red button again and click start.
Imaging was performed at the Nikon Imaging Center
Nir Lab 