TIRF alignment guide

Aligning TIRF lasers

Goal

To ensure that laser critical angles are always critical, independent of chosen laser direction (rotation orientation)

1. Bertrand lens switch: Rotate to "B" (counterclockwise) - will need to adjust small rotatory button on this switch in a later step
2. Change side switch: From Eye -> Camera (rotate) (on the right of the microscope, under the eye pieces)
3. Switch Box-lights on (proximity sensor button top right of big incubator box on microscope)
4. Set Observation mode Camera L (left button out of 4 round buttons, front of the microscope)
5. Move Condenser down (inside black box, above sample) as low as reasonably possible (not hitting the sample...) by adjusting one of the rotary buttons on either side of condenser

Placing the sample and finding it

1) Select the lens (e.g. 100X)

2) Lower the lens with ESC, hold ESC to make that the current position

3) Put oil on the lens

4) Center the sample holder

5) Place the sample on the holde

6) Switch on an illumination mode (e.g. brightfield, or epifluorescence in the software)

7) Find focus on the sample (lens up, until image = sharp)

8) (optional) switch-on PFS in the software to keep this focus

Performing the TIRF alignment using the NIS Elements software

1) Select TIRF Prime BSI mode, pick an OC to be aligned

2) Go "LiveAllCameras" and select the Fi3 view window

3) Press "Eyes only" on the front of the microscope (top button / observation method) to direct all light to the Fi3 camera

4) Using the Fi3 window, adjust brightness/contrast of this view, done in the LUTs window, to clearly view the laser spot (if laser is not centered, move the TIRF angle a bit in the software, and move it back to 0)

5) In the Ti2 E-TIRF Pad, hit Align and provide it with the correct fully reflecting critical angle for about 5-6 different rotations

6) Press "compute zeo from critical angles" and close window with "OK"

7) (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser