Biology Reference
In-Depth Information
Enter the plate calibration mode of the AxioVision Mark&Find module.
Select the multiwell plate geometry template matching the microplate.
Calibrate the plate geometry using the provided seven-point routine.
15.8.5
Step 5: adjust TIRF excitation
Select the TIRF focus adjustment tool in the objective turret.
Change the filter cube turret to the TIRF adjustment cube.
Turn on the 642 nm laser and set the power to 5 mW.
Observe the beamwith the AxioCamMRm and adjust the TIRF focus knob on the
TIRF3 slider to minimize the spot size.
Select the TIRF angle adjustment tool in the objective turret.
Observe the beam with the AxioCam and adjust the TIRF angle micrometer to
move the spot to a position approximately 20% close to the image bottom.
(The exact position has to be determined from a sample with fluorescent beads
and should be used to set this angle. Refer to the Zeiss TIRF3 manual.)
15.8.6
Step 6: sample for subpixel alignment of color channels
Prepare a sample with immobilized 100 nm TetraSpec beads (Invitrogen) on
poly-
D
-lysine (PDL)-coated glass-bottom microplate dish (SensoPlate) or culture
dish (CellView) (Greiner).
Image surface-immobilized beads with simultaneous laser (TIRF) excitation (488
and 561 nm). Use images to calculate image registration transformations
described in Step 11.
15.8.7
Step 7: alignment of the CRISP focus-stabilization
(autofocus) system
The extension tube adjustment and prism adjustment typically have to be
performed at the installation of the CRISP unit only.
Start the ASI Console software, connect to the CRISP controller, and start the
three-step adjustment routine.
Manually optimize the detector lateral adjustment to maximize the dithering
difference signal on the ASI controller.
Finish the adjustment routine with the Gain Setting step.
Generate the Focus Curve using the control button and check if the vertical axis is
in the linear part of the curve and well separated from the extremes.
15.8.8
Step 8: preparation of antibleaching and antiblinking
imaging buffer system
15.8.8.1
Materials
Trolox, (
)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Aldrich
238813, 97%, MW
¼
250.29 g/mol)
