Use the TIRF Illumination Settings window to specify TIRF settings for an experiment.
Locate the sample through the eyepieces using widefield fluorescence.
From the Resolve3D Settings window under the Lasers tab, click the TIRF Illumination Settings button near the bottom of the window. The TIRF Illumination Settings window is displayed.
Activate the radio button next to the laser you want to adjust.
(Advanced only – not required): In the Back Aperture Focus For Current Lens field, deactivate the Locked check box to release the slider tool for use. By default, this check box is activated (slider is locked).
From the Resolve3D main menu, use the Acquire button with the Z control to adjust the focal plane. When you control the incident angle of illumination, an evanescent field is generated. Adjusting the angle changes the depth of light penetration into the sample.
Adjust
the TIRF angle as follows:
• Coarse adjustments are made to the TIRF angle by dragging and dropping
the box in the slider bar using the left mouse button. An image is
acquired when the mouse button is released.
• Fine adjustments are made to the TIRF angle by clicking the arrows
on either end of the slider bar or by using the left or right arrows
on the workstation keyboard.
To illuminate the sample and not generate an evanescent wave, click the Epifluorescence button.
To return the laser to that last incident angle used, click the Restore TIRF Depth button.
Adjust the slider for the Laser Path Splitter to determine the amount of light to send to the TIRF light path.
Activate the Use Beam Concentrator check box to enable the laser beam concentrator.