Verify that the Laser Module is correctly installed on the Fiber Optic Module and that the cover is installed over the beam expander turret.
Turn on the AC power to the system by using the PKC POWER rocker switch on the right side of the front panel of the Laser Source Chassis.
The green “POWER” light indicates that power is applied to the laser control electronics. At this point, any lasers are in Standby mode, all of the power supplies are energized, and the thermal-electric coolers are active in each laser head.
Rotate the key on the left side of the Laser Chassis to the LASER ON position.
The Chassis checks the current interlock status. When the interlock circuit is closed, the “EMISSION” indicator light on the front of the Laser Source Chassis and the indicator light next to the Beam Selector are on. The lasers and shutters cannot be activated until these indicators are on (i.e., until the key is on and the interlock circuit is closed).
Click the Settings
icon from the Resolve3D window to open the Resolve3d Settings window.
Then click the Lasers tab.
The Laser field shows which
lasers are installed in the Laser Chassis. The border of each heading
displays the approximate color of each laser.
Click on the lasers that you want to activate. You can turn on all or none of the available lasers.
If PKC POWER is on, the Key is in the ON position, and all of the interlocks are engaged, selecting a laser supplies power to the laser diode, the laser starts to lase, and the shutter for the laser is enabled.
The Status field alerts the user whether a laser is Off, Ready, or in the process of Warming Up. A laser is not available for use in an experiment until the Status displays READY. The time between supplying power to the laser and when the laser is ready depends on the laser type and manufacturer.
In the Power and Duration fields, enter the minimum values for the laser that you are using for your experiment. The minimum Duration field value is about 0.005 seconds.
On the Fiber Optic Module, set the Mirror Switch to the Mirror In position.
Set the laser spot size by rotating the Beam expander on the Laser Optic module to one of the four positions on the Laser module. You will feel the beam expander snap into place when it is in position.
Place the mirror slide (PN52-502742-000) that is included with your system on the slide holder. Set the Excitation filter to RD-TR-PE, the Eyepiece filter to FITC, and the ND filter to .1% light transmittance. Then use the eyepieces to focus on the slide.
In the Resolve3D Settings window, under the Lasers tab, activate the Show spot alignment target check box.
Set the Beam Selector to direct light to the camera and set the Emission filter to POL.
Click Image Spot to acquire an image and view it on the monitor to make sure that the laser is properly aligned. To align the laser, click Continuous to acquire a series of images and use the tilt screws on the Laser Optic module to adjust the tilt until the laser is aligned with the center of the field. If you are unable to align the laser, notify your lab administrator or someone who is trained in alignment procedures.
Test the magnitude of the
event by moving a test sample into the field of view. Select a point
of interest and click 
in
the Resolve3D window to center the selected point in the field of
view.
Click Do Test Bleach in the Settings window.
A pre-event image is acquired. Then a laser pulse of the specified duration and power is applied, and a post-event image is acquired. The magnitude of the effect is displayed in the % Bleach field. The background intensity is displayed in the Background field (Background is needed for FRAP recovery analysis).
If the test bleach is not centered, repeat Steps 12-14 to realign the system.
In the Power and Duration fields, enter values for the laser that you are using for your experiment. Typical values for these fields are 20% and 0.10 seconds, respectively. The minimum Duration field value is about 0.005 seconds. (The shutter timing error is ± 0.002 seconds, regardless of the duration of the shutter opening.)