To create and run a Photokinetic experiment:

 

  1. Configure and align the lasers.

  2. From the main Resolve3D window, click the Experiment Icon_Experiment.jpg icon to open the Design/Run Experiment window. Then click the PhotoKinetics tab. These tab settings define the nature of the photokinetic event, the timing of the event, and the imaging conditions to associate with the event. They also define the locations of the photokinetic events.

  3. In the Experiment Name field, enter the name of the macro. The name “Resolve3DPK” is reserved, much like “Resolve3D.” It is a default name that can be overwritten without warning. All other names will generate a warning message when overwriting a preexisting macro.

  4. Click the Laser Events tab. In the Laser Channel list, select which laser and shutter to activate and use. This list includes the lasers that are installed in the X4 Laser Module chassis and defined in the ic540.ini file in the Instrument Controller.

  5. If you want to repeatedly pulse the spot, enter the number of pulses in the Number of Events field. (If a pattern or list of points is defined in PK Bleach Setup and selected in Event Pattern, each of these points are repeated the number of times defined in the Number of Events field.)

  6. In the Bleach event motion field, select whether to optimize for speed or for position accuracy. (When you select to Optimize for Speed, minor stage position corrections are not performed.)

  7. In the Event pattern list, choose the area of the sample that you want to bleach. To define the event on a single point, choose Use the Current Center Point (no additional settings are necessary). To define more than one point for the event, choose Use Bleach Event Specification and use the icons under Create Bleach Events to define the event.

    Use

    To

    select_polygon.gif

    Select a point to delete

    addABleachPoint.jpg

    Add a point to the event

    addALineSegment.jpg

    Add a line of points to the event

    create_polygon.gif

    Define the event by creating a polygon area.

    deleteAPoint.jpg

    Remove a selected point

    delete_polygon.gif

    Remove a selected polygon

    deleteAll.jpg

    Remove all

  8. Click the Imaging tab and select the exposure and filters to use for data collection throughout the event. Select the imaging conditions in the Design tab.

  9. Click the Repetition tab and define how many times to repeat the entire experiment in the Entire experiment repeats field. Then define the time delay between these experiment repetitions in the Time lapse between repeats field.

  10. Choose File | Save As and fill out the fields in the Prompt window. The file size is calculated and reported in the Estimated File Size field, based on the image size selected in Resolve3D. If you change the image size, the Estimated File Size feature will over or under estimate the actual file size unless you recreate the macro.

  11. Click the Run tab and select Start Scan Run_icon_sm.JPG to run the experiment.

    Notes:

    • After you create and save an experiment macro, you can reload and run it later.

    • DeltaVision prevents light from reaching the eyepieces when the laser is on, but you can still view the sample through the camera.

    • Unexpected FRAP results can often be explained by reviewing the output image. Unlike most DeltaVision images, this image consists of three channel, 32-bit, floating-point pixels and is therefore six times the size of a single channel, 16-bit input image.

     

    Tip:  You can change the length of time (in seconds) that the shutter is open in the Duration field. To display or change Laser Power, select Settings | Lasers tab and change the value of the Power field for the laser that you are using.