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Description...
Experiment name
Enter the name of the PhotoKinetics experiment macro to run.
Laser Module Settings
Open the Resolve3D Settings window with the Lasers tab selected.
Laser Events Tab
Use the Laser Events tab to define which laser to use, the pulse duration, the number of pulses, and the bleach pattern. You can also select when to mark the event time and whether to optimize for speed or position accuracy.
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Description...
Laser channel
Allows you to select the laser channel by wavelength.
Duration (secs)
Allows you to set the laser pulse duration in seconds. In many situations, short pulses are better than long, but the appropriate duration depends largely on the type of PK experiment.
Refresh
Updates the active laser and duration to the values most recently used in the Lasers tab of the Resolve3D Settings window.
Number of Events
The number of times to repeat the laser pulse event before collecting the images that contain the recovery data. For quantitative FRAP experiments, 1 short laser pulse is best.
Bleach event motion
When bleaching (or activating) multiple locations within the target, the XY stage moves sequentially between locations. You can choose one of the following options to optimize for position accuracy or for speed.
Optimize for Position - Choose when moving to exact XY locations is essential. For example, when the targets are small and precise bleaching is required.
Optimize
for Speed - Choose when it is more important to finish
the bleaching procedure as rapidly as possible, which is often
true when the fluorescence recovery time is relatively short.
Selecting Optimize for Speed moves the XY stage as rapidly as
possible between bleach locations, skipping some of the sophisticated
positioning techniques that require extra time.
Event pattern
Allows you to specify how to set the event pattern.
Use
Current Center Point - Bleaching the Center Point is often
the most effective method of studying fluorescence recovery, when
combined with Resolved3D's Center Object 
feature. By aligning the laser
spot with the center of the image, minimal effort is required
to position and start a single point FRAP experiment. Many FRAP
experiments can be collected when the event pattern is set to
Center Point.
Use Bleach Event Specification - For experiments that require multiple laser points, select Use Bleach Event Specification. Refer to Create Bleach Events for various methods of defining multi-point bleach events.
Create Bleach Events
When specifying a custom bleach event (the opposite of bleaching the center point), use the tools represented by the following icons to define the list of points at which laser pulses will occur. Bleach events are defined within an image acquisition window (often Window 21), so a recently acquired image of the target is necessary before a custom bleach event can be prepared. Points, lines, and areas can be combined to form complicated bleach patterns. The ability to delete bleach points (within lines and areas) adds even more ways to control the shape of the bleach pattern.
Bleach
Event Tools:
From left to right, the bleach event tools are:
Point,
Line, or Area Selection - To select existing bleach locations
for modification, click on the arrow and then select events within
the image window. Selected objects change color from red
to cyan. In the case of lines and areas, it is possible to select
both the larger object and a single point within the object.
Add Bleach Point - To add laser
spot locations, select this icon and then select bleach locations
by clicking at appropriate locations within the image acquisition
window.
Add
Bleach Line Segment - To add a line bleach, click on two
locations within the image acquisition window. As indicated within
the image window, line bleaches are accomplished as a series of
evenly spaced point bleaches. The spacing between spots can be
controlled by adjusting the Pattern Spacing.
Add
Bleach Area - To bleach a polygon shaped region, select
as many boundary points as necessary within the image acquisition
window. The polygon will be automatically closed when the selected
location matches the first boundary location. At least three boundary
locations are required. As shown in the image window (see below),
area bleaches are accomplished with evenly spaced point bleaches. The
bleach spot spacing within the area can be controlled by adjusting
the Pattern Spacing.
Delete
Selected Point or Line - To delete a selected point
or line, first use the Point or Line Selection tool and then click
on this icon.
Tip: It is possible to delete individual points within a bleach line or area, which enables one to bleach large regions while avoiding substructures of interest (see Point Selection above). For example, the picture below shows the result of removing five laser spots from the bleach area above.
Delete
Selected Area - To delete a selected bleach area (i.e.
a polygon), first use the Point or Line Selection tool and then
click on this icon.
Delete
All - Deletes all bleach events within the image window.
This is the only bleach event deletion tool that will prompt the
user before responding.
Pattern Spacing
Controls the separation between laser spots within a bleach pattern. For convenience, the units are image pixels. Choosing an appropriate pattern spacing can be difficult, because the effective size of the laser spots is determined by many parameters including laser power, pulse duration, objective NA, and beam expander position. It is often necessary to perform trial experiments before finding an optimum pattern spacing. The factors that influence the choice between coarse and find spacing include fluorescence recovery rate, optimum percent bleach, desired bleach profile (uniform or spotted), and cell viability.
Show Event Graphics
If requested, the data collection software will generate a graph of the fluorescence intensity during the experiment.
Imaging Tab
Use the Imaging tab to select the information for the acquisition channel and specify time course parameters for the experiment.
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Description
Channel Setup fields
Allows you to select the appropriate information about the acquisition channel. That is, the exposure time, filters, and shutters to use throughout the photokinetic event.
Time course style
Allows
you to specify one of the following experimental timing parameters.
Adaptive Time Intervals - This is often the best choice
for quantitative FRAP experiments in which the fluorescence recovers
according to the conventional 2D diffusion models. Data are
collected more rapidly at the start of the experiment (where the
fluorescence is changing rapidly) than at the end (where the fluorescence
is stabilizing at an asymptote). The data collection rate and
experiment duration are both controlled by the expected half-time,
so it is important to provide a reasonable value for Expected
Half-time when using this time course style.
Uniform Time Intervals - Certain FRAP experiments work
best with uniform measurement intervals.
As Fast As Possible - This is a special acquisition
mode that is similar to the Fast Acquisition technique used for
Z stacks and time-lapse imaging. In this mode, the exact
image acquisition interval is determined by the combination of
exposure time and other camera settings. Because the shutters
stay open while recovery images are acquired, this mode may not
be inappropriate for sensitive cell types. The duration of the
experiment will be a result of the number of requested post-event
images multiplied by the image acquisition interval.
Mark event time
Allows you to mark the exact time for the event At the Start of the Event, In the Middle of the Event, At the End of the Event, or Never. Marking the event time can be critical for determining accurate results when an event is comprised of multiple actions or a single action of longer duration.
Pre-event images
Allows you to specify the number of images to take before the event. The default of three images is sufficient in most cases. For better pre-bleach statistics, however, it may be helpful to increase this value.
Post-event images
Allows you to specify the number of images to take after the event. When using adaptive time intervals, the default value of 32 images provides a good measurement of the fluorescence recovery curve. To more fully sample the asymptotic recovery and extend the length of the experiment, increase this value.
Pre-event image time interval
Indicates the amount of time between pre-event image exposures.
Post-event image time interval
Applies to experiments in which the time interval is uniform and indicates the amount of time between post-event image exposures.
Expected Half-time
For experiments that use Adaptive Time Intervals, the expected half-time is the essential parameter used for determining an appropriate data collection rate. In most cases, trial experiments are necessary before it is possible to precisely set this parameter.
Tips:
To more finely sample the fluorescence recovery, set the expected
half-time shorter than the actual half-time. The time course
will then be adjusted to acquire data points at shorter intervals.
Then add more post-event images to extend the duration of the
experiment and ensure that the final recovery is adequately measured.
For sensitive samples, it may be necessary to sample the fluorescence
recovery more coarsely, in which case the expected half-time can
be set slightly longer than the actual value. In all situations,
make sure that there are enough data points along the rapidly
changing part of the FRAP curve and that the experiment lasts
long enough to properly measure the recovery. Excess extrapolation
or interpolation will lead to noisy and potentially inaccurate
results.
Experiment duration
Applies to experiments in which the time interval is uniform. The number in this field represents the total amount of time the experiment will take to complete.
Repetition Tab
Use the Repetition tab to set the number of times to repeat the experiment and the time lapse between the repeats.
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Description
Entire experiment repeats
Allows you to select how many times to repeat the entire experiment. Repeated PK experiments can be useful for monitoring changing kinetic conditions within live cells.
Time lapse between repeats
Allows you to specify the time delay between experiment repetitions.