More PK Analysis Options

Field

Description

Normalize to Mean Intensity

In most cases, the mean (or total) image intensity of a Photokinetic time-lapse image is expected to remain constant. (In fact, recovery models often assume that the total amount of photobleached probe is zero compared to the surrounding pool.)

There are a number of artifacts, however, that prevent the fluorescence intensity from being perfectly consistent. The most likely cause of an intensity drop is photobleaching caused by a combination of imaging and excess laser bleaching. Photobleaching suppresses the apparent fluorescence mobility (i.e. fractional recovery) and leads to an apparently shorter half-time and higher diffusion coefficient.

A second source of inconsistency is arc lamp flicker, which causes sporadic illumination intensity changes. Such intensity changes add noise to the resulting data and can skew the results, depending on when the intensity changes. By default, this option is always on. When analyzing photobleaching experiments that involve large areas or numerous bleach sites, this option should be turned off.

Use Photosensor Values

Each image is recorded with a photosensor value that represents the illumination intensity. The photosensor value can be used to correct for arc lamp flicker. Because the mean intensity normalization procedure cancels the effect of the photosensor correction, this option cannot be enabled at the same time as the mean intensity normalization.

Post -event Corr Factor (X)

A constant scaling factor that is applied to the data after the event. By default, this value is 1.0. Adjusted values (typically > 1.0) should be determined by external calibration methods.  

Time Zero Offset (secs)

Proper analysis of certain types of Photokinetic experiments may require adjustment of the zero time point. Negative time values are treated as pre-bleach measurements, whereas positive time values are treated as post-bleach measurements. The value provided in this field is subtracted from the time values stored within the image.

Spot Search ROI (um)

This is the size of the area about the center of the image within which to search for the laser bleach spot. This applies to the "Bleach Profile" method of extracting recovery data.  By convention, the Photokinetic analysis searches for bleach spots near the center of the image.

Spot ROI Sampling Interval (pixels)

Set this value larger than 1 to reduce computation time. A value of 1 causes every pixel to be processed during the analysis. A value of 2 causes every other row and column of pixels to be included in the analysis, and thereby reduces the pixel count by a factor of 4.  If the computation time is acceptable when using a sampling interval of 1, this value should remain at 1.  Values larger than 1 are most useful when the recovery data are calculated from the “bleach profile,” because of the computation time required to calculate the best-fit Gaussian intensity distribution.  See Recovery Data From on FRAP Analysis Options.

Calculate Vel. & Accel Maps

A series of images that show the first (velocity) and second (acceleration) derivative of intensity with respect to time. These images are useful for showing flow patterns in photoactivation experiments.

Velocity Map Smoothing

A smoothing function for velocity and acceleration maps.

Results File Types

• Log File (.log)

• Tab Delimited (.txt)

• Comma Separated (.csv)