Photokinetic Data Analysis

Field

Description

Input Image

The image to analyze. The image must have at least one pre-bleach time-point and two post-bleach time-points. Pre-bleach time points must be identified by negative time values.  If the data do not have proper time stamps, see More Options | Time Zero Offset.

Output Image

An image containing the photokinetic analysis results in the form of a three channel image. This image file includes the following three channels:

• The first channel is processed image data. If appropriate, the intensities are normalized according to the photosensor and mean intensity normalization options.

• The second channel, (/), is the FRAP Ratio Image. The ratio of the current time-point to the average of the pre-bleach timepoints. At the location of the laser spot, the ratio represents the fractional recovery. Motion artifacts are clearly visible in this image.

• The third channel, (-), is the Spot Region of Interest Image. The ratio data at the location used for analysis. Look at this image to determine whether cell motion or other artifacts are affecting the results. Pixels that are not involved with the analysis are set to 1.

Results File

The name of the photokinetic analysis results file. Details about the analysis are recorded in this file, as well as tabulated FRAP results that can be exported to a spreadsheet or a graphing program. Certain results are also recorded in the FRAP results graph.

Response

Specifies the type of response to analyze:

• Bleach is used for Fluorescence Recovery After Photobleaching (FRAP)

• Activation is used for Photoactivation

• Auto Determine specifies to allow the software to determine the nature of the event (FRAP or Photoactivation)

Recovery Model

Fluorescence recovery may occur by a number of different underlying mechanisms, some of which have been described and modeled in the literature. You can select:

• None - The recovery data are not curve-fit in any way. No smooth curve is included with the graph or results file.

• Smooth Curve - The recovery data are curve-fit with a flexible model that works well for a variety of different diffusion recoveries. The curve-fit yields a fractional bleach, fractional recovery, and half-time.

• 2D Diffusion - The recovery data are curve-fit with Axelrod's two-dimensional diffusion model [See Axelrod, 1976]  and the resulting half-time is used to calculate the 2D diffusion coefficient. Note that the diffusion coefficient is only valid if the fluorescence recovery is indeed coming from 2D diffusion (e.g., if the recovery arises from 2D diffusion within a membrane of some kind). Note also that the beam spot size must be reasonably accurate, because errors in beam spot size will propagate to the diffusion coefficient as the square of the error. See Spot Radius.

• 2D Diffusion, 2 Component - This is the same as the 2D Diffusion option, except it assumes that the recovery comes from two diffusing components.  For a multi-component version of Axelrod's 2D diffusion model, see Tsuji 1986.

Recovery Data From

• Site ROI - By default, the photokinetic analysis extracts data from the fractional recovery image (channel 2) using a region-of-interest ("ROI"). The size of this region-of-interest is controlled as a function of the expected beam size. The location of the bleach spot corresponds to the minimum intensity fraction in the first postbleach image. To control the ROI size and shape, use "Bleach ROI Type".

• Beam Profile - Because the bleach profile is often Gaussian, the analysis can determine the recovery data from a best-fit to either a symmetric of elliptical Gaussian intensity distribution. See "Beam Profile Shape". This method is not recommended for use with bleaches that have 100% recovery, because the bleach profile may "disappear" after the first few time-points. A benefit to this method, however, is that it can track the bleach location and yields information about the beam size and shape.  In the case of a slow recovery, the best-fit results of the first, post-bleach time point provides a reasonable estimate of the laser spot radius.

Site ROI Type

When using a bleach ROI to extract recovery data, the size and shape can be adjusted to best match the experiment. The analysis uses only the first post-bleach image to locate the bleach ROI. Subsequent time-points are analyzed with this fixed ROI. Look at channel 3 of the output image to view the resulting ROI. ROI type selections are:

• Box - A square box centered on the bleach location. The parameter at right determines the box width and height as a multiple of the beam radius. By default, the ROI width is "1.00" times the beam radius.

• Circle - A circle centered on the bleach location. The parameter at right determines the circle radius as a multiple of the beam radius. By default, the ROI radius is "1.00" times the beam radius.

• % Change- Bleached locations are determined by percent bleach on a pixel-by-pixel basis. Use this setting for arbitrary pattern type bleaches. The first parameter at the right shows the percent bleach threshold, below which pixels will not be included in the recovery analysis. Bleach locations are limited in size by the second parameter at the right, which is in units of the beam radius. By default, the analysis looks for pixels that are bleached at least 30% and within 4 beam radii of the bleach location.

Beam Profile Shape

When analyzing recovery data from a "beam profile", either a "symmetric" or "elliptical" Gaussian profile can be applied. In most cases, the ideal beam is circular. In reality, all beams are somewhat elliptical. Symmetric analysis is faster than elliptical.

Number of Laser Sites

By default the analysis looks for the site that exhibits the largest fractional bleach. In the case of a multi-spot bleach there will be other legitimate bleach sites. Use this parameter to direct the program to find and analyze other potential bleach sites. The order of the bleach locations will correspond to the rank of the fractional bleaching. That is, higher percent bleaches will be identified before lower percent bleaches.

Spot Radius (um @ 1/e2)

The expected beam radius in um at 1/e2 (“one over e squared”), the central intensity. This is the standard method of describing the size of a Gaussian shaped laser spot.  When the recovery is a result of 2D diffusion, the diffusion coefficient can be estimated from:

 
D = w2 / (4 td)
where w is the laser spot size radius at 1/e2 and td is Axelrod's recovery time constant [See Axelrod 1976].  D has units of um2/sec.  Note that D is a function of w squared.

Background Intensity

The background intensity within the time-lapse data. Many samples have an unbleachable or high-speed fluorescent component that adds background intensity to the data. An estimate of this value should be obtained during data acquisition, because accurate fractional bleach and fractional recovery values depend on this value. Test bleaches are a good way to estimate the background fluorescent intensity. See "Image Bias".

Image Bias

Intensity bias within the time-lapse images.  This value is subtracted from the image data before analysis.  Accurate fractional bleach and fractional recovery calculations depend on this value.  If the bias values in the user interface show up as "0", a valid measurement should be provided. Most CCD cameras have a bias value between 50 and 150 counts. In recent versions of the acquisition software, the image bias is stored within the image and automatically retrieved by the analysis program. To measure the image bias, acquire a dark image from the data acquisition microscope using similar or identical camera settings to those used for the FRAP experiment.  Ensure that no light is reaching the CCD detector.  The mean intensity of the dark image is a good measure of the image bias. See "Background Intensity".

Z Section

Use this parameter to select the Z section number to process when the image contains more than one Z section. For simple time-lapse images, the Z section number is always 1.

Wave Num

Use this parameter to select the wavelength number to process when the image contains more than one wavelength. For simple time-lapse images, the wavelength number is always 1.

Last T Num

The last time-point number to include in the FRAP analysis. This is often useful for

excluding time-points affected by cell motion or other artifacts. To determine the exact point number, open the input image in an image window and scroll to the time-point where the problem occurs. Alternatively, press the middle mouse button over the corresponding point in the recovery graph. A label containing time-point information is superimposed on the graph. By default, all time points are used by the analysis.

Do It

Runs the analysis. The following three files are output: a three channel output image file, a JPEG file that contains a time point fluorescence recovery graph, and a log file (in a text format) that contains the analysis results.

More Options

Opens the More PK Analysis Options window.