Localization Phase Input Parameters

To determine the sub-pixel positions of the fluorophores in a data set:

1. From the softWoRx main menu, select Measure | Localization Image Analysis. The Localization Image Analysis window is displayed.

2. Click the Localization tab to activate the Localization portion of the window.

3. Click the Input Image button to browse and select the image file to use for analysis. Alternatively, you can click and drag an image window number into this field. This file must be a DeltaVision image file (*.dv)

4. To use only a portion of the input image, click the Select Region button and then click and drag the mouse to specify the part of the image you want to analyze. Alternatively, you can use the Details button to define a subset to analyze.

5. Activate the Auto-increment file names check box to automatically increment the _LOC.txt file names for the experiment.

6. The Localization Data field is automatically filled with your original localization file name (_LOC.txt). Click the Localization Data button if you want to browse for and select a different localization file.

7. In the PSF size factor field, enter the PSF size factor in units of pixels. The PSF size factor is the predicted, or measured, value of the standard deviation of the Gaussian fit to the point spread function of the microscope for the experimental conditions. It may be necessary to measure the PSF size factor directly using the PSF Width tool under the Measure menu. Use the Set to default button to set this field to the theoretical value for the chosen experiment conditions.

8. Enter the desired local maximum factor into the Local maximum factor field. The local maximum factor should be a value from 0.0001 to 0.5. The local maximum factor is 1 minus the probability that a detected peak is significant above the background distribution (also known as an alpha value). Therefore, the local maximum factor will have typical values of 0.1, 0.05, and 0.01. The default value is 0.05 and larger values lead to more detected peaks.

9. Click the Test Localization Parameters button to test the selected parameters on a small sample of the data. To use the Test Localization Parameters button, the raw DeltaVision image file must be open and used as the input to the Input Image field (drag a window number into the Input Image field as in Step 3 above).

10. By default, the input localization parameters will be tested on frames 1-20 of the dataset. To change these defaults, click on the Test Options button. The test options window is displayed.

Use the Test Start Frame field to select the frame where the test of localization parameters will begin. Use the Number of Test Frames field to enter the total number of frames in the localization parameters test. If desired, deselect the Start test at current frame check box to have the test start at the Test Start frame. By default this box is activated meaning that the test will start on the current frame of the raw data and proceed for the number of test frames. Click the Done button to close the Test Options window and return to the previous window.

11. If desired, activate the Show Localization Viewer check box, which will display the Localization Viewer once the data has been analyzed.

12. When you are satisfied with the selected detection parameters, click the Run the Localization button to process the indicated frames.

13. The selected detection parameters can be used to analyze data sets in a queue. To run the localization as a background task, click the Add Localization to the Queue button.

To evaluate the selected parameters, you can compare the detected fluorophore positions to the original acquired image using The Localization Results Viewer.