Running a Localization Imaging Experiment

To run a previously configured localization experiment:

1. To start the localization experiment, select the Run tab on the Design/Run experiment window.

2. In the Image file name field, enter a file name for the image that will be generated by this experiment.

3. In the Image title field, optionally enter a title or label to put in the image file header.

4. In the Add note to log field, you can optionally enter any text for a note to be added to the experiment log file.

5. Activate the Show images during acquisition check box to view preview images in the Data Collection window during acquisition.

6. Activate the Launch viewer after acquisition check box to view the resulting image in an image window after the experiment is finished.

7. Click the More Run Options button. The More Run Options window is displayed.

8. From the More Run Options window, activate the Show monitor check box to display the Localization Imaging Monitor during the collection process.

9. In the Skip frames before monitoring field, enter the number of frames to skip before any results are collected.

10. In the Monitor update frequency field, enter the frequency at which the localization monitor should update.

11. In the Monitor detection sensitivity field, adjust the location sensitivity for the localization monitor.

12. In the Maximum frame increment field, enter the maximum number of frames to skip when you are acquiring images as fast as possible with Display Images set to On.

13. Click on the green Start button at the top of the Design/Run Experiment window. The Activation Power/Duration window is displayed.

14. At this point, the researcher will visually inspect the data. The sample should be exhibiting some blinking or photoswitching. If there are too many fluorophores “on” (i.e. multiple fluorophores in a single diffraction limited spot, or adjacent spots), then you should wait until only a few fluorophores in each frame are observed before increasing the activation laser power.

15. From the Activation Power/Duration window, use the Power (%) sliders to adjust the percentage of power to use for each laser. This is used to control the blinking and should only be adjusted if (1) the fluorophore can be activated, and (2) the number of fluorophores on in a field of view is very sparse.

Related Topics

Using the Beam Concentrator

Setting Up Localization

Using the Localization Progress Monitor

Localization Phases

The Localization Results Viewer