Setting Up Localization

To set up and prepare for a localization experiment:

1. From the Resolve3D window, click the Experiment icon. The Design/Run Experiment window is displayed.

2. Select the Design tab to activate it.

3. Select the Localization tab on the left side of the window.

4. In the Experiment name field, you can either accept the default name, or rename the experiment in order to retain the settings for future experiments.

5. Click on the Laser Module Settings button. The Resolve3D Settings window is displayed with the Lasers tab activated. Use this window and the TIRF Illumination Settings window to set up the DeltaVision system as you normally would to perform a TIRF-based experiment. This procedure is described in the “Imaging Using TIRF” section in Chapter 8 of the DeltaVision Imaging System User’s Manual.

Note: In the TIRF Illumination Settings window, move the Laser Path Splitter slider all the way to the right (for 100% TIRF).

6. In the Reporter Imaging Setup portion of the Design/Run Experiment window, use the drop-down menu in the Exp field to set the exposure time. Then enter your selected EX/EM pair using the drop-down menus for the EX Laser and the EM Filter.

Note: When you make a selection from the EX Laser drop-down list, the rest of the fields in the Reporter Imaging Setup portion of the window are populated automatically with the last values used.

7. To leave the excitation laser turned on throughout the experiment, activate the Continuous Illumination check box. Leaving the laser on will help to keep photoswitchable dyes in their “off” states during acquisition so that only a few fluorophores are on in any given frame. Though the photophysics are not completely understood, it may be useful to pulse the excitation laser when using certain fluorophores, such as photo-convertible fluorescent proteins, to help avoid photobleaching or unwanted transitions into triplet states.

8. Activate the Switch fluorophores Off check box to use the Reporter Imaging laser to switch all fluorophores to an “off” state before the first activation. With this check box activated, you can adjust the Laser power, Pre-Activation Images, and Event duration (secs) fields as desired.

Note: Pre-activation images are used to confirm that the majority of the fluorophores have been switched to the “off” position.

In some cases, it may be useful to initially switch the fluorophores to an “off” state prior to data acquisition. This can be very useful when dyes have been used for labeling. The user must be careful not to subject the sample to an excessively long pulse duration, which could photobleach the sample. It may be necessary to empirically determine the optimal duration for each sample type.

9. In the Activation/Imaging Sequence portion of the Design/Run Experiment window, use the drop-down list in the Activation field to select each laser wavelength (up to three lasers) for the activation laser(s). To the left of the Activation field, click the + button to add another laser or click the - button to remove a laser.

10. In the Power field, enter the percent of laser power to use for this wavelength. This field should be set at a minimum of 10% for laser stability.

11. In the Duration field, use the drop-down menu to select the duration for each laser pulse.

Note: If you have the Continuous Illumination check box activated, you cannot set the duration for any of the activation lasers to Continuous.

12. In the Sequence repeats field, enter the number of times you want to repeat the entire sequence.

13. Use the settings in the UltimateFocus Setup portion of the window to set up UltimateFocus for the experiment. Enter your selection for how often you want UltimateFocus to perform a Z-focus maintenance correction in the UltimateFocus every field. This is the number of sequences to wait before performing UltimateFocus.

14. In the Move threshold field, enter your selection for the move threshold. Any drift greater than the value set in this field is corrected.

15. In the Maximum iterations field, enter the maximum number of iterations for UltimateFocus. This selection represents the maximum number of times the UltimateFocus function will be performed to find optimal focus during a single correction event.