Now you can transfer (Shuttle) the sample and the LM (Light Microscope) image file (.czi) to the SEM (Scanning Electron Microscope). There you can easily relocate (Find) the same sample positions and acquire a corresponding image within the ZEN SEM software. Therefore exactly the same steps have to be done as for the light microscope.
Shuttle & Find Sample Positions at the Electron Microscope
Mounting the Sample Holder to the SEM
For imaging your sample in the SEM, insert the sample holder (2) in the special SEM adapter (1) and mount it to the SEM.
The arrow of the sample holder has to face the arrow of the SEM adapter.
Starting the ZEN SEM Software
For correlative microscopy with scanning electron microscopes SmartSEM and ZEN SEM have to be installed. SmartSEM is still the control software of the scanning electron microscope. ZEN SEM comes as an add-on for SmartSEM to perform correlative microscopy and using Shuttle & Find on a SEM.
- You have started SmartSEM.
- Start the ZEN software by clicking on the program icon on your desktop.
- The application selection window appears.
- Click on the SEM button to start.
- You will see the program interface with a reduced user interface comparing to the software. In the Left Tool Area, the SEM Acquisition tab and the Processing tab are available only. On the SEM Acquisition tab you will find the Shuttle & Find tool which has 3 additional buttons at the lower part of the tool.
Selecting the Sample Holder
This step is exactly the same step like for the light microscope, so please read the chapter Selecting the Sample Holder if you want to know the exact steps which you have to perform.
Calibrating the Sample Holder
Like the step before this step is exactly the same like for the light microscopy, so please refer to the chapter Calibrating the sample holder for details.
- The calibration of the sample holder has to be done on both systems the LM and the SEM. Otherwise the relocation of your sample positions or ROIs/POIs stored in the image won`t be successful.
- Note that for Shuttle & Find the beam shift must be switched off. The beam shift is deactivated in SmartSEM as follows:
- Call up the shortcut menu Center Point/Feature by right-clicking on the Stage property page.
- Select Center Point/Feature and select Stage only.
Acquiring an EM Image
- Load your LM image to ZEN SEM (.czi).
- The image will be displayed in the center screen area.
- Activate the Live mode.
- You will see the Live image from the SEM. Notice that all settings for the SEM image have to be done within the SmartSEM software.
- Activate the S&F View in Center Screen Area.
- Go to the S&F tab.
- Check if the Double click in image to move stage and Show splitter view checkboxes are activated (default setting).
- In the left image container you see the live image from the SEM. The right image container is empty.
- Drag the loaded LM image from the Images and Documents gallery into the empty image container.
- Now you can easily relocate sample positions by double clicking within the image or on the ROI/POI button (if ROI/POI are drawn in and selected) on S&F tab.
- For image acquisition you have to use the Snap button within ZEN SEM. Notice that we will not describe setup and image acquisition with the SEM. Please read the online help or user guide for the SEM software.
Fine Calibration of the Sample Holder
The precision of relocation can be improved by determination of an offset value. This value describes the position offset between the loaded image and the live image. The defined offset value is only valid for the loaded image. If another image is loaded or if you close the dialogue, the offset value will be deleted.
- An offset is visible when you try to relocate marker positions on the live image comparing to the LM image.
- Click on the Set Offset button.
- The stage moves to the selected marker position. Then a message appears which asks you to move the stage to the correct position.
- Move the stage manually to the correct position by using the joystick.
- Confirm the message by clicking on the OK button.
- Now you can repeat the relocation. The positions should be identical now.