Last Updated: 2024/05/27

Stellaris-STED Confocal Microscope Instructions Manual

Starting the System

1. Power On the Controller:

   • Turn on the power switch on the left side.
   • Turn on the laser switch.
   • Turn the key to the "on" position.

2. Log In to Bookit booking System:

   • Use your credentials:
     • Username: Your email address.
     • Password: Your email address password.
   • Verify that your booking is approved. Contact a facility member if your appointment status is pending.
   • Click "Log In" and then click "Activate Service."

Configuring the Microscope

1. Select Configuration:
   • After logging in, wait for the dialogue box to appear.
   • In "Configuration" choose: Machine.
   • In "Microscope" choose: DMI8.
   • Click "OK."

Caution

Ensure the objective is set to the smallest magnification (10X) to avoid damage to the microscope.

2. Motorized Stage Setup:

   • When prompted about the motorized stage, click "Yes" to enable X & Y movement.

3. Software Interface:

   • The interface will load in about 1-2 minutes.

   • The interface is divided into widgets:

     • Left Side Widgets: Scan mode (Left) and Z stack (Right) settings.
     
     • Right Side Widgets: Objective selection (Left), laser, and detectors settings(Right).
     

Setting Up for Imaging

1. Verify Objective Magnification:

   • Ensure the microscope is set to 10X magnification. If not, inform the staff and change it.

2. Detector Settings:

   • Detectors: HyD S & HyD X.
     • HyD S: Efficient for UV to yellow spectrum.
     • HYD X: Efficient for yellow to far-red spectrum.
   • Use the fluorophore panel to assign fluorophores to the appropriate detectors by dragging them from the search results.

3. Turning On the White Light Laser (WLL):

   • When prompted, click "Yes" to turn on the WLL (White Light Laser).
   • Ensure no emission spectrum overlap to avoid crosstalk.

4. Default Parameters:

   • Lasers: 5%
   • Detectors: 10%
   • Adjust settings as needed based on your experimental requirements.

5. Image Acquisition Settings:

   • Avoid taking too many or too few pixels to ensure manageable image sizes and useful resolution for analysis.
   • Your object of interest (e.g., nuclei) should be defined with 5-10 pixels in each axis; you can adjust the pixel size accordingly in the scan mode widget (by increasing pixel density or by switching to a higher mag. objective).
   • Use the "Projects" tab to properly name your files, including dates, magnification, and fluorophores used.

Tip

Name your project as YYYY-MM-DD-your-project-name. In case you acquired multiple images (nImages>10), it is recommended to split the .lif project to multiple .lif files that contain up to 15 images. In case of missing data, you don't lose an entire dataset. You can start naming your images while acquiring them. It is a good way to avoid confusion at the end of the session.

Important

Save images to the image analysis server: Z:/projects/your_lab_folder

Shutting Down the System

1. Save and Close Files:

   • Save all your files.
   • Right-click on the project line and select "Close All."
   • Reset acquisition parameters to default.
   • Delete detector settings by clicking the "X", reset (right-click) or trash button.

2. Clean and Remove Sample:

Caution

Ensure the objective is set to the smallest magnification (10X) to avoid damage to the microscope.

• Remove your sample and clean the microscope room.

3. Close Software and Log Off:
   • Close the software (this may take up to 2 minutes).
   • Log off from Bookit system.

Important

Log off only when you are sure everything is properly closed and your data is saved in your folder.

4. Turn Off the Controller:
   • Switch off the key.
   • Turn off the laser switch.
   • Turn off the power switch.

Following these instructions will help ensure proper use and maintenance of the microscope, as well as facilitate smooth transitions between users.