1. On the microscope, gently push the transmitted light arm backward

2. In NIS-Elements, click Escape Z to move the objectives to a safe position

3. If not done already, click on the lowest magnification objective

   The lowest magnification is the safest to use due to its long working distance: the sample will appear in focus well before the objective lens gets close to it. It is recommended to always perform the initial focusing with the lowest magnification objective. Since the objectives are parafocal, focusing with le safest objective will make it easier to locate the sample when switching to higher magnification objectives

4. Place the test slide on the microscope stage, with the coverslip toward the objective
   

   Using a test slide will significantly reduce the time needed to set up the instrument

5. If necessary, use the joystick to move the stage so the sample is centered under the objective

6. Gently return the transmitted light arm to the vertical position

7. In NIS-Elements, click Escape Z again to return the objectives to their normal position

8. Click on the desired optical configuration (BF, DAPI, GFP, etc.)

9. Click Live (») to activate the light and display the camera image

10. Adjust the light intensity and exposure time to obtain a well-exposed image

11. If necessary, use the image autoscaling to adjust the display settings

12. Adjust the focus until the image is perfectly sharp

13. Click Stop to stop the Live or on Off to turn off the light

It is possible to scan an overview image to then easily navigate your sample. It is strongly recommended to use the lowest magnification objective and whenever possible use the BF optical configuration not to bleach your sample.

After completing the initial focus:

1. In NIS Elements, click on the desired optical configuration (BF, DAPI, GFP, etc.)

2. Click Live (») to activate the illumination and display the camera image on the screen

3. Adjust the light intensity and exposure time to obtain a properly exposed image

4. If necessary, use the image autoscaling to adjust the display settings
5. Using the Joystick navigate to the top left corner of your sample
6. In the the XYZ overview click + to add a point and save this position
7. Using the Joystick navigate to the bottom right corner of your sample
8. In the the XYZ overview click + to add a point and save this position
9. In the XYZ Overview right click and under Large Image Area select Define Area
10. Draw a rectangle around the two points
11. Right click again on the created region of interest and select scan preview
12. Wait until the preview acquisition is completed

You can now directly click on the overview to navigate your sample !