TITLE: Insert Alignment ELYRA7
AUTHORS: Rafael Camacho

[DESCRIPTION]
Alignment of the stage insert to ensure that the sample is perpendicular to the optical axis. This protocol is based on material provided by ZEISS' application specialists. 

What is needed before to start:
    - This protocol was designed for the ELYRA7
    - You will need 2 microscopy slides, one of standard size and one of about 2x2cm
  

[STEPS]
SECTION: Find sample plane via the eye pieces
2. 
 
ZEN black -> Locate Tab,
  
and set an imaging protocol for transmitted light so you can see the sample via the eye pieces. 
  
 


SECTION: Find sample plane via the eye pieces
3. Change the objective to the lowest magnification, typically 10X
  

SECTION: Find sample plane via the eye pieces
5. Place the marker-slide on the sample holder so you can focus on the appropriate sample plane
  
SECTION: Find sample plane via the eye pieces
6. Focus on the sample via the eye pieces
  
SECTION: Place glasses for reflection mode
7. Remove the sample holder carefully and place it a side
  
SECTION: Place glasses for reflection mode
8. Remove the objective lens and carefully place it a side
 
SECTION: Place glasses for reflection mode
10. Place the 2x2cm clean glass plate on the objective turret. 

 

  

SECTION: Place glasses for reflection mode
11. Place back the sample holder (which still has on it the marker sample)
  
SECTION: Acquisition Settings
13. Go to ZEN black -> Acquisition Tab
  

14. Set the aquisiton settings

15. Load the acquisiton settings by using the Experiment Manager
In our case these settings are stored as "insert alignment"
SECTION: Image Acquisition
18. While in Live mode, carefully take out the fibre from the HXP that is connected to microscope body until you see the sharp edges of the fibre in the field of view
  
SECTION: Image Acquisition
16. Go to Live mode and make sure image is not saturated
  
SECTION: Image Acquisition
19. Use the levelling screws of the holder to match the 2 sets of reflections to one another  

SECTION: Image Acquisition
20. Take an image snap, and save the image for your own record
SECTION: Place objective back into the turret
22. Remove the sample holder carefully so you don't touch/move the levelling screws

 

SECTION: Place objective back into the turret
23. Remove the glass slide placed at the base of the objective port
SECTION: Place objective back into the turret
24. Place back the objective
SECTION: Place objective back into the turret
25. Place back the sample holder

 


SECTION: Place glasses for reflection mode
12. Close the imaging chamber
  

16. Go to Channels, and set the exposure time to a short value, such as 10ms, make sure not to over expose your camera.
SECTION: Find sample plane via the eye pieces
4. Prepare a clean microscopy slide having a few marker points, marker-slide. You can keep track of the surface on which you made the marks by writing TOP and DOWN
 

  

SECTION: Place glasses for reflection mode
9. Prepare a clean microscopy slide of size aproximately 2x2cm
  
15. Example imaging setup:
  
18.    and select you have settings

17. Save the current experimental settings via the Experiment Manager
SECTION: Image Acquisition
17. Find where the fibre of the HXP is connected to the microscope body. This will be on the left hand side of the microscope's body, after the on/off button
  

SECTION: Image Acquisition
21. Place back the HXP fibre into position so you do not see the edges of the fibre while in live view
SECTION: Turn on the microscope
1. Turn on the ELYRA 7
SECTION: Turn on the microscope
1.1. Turn on Main Switch
  
SECTION: Turn on the microscope
1.2. Turn on Components
  
SECTION: Turn on the microscope
1.3. Turn on the Microscope Computer

 

SECTION: Turn on the microscope
1.4. Open ZEN black, and choose start system