T517_ImagingManual

1. 1. Cut the expanded gel to fit the imaging chamber using a razor blade, scalpel, or a similar tool.
2.      ※For example, when using a 35‑mm dish, dividing the gel (approximately 40 mm in diameter) into four pieces allows up to four imaging sessions from a single gel.
3. 2. Transfer the cut gel using a spatula, taking care not to damage it.
4. 　※Using a micro-spatula with a slightly bent tip on the handle can make handling easier.
5. 　 Example: When using a 35‑mm dish as the imaging chamber and dividing the gel into four equal parts
        
6.  *When using an oil‑immersion objective, you may need to slice the gel. Handling becomes easier if the gel is pre‑trimmed to a smaller size.

Cut the expanded gel

1. 1. Using low magnification (10×) and a digital zoom (0.5×), locate the nuclei from the bottommost plane using the 405‑nm laser for DAPI.　
   ※ Because fluorescence becomes weaker compared to the pre‑expansion state, increase the gain during imaging.　Example: Using an oil‑immersion objective on a Zeiss LSM800. Pre-expansion: Laser 1–2%, Gain 550–650 V; Post-expansion: Laser 20–80%, Gain 700–800 V.
    
2. 2. Once the nuclei are located, reset the digital zoom to the normal setting (1.0×) and confirm that the nuclei appear enlarged.
    
3. 3. Switch to a higher magnification (20× or higher) and locate the nuclei again (this step is essential because changing magnification causes a shift along the Z‑axis).
   
   While visually confirming the distance between the objective lens and the imaging chamber, gradually increase the magnification (e.g., from 1× to 10×)
   
   If the objective does not contact the imaging chamber even at higher magnification, proceed as is.
   ※ If the objective lens comes into contact with the imaging chamber, refer to “③ Supplement: When the lens contacts the imaging chamber.” 
    
4.  4. Once the nuclei are located, switch to the laser appropriate for the target structure and search along the Z‑axis near the identified nuclei.
   ※ If the target structure cannot be located, refer to “③ Supplement: When the target cannot be found.”
    
5. 5.  Perform imaging.
   ※ If the gel moves and imaging cannot be performed, refer to “③ Supplement: When the gel moves and imaging cannot be performed.”

When the lens contacts the imaging chamber:

1.  
2. 1. Flip the gel carefully
   　
3. ・Carefully flip the gel within the imaging chamber using a spatula or a similar tool.
4. ・Take care not to tear or damage the gel during this process.

5. Examples of cases where cells in the gel cannot be imaged due to their position, and corresponding countermeas

6. 

7.  
8. 2. Slice the gel in half (if the gel still contacts the imaging chamber after Step 1)
    
9. ・Transfer the gel from the imaging chamber onto a separate flat surface, and slice it near the center using a razor blade, scalpel, or similar tool.
10. ・Place the sliced gel back into the imaging chamber with the cut surface facing downward, and then perform imaging.
11.  
12. 　Case: Cells are located near the center of the gel and cannot be imaged even after flipping — how to resolve it   
         
13. When the target  cannot be found:
14.  
15. 1. Increase the microscope's laser power and gain during imaging.
16. 　※Example (using an oil‑immersion objective on a Zeiss LSM800): Pre-expansion: Laser 1–2%, Gain 550–650 V /Post-expansion: Laser 20–80%, Gain 700–800 V
     
17. 2. Perform additional staining (if the target still cannot be observed even after increasing the laser power and gain in Step 1).
18. 　※For details on the post‑staining procedure, please refer to the supplementary notes under [Step 24: Fluorescence Observation] in the instruction manual.
19.  
20.  
21. When the gel moves and imaging cannot be performed
22.  
23. ・If water remains between the imaging chamber and the gel, the gel may slip. Wipe off excess moisture with a Kimwipe or similar material before imaging as needed .
24. ・If a significant amount of water is present, remove it using an aspirator or a micropipette, taking care not to aspirate or damage the gel.
25.     ※You may also place a Kimwipe or similar material between the imaging chamber and the gel to increase friction and reduce gel movement during imaging.
26.  
27. 　
        To prevent the gel from shifting, place Kimwipes around its perimeter.