Metamorph Journal for Time-Lapse Microscopy in multi-well plates
(to Move to desired positions, Auto-focus, Take pictures, and Save)
- Place the plate on the microscope stage and set up Koehler illumination as usual.
- Memorizing different positions in the plate:
On Metamorph, go to >Devices>Stage>Move Stage to Absolute Position.
Click Memory List , and it will open the memory list.
Move to the first desired position in the well and focus.
Click Memorize, and give it a name.
Repeat for all the positions you want to observe.
- Edit Z-positions for all the memorized positions: (While memorizing positions, z-positions shift a lot, and the memorized positions in Step 2 can get out of focus. Therefore, you need to check all the positions again, re-focus, and edit z-positions of all the memorized positions.) I find that when this is done 2-3 times, shifts in z-directions become smaller.
Move to each memorized position, re-focus, and click Memorize, again.
At the same time, you can test Auto-Focus , to choose the best Number of Steps, Increment, and Range for your study (In Sooyoung’s case, 100, 0.3, and 15 (respectively) worked fine.)
On Metamorph, go to Auto-Focus: >Devices>Auto-Focus Via Software>Adjust Focus.
- Recording Journal.
Open all the windows needed to record the journal (Memory List and Adjust Focus)
Set up sequential file names you want to save the images with: Go to >Files>Set-Up Sequential File Names,
Begin recording the journal.
①Go to >Journal>Start Recording. All the recordable keys turn blue.
②Select the first memorized position on Stage Memory List, and click Move.
③Open Bright Field : Opens bright field
④Auto-Focus : Focuses on the object.
⑤ACQ-BF: Acquires bright-field image.
⑥Ctrl-u. Saves with the sequential file name you chose in a previous step.
Stop recording the journal: Go to >Journal>Stop Recording, and save the journal.
- Edit the journal to write journals for all the positions.
①Go to >Journal>Edit Journal.
②Select all the entries appearing on the left side of the window.
③Copy from Journal
④Paste to Journal
⑤Repeat paste for the number of positions you have.
⑥Edit stage position entries: Click each “Stage (Move to Description),” from journal entries. Enter your next positions, and click Move.
⑦Save the edited journal.
- When running the journal for every interval of your choice, loop the journal by:
>Journal>Loop>Loop a journal.
Press to select the journal to loop, and choose your journal.
Enter # of loops, interval, and Continue
- After all the images are taken and saved automatically, you need to collect images for the different positions in the different folders.
- Then build stacks for the different positions, and save!
SYK 11/9/01