Metamorph Journal for Time-Lapse Microscopy in multi-well plates

(to Move to desired positions, Auto-focus, Take pictures, and Save)

  1. Place the plate on the microscope stage and set up Koehler illumination as usual.
  1. 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.

  1. 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.

  1. 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.

  1. 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.

  1. 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

  1. After all the images are taken and saved automatically, you need to collect images for the different positions in the different folders.
  1. Then build stacks for the different positions, and save!

SYK 11/9/01