Making multiple-position movies is really quite simple.  You use the “Multi-time Macro”, which stores the XY co-ordinates and focus for each position you select.  You can do Z-stacks at each position, or single images, or even do FRAP.  The macro can also perform autofocus if you want, and you can get really fancy and set up loops within loops…

Note that you need to use MULTITIME version 32.z15.  Earlier versions do not have the full range of functions or have bugs.  If you don’t have this Macro in your personal LSM log-in window, ask me and I’ll install it for you.

A. Simple multi-location time-lapse (no autofocus, no Z-stacks)

  1. Turn on the scope, turn on the heater for the stage, set up the lasers you need, etc, all as usual.
  2. Make sure to store the exact Configuration that you need for imaging – e.g., save the Config as “MyGFPConfig” or something.  Do it as Multi-track. This is important, because it stores the pinhole diameter, gain, etc – and the multi-time macro looks up this info for each position!
  3. Open the Acquire window.  Open the Stage window.  (Both listed in the banner at the top of the screen).  Move to the first position you want to image and focus on your cells.
  4. Click Z in Focus Position (Stage window, top panel); and click Zero in Stage Position (Stage window, middle panel); then click Mark in Stage Position.
  5. Using the XY stage joystick, move to the next position (#2) and focus on your cells.  Click Mark in the State Position panel.  Continue for as many positions as you want (up to 999!).  Xs will appear on the small window, showing the various locations you have marked.
  6. Open the Macro window (Listed in the banner at top of screen).
  7. Open Multitime.  (This should be version 32.z.15). Click Update (yellow smiley-face icon). Click on Multiple Locations, top right of panel.  All the positions you marked should be listed here.
  8. Set the number of Experiment Repetitions (i.e., the number of frames you want for the time-lapse movies); then under Block Parameters uncheck All Locs and enter the Wait Delay between scans for the time-lapse (e.g., 10 minutes).
  9. Make sure Autofocus and Bleach are unchecked!
  10. Set the Configuration – e.g., to “MyGFP Config” or whatever.  You need to be sure that this is the same for each position!! Set Number of Scans = 1.
  11. Click on Image Database and choose the folder in which you want to store your movies.
  12. OK, you are ready.  Click on Start Time icon (right icon list, near top).  Note that unless you are using the 10x objective the image is likely to drift out of focus quite quickly.

B. Using the Autofocus for multi-location time-lapse.

This is much easier than it might appear at first glance.  It works well.  You can do time-lapses for many hours and retain focus.   It works by detecting the reflection off the surface of the coverglass, then offsetting the z position by a calculated amount to bring your cells into focus.

  1. Set up the sample and microscope as in A, and open the Multitime macro.  It must be the 32.z15 version!  As I have set it up, this procedure uses the red (543nm) HeNe laser for the autofocus, so turn it on plus whatever other lasers you need.
  2. Find your cells, adjust the gain, pinhole, laser power, zoom, etc so you get a nice image – then SAVE the Configuration as a Multitrack, e.g., as MyGFPConfig.  This will save all the settings, to be used in the time-lapse.
  3. Open the Stage window, move to the first location, focus on the cells.  Click on Z to zero the focus; click on Zero to zero this XY location; click on MARK POS to mark location #1.  Now move around and find as many other locations as you want (up to 999) – focus on the cells at each location and click MARK POS.
  4. In Multitime click Update, to upload your locations.  Now make sure Autofocus is unchecked.  For location #1, load your Configuration – MyGFPConfig or whatever.  Switch to “Single Location” then back to “Multiple Location” using the buttons at the top of the window.  This will copy the Configuration parameters into each location.
  5. Now use the ‘Go to Position’ in the Stage window to return to location #1.
  6. In the Multitime window, make sure you are on location #1, then click on the Define Autofocus, check “Cnf” and Autofocus and load the Multitrack AUTOFOCUS Configuration. (If it does not exist on your configuration list, either ask Ian Macara or go to the notes on “Setting up an Autofocus Configuration”).
  7. The next step is to set up the autofocus. In the Stage window, make sure the Z position is zero.  In the Multitrack window, set the “Z Offset” value to zero.  Now run the Autofocus routine (button on right of Multitrack window).  You should see a new image window open, and a single horizontal red line should appear.  In the Multitrack window the bottom panel should turn green as it runs the procedure.  When it is finished it will turn yellow/brown.  Now check the Z value in the Stage window.  It should read a negative value (e.g., -8.4 um), because it is the focus position of the coverglass surface, below the cells.  Click on Define Autofocus in the Multitime window and type this value into the Z-Offset box.  This tells the microscope how far above the coverglass your cells are, in location #1.  OK, you are now ready.
  8. Choose the folder (Choose DB) in which you want the data to be stored.  You can choose a temp file as well if you want, otherwise it uses a default one.
  9. OK, now just set the number of Experiment Repetitions to whatever number of frames you want to acquire in the timelapse; set the Wait Delay in the Block Parameters to the time between acquisitions (e.g., 5 min).  If you want to save this whole process, hit SAVE/APPLY and give the setup a name.  Now, you can hit Start Time.
  10. It is best to now close all the windows except for the Multitime and the image window.  This prevents an error message from crashing the system after about 7 hours.  Go for coffee.



a.  If you want to do z-stacks at each position and time point, you need first to set up the number of sections (in the Acquisition window).  Then just click on the “z-stack” button in the Multi-time macro window.  That’s it.

b. Note that doing this will use up gigabytes of disk space!  Currently the disk on the Meta is too small to run major experiments.  We hope to acquire a bigger one soon.

c.  If you stop the timelapse early, by clicking on STOP instead of FINISH, the images will not be concatenated properly and moved to your database.  Instead they will be stored in the temp database.  You can concatenate them yourself using the CONCATENATE macro.

Just open the MACRO, then open the TEMP database and select the images you need concatenated (NOTE that there might be other images from previous experiments in the same DB!!!.  The process is pretty straightforward.

d.  Delete your files from the TEMP database when finished – they use up vast amounts of disk space.