Always tick 'Save Sequence to Disk' and set a path under 'Directory' (ideally save it directly to the Hive or Fileshare). Only tick 'Separate Files' if you want to save each recording individually (meaning each color channel etc.). **Important:** Sometimes the box next to 'Save Sequence to Disk' or 'Separate Files' disappears (which is a bug). Solution: untick 'Time-lapse', 'Wavelength', 'Z-series' and 'Stage positions', then you should see it again. Tick the box next to 'Save Sequence to Disk' or 'Separate Files' and then tick again 'Time-lapse', 'Wavelength', 'Z-series' or 'Stage positions'. Even if the box disappears again now, it will remain active.
Tick Time-lapse and choose your number of time points, your time interval or your duration. You only have to choose two of them and the third one will adapt automatically. If you are only doing a time-lapse (no other wavelengths, z-series or stage positions), you can also tick 'streaming' for a faster recording. The time interval is only the waiting time between two time points, after which an image is recorded and needs to be read out from the camera chip (so there will always be a couple of 10-100ms added to your time interval, which you can read out from your metadata). If you set a time interval of 0 the recording will be done as fast as possible (meaning the time interval is then only the illumination time plus the readout time of the camera - multiplied by the number of wavelengths, z-planes and positions, depending on your experiment: also switching in between wavelengths or moving to different z-planes or positions takes time!). If you set a time interval >0 then it will automatically be increased, in case you have chosen it too short for your number of wavelengths, z-planes and/or positions. If you still want to further decrease your time interval, you have to reduce your number of wavelengths, z-planes and/or positions per time point.
In the Time-lapse tab you can also tick Autofocus and FRAP (for a detailed explanation see further below).
@@ -170,9 +191,11 @@ To use a different offset, turn off the autofocus, focus manually on a different
...
@@ -170,9 +191,11 @@ To use a different offset, turn off the autofocus, focus manually on a different
#### B. Stage positions with multiple offsets
#### B. Stage positions with multiple offsets
The following procedure describes the setup of a multiposition time series with multiple offsets (i.e., positions may have different z-coordinates above the cover glass).
The following procedure describes the setup of a multiposition time series with multiple offsets (i.e., positions may have different z-coordinates above the cover glass).
1. In the Time lapse tab, check "Time-lapse series" and the "Autofocus" option. Click the "Config" button right of the "Autofocus" option
1. In the Time lapse tab, check "Time-lapse series" and the "Autofocus every x time points" option ("every 1 time points" is recommended for multiple stage positions. **Important:** Sometimes the box 'every x time points' disappears (which is a bug). Solution: untick 'Time-lapse', 'Wavelength', 'Z-series' and 'Stage positions', then you should see it again. Change the value in 'every x time points' to 1 and then tick again 'Time-lapse', 'Wavelength', 'Z-series' or 'Stage positions'. Even if the box disappears again now, it will remain active.
Click the "Config" button on the right of the "Autofocus" option
2. In the "Configure AutoFocus" dialog, check "Hardware Autofocus" and "use Continuous Focus". For very distant stage positions, additionally check "during stage move". Close the dialog ("x" on the right upper corner)
2. In the "Configure AutoFocus" dialog, check "Hardware Autofocus" and "use Continuous Focus". For very distant stage positions, additionally check "during stage move". Close the dialog ("x" on the right upper corner)
