I’m trying to use MM 2.0.0 beta3 to do triggered time lapse z stacks with our spinning disk, TiE PFS and ASI MS-2000 piezo z stage.
Here is what I expect to happen when setting the “CoreFocus” motor as “Z stage”:
1. -Lock focus on the center of the specimen with PFS
2. -Use “Relative z” to set a range around that focal plane, i.e. “Start z” = -5 um and “End z” = 5 um for a 10 um z stack.
3. -PFS should turn off, piezo move to –5 um, acquire the 10 um z stack with the piezo, PFS relocks at same center position, turns off for next z stack, piezo moves to -5, etc., repeat.
4. -The PFS offset position is always used as the center of the z range
What I observe is different. The above works for the first point in the timelapse. PFS turns off and a 10 um stack is acquired with the sample in the center and then PFS turns back on. However, for the
second and subsequent z stacks, after PFS turns off each time there is movement of the Ti motor to -5 um, then a -5 to 5 um z stack is acquired with the piezo. But that -5 um shift of the Ti motor moves the focus for a total of -10 um so that the sample is
now shifted to the end of the z range.
Is my expectation of how autofocus is integrated into the acquisition wrong? Why would it behave one way for the first time point but different for the rest? I should point out that this problem only happens
with the piezo set as the CoreFocus. When the Ti motor is used it works as expected. Any insight is appreciated.
Yes, If I uncheck "Autofocus," center the sample with the Ti motor, and then use the piezo to section a relative range from -5 to 5 um around that it works perfectly for each time point and there is no change to the Ti motor position.
Chris O'Connell, PhD
Director, Advanced Light Microscopy Facility
University of Connecticut
Department of Physiology & Neurobiology
75 North Eagleville Road, U-3156
Storrs, CT 06269-3156
[hidden email] www.confocal.uconn.edu
From: JonD [mailto:[hidden email]]
Sent: Friday, March 16, 2018 1:34 PM
To: [hidden email] Subject: Re: [micro-manager-general] Autofocus with triggered piezo z stack
Chris O'Connell wrote
> ... after PFS turns off each time there is movement of the Ti motor to
> -5 um ...
It seems to me that this is crux. Can you make the Ti motor stay put when PFS turns off?
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