Re>Quantifying Z motion of my home-brew stage

[Richard Cole]

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-----Original Message-----
From: Rob Campbell [mailto:[hidden email]]
Sent: Saturday, July 13, 2013 10:52 AM
Subject: Quantifying Z motion of my home-brew stage

 

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Good day, microscopists. I wonder if you can help me quantify how smoothly
my stage is moving?

 

I've built a 3-axis stage around my 2-photon using breadboards and
translation stages (e.g. Thor PT1). I am actuating the stages with
micrometers. All three axes will be motorised by coupling the micrometers to
stepper motors. At the moment I've only motorised Z, to check that
everything works roughly as planned. My immediate concern is quantifying how
smooth the motions are in all three axes. I've not done anything like this
before, so I wanted to bounce ideas off you more experienced people.

 

The steppers won't have encoders, since I can't be certain these would
reliably convey what's happening at the stage. Quantifying smoothness in X/Y
should be simple: I'm thinking of translating the X/Y stage at a constant
speed whilst imaging a target (e.g. pollen grains). Since I know how many
microns per pixel there are, I can extract velocity and hence jerk in real
units. I'd base measurements on the whole image, so imaging-related noise
should have a negligible impact.

 

I'm less sure what to do about z motion, however, since targets will enter
and leave the image plane. Perhaps fluorescent nano-beads are the way to go?
Using these I could measure PSFs in Z. If I move at a constant speed in Z,
intensity as a function of time should produce a Gaussian. Maybe I can use
the known PSF of the objective to figure out velocity in real units? One
problem might be that, since the target is small, imaging noise could
contaminate the velocity measurements. Anyone have suggestions on this
procedure or ideas for better targets?

 

Thanks!

 

 

--

Rob Campbell

turnerlab.cshl.edu

 

 

 

Bob,

 

Measuring the PSF has work well for me in the past; if the motor is "jerky"
you will see it clearly.  Additionally, if you recollect the same bead twice
you will have any idea about the repositionability.

 

Happy imaging...

 

Rich

 

 

Richard Cole
Research Scientist V
Director: Advanced Light Microscopy Core Unit
Wadsworth Center

 

Research Assistant Professor
Dept. of Biomedical Sciences
School of Public Health State University of New York


P.O. Box 509 Albany N.Y. 12201-0509
518-474-7048 Phone
518-474-4430 Fax

 

Email  <mailto:[hidden email]> [hidden email]

Website  <http://www.wadsworth.org/cores/alm/index.htm>
www.wadsworth.org/cores/alm/index.htm

 

 

[Craig Brideau]

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I have done the same thing as you using high-torque stepper motors. A
sufficiently strong motor will always step and thus remain accurate. I was
getting <1µm xyz position repeatability with my rig. As long as the load of
your stage and sample is less than the load of the stepper motor you should
be OK. I wrote some LabView code to control mine with a playstation
controller and the users are quite happy with it. as for stability, just
get a sample with lots of fine features, pick several regions at different
×yz locations, and then image them over and over. Compare the pictures and
if they look identical after numerous visitations you should be good to go.
You can quantify this if you need to by doing a correlation of the multiple
pictures. The better the correlation, the better the repeatibility
(relative to small features in your image). If you need 3-D, do Z-stacks
and 3-D correlate them. Note that the smaller your sample is (feature-wise)
the better this will work.

Craig
On 2013-07-15 5:27 AM, "Richard Cole" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> -----Original Message-----
> From: Rob Campbell [mailto:[hidden email]]
> Sent: Saturday, July 13, 2013 10:52 AM
> Subject: Quantifying Z motion of my home-brew stage
>
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
>  <http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
>
>
> Good day, microscopists. I wonder if you can help me quantify how smoothly
> my stage is moving?
>
>
>
> I've built a 3-axis stage around my 2-photon using breadboards and
> translation stages (e.g. Thor PT1). I am actuating the stages with
> micrometers. All three axes will be motorised by coupling the micrometers
> to
> stepper motors. At the moment I've only motorised Z, to check that
> everything works roughly as planned. My immediate concern is quantifying
> how
> smooth the motions are in all three axes. I've not done anything like this
> before, so I wanted to bounce ideas off you more experienced people.
>
>
>
> The steppers won't have encoders, since I can't be certain these would
> reliably convey what's happening at the stage. Quantifying smoothness in
> X/Y
> should be simple: I'm thinking of translating the X/Y stage at a constant
> speed whilst imaging a target (e.g. pollen grains). Since I know how many
> microns per pixel there are, I can extract velocity and hence jerk in real
> units. I'd base measurements on the whole image, so imaging-related noise
> should have a negligible impact.
>
>
>
> I'm less sure what to do about z motion, however, since targets will enter
> and leave the image plane. Perhaps fluorescent nano-beads are the way to
> go?
> Using these I could measure PSFs in Z. If I move at a constant speed in Z,
> intensity as a function of time should produce a Gaussian. Maybe I can use
> the known PSF of the objective to figure out velocity in real units? One
> problem might be that, since the target is small, imaging noise could
> contaminate the velocity measurements. Anyone have suggestions on this
> procedure or ideas for better targets?
>
>
>
> Thanks!
>
>
>
>
>
> --
>
> Rob Campbell
>
> turnerlab.cshl.edu
>
>
>
>
>
>
>
> Bob,
>
>
>
> Measuring the PSF has work well for me in the past; if the motor is "jerky"
> you will see it clearly.  Additionally, if you recollect the same bead
> twice
> you will have any idea about the repositionability.
>
>
>
> Happy imaging...
>
>
>
> Rich
>
>
>
>
>
> Richard Cole
> Research Scientist V
> Director: Advanced Light Microscopy Core Unit
> Wadsworth Center
>
>
>
> Research Assistant Professor
> Dept. of Biomedical Sciences
> School of Public Health State University of New York
>
>
> P.O. Box 509 Albany N.Y. 12201-0509
> 518-474-7048 Phone
> 518-474-4430 Fax
>
>
>
> Email  <mailto:[hidden email]> [hidden email]
>
> Website  <http://www.wadsworth.org/cores/alm/index.htm>
> www.wadsworth.org/cores/alm/index.htm
>
>
>
>
>