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pauline yu wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Listers,
> Ms. Foster and Ms. Puhalla bring up two good points about calibration that I've been wondering about myself. While using a stage micrometer is the best way to calibrate measurements of objects in brightfield, how can one do this for confocal? I don't know of any "fluorescent micrometers" and I'm not sure how I would capture an image through the detector in brightfield. Alternately I suppose one would need to have a camera port parfocal with the confocal imaging plane, but I don't have that on my old MRC-600.
>
> So am I stuck with using pixel dimensions? I assume this in invariate for the raw files, but when we open the files in other programs (ImageJ, Confocal assistant etc.) will this always be invariate before we perform any transformations?
> (Additionally I can't get the on-screen scale bar saved onto the image files--I was told to make a printout of the scale bar for reference.)
>
> Thanks!
> Pauline Yu
> [log in to unmask]
> Graduate Student
> Manahan Research Lab

Pauline, Barbara and others,

Regarding calibration with a stage micrometer:

Yes, indeed an important issue, as we all assume the instrument is
correctly calibrated when it arrives in our lab, but over the past 10
years I have experienced at least two instruments (different companies,
different continents) with galvo scanning systems "not perfectly"
calibrated ! [One was out by as much as 10% in one direction !].

Hence I would advocate checking linear measurements against a stage
micrometer, and to do this for both X and Y directions if you have a two
mirror spot scanning system (No, I don't have shares in a graticule
manufacturing company). Incorrect calibration leads not only to
incorrect measurements, but also drifting of the zoom area as you zoom
up (but the two are not necessarily co-dependant). Setting the
potentiometers that control the galvo systems was very simple for my
systems, (and may even be software controlled in more modern systems for
all I know). However, I would generally caution that anyone doing this
should do so in conjunction with a company service engineer.

You can easily check the calibration by using a stage graticule, which
is imaged in reflection mode on the confocal microscope (hence you don't
need a fluorescent sample). You would need to perform the calibration
for both the x and y directions by simply rotating the stage micrometer
(assuming your scanning system has independant x and y galvos).

Question: how do people calibrate the Z axis, or do we all assume the Z
stage devices are in perfect working order regarding (1) accuracy, and
(2) repeatability ?

Ian.


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Dr Ian S. Harper, Director
Monash Micro Imaging
The Microscopy & Imaging Research Facility
Building 13C, Monash University,
Clayton, VIC 3800, Australia

Email: [log in to unmask]
Tel: +61 3 9905 5635;  Fax: +61 3 9905 2766
Mobile: 0408 314168

General enquiries: [log in to unmask]
http://www.microimaging.monash.org
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