Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Quoting Ian Harper <[log in to unmask]>:

> pauline yu wrote:

> > 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.

Most MRC 600s have a reflection filter block, so as Ian says below you
can do it in reflection.  If you don't have that you can use the old
fibre-optic transmitted light device to measure it in transmission
mode in detector 2.  Since it is the scan you are measuring this is
just as accurate.

> > 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?

So long as you have recorded the pixel values - with the old generation
of Bio-Rad machines you cannot trust 3rd-party software to read the
image dimensions correctly from the file header.  So long as you keep
your image at 512 x 512 or 768 x 512 as originally collected these
cannot change.

> > (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.)

Unless you have a very old version of the software you can save the
scale bar into the image BUT you have to do it in MPL.  I'm afraid
I'm 10,000 miles away from my old MRC 600 and 500 and I can't recall
the command from memory but maybe someone else can help out here?

and Ian replied:

> 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 !].

I've also had the wrong scale factor entered into the software by
an installation engineer who just assumed that a Zeiss Axiophot would
be the same as an Axioskop (it isn't)!

> 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 ?

Use a mirrored coverslip (preferably with a few scratches for ease
of finding focus).  You'll be able to get focus with this to within
a small fraction of the actual Z-resolution so it is very accurate.
Focus up and down and see if bringing it back to the original displayed
value brings it back to focus.  (It won't, but you can find out how
far off it is!)

To check the accuracy of the z-steps you ideally need a sample with
known steps on it - these are expensive so try to borrow one before
you think of buying.  If you know anyone with a tally-step type of
profilometer they'll probably have one.

If you can't get one you can check against the focus control on the
microscope, which is usually calibrated.  Or you can use 5 or 10 micron
fluorescent beads - they should appear the same in Z as X & Y.

For a very cheap but really quite accurate measurement use a slide
which you've raised at one end (by a known amount, of course) and
measure the lateral shift in the reflection line of focus as you
focus up and down.

                                                          Guy

--
Associate Professor Guy Cox
Electron Microscope Unit, F09
University of Sydney NSW 2006
+61 2 9351 3176

Until 25th July:
Instituto Gulbenkian de Ciencia, PT-2780-156, Oeiras, Portugal.
+351 21 446 4638 (office) or +351 91 401 5726 (mobile)
Fax: +351 21 440 7970