Hi,

Don't know if the following is off any help at all [or whether you are
asking a Leica SP5 stage software specific question], but I this response to
similar query a while ago and it may or may not be useful [i.e. I always
found it a good idea to visually check what the software is doing physically
with the stage, and the following was how I generally did it]:


" I know it's going back 15 years since I regularly used a motorised stage*
on a standard microscope [other than a PALM micro-dissection system], but I
always used my England Finder to ensure that the stage origin 0,0 was always
in the same place - assuming the slide & slide holder is fixed/moved to the
same point on the stage. The England Finder slide has a complete reference
grid etched across its surface. I always aligned the stage origin to a
specific point, e.g. a cross or the tip of a letter/number. It's nice to see
the 0,0 origin co-ordinate always confirmed as the same place on-screen
visually [and you can use it to confirm co-ordinates of objects of interest,
although you have to take the specimen slide out and replace it with the
England Finder slide]. Our main Magiscan image analyser microscopes had 8
and 16 slide stages so things got complicated. The software allowed us to
define any point as the stage origin. Our Magiscan software could even
optionally report stage positions in England finder co-ordinates. You can
buy slide cover-slips and Mattek dishes with these grids etched on them as
well, but only the England Finder has them etched over the entire slide. 

Once our stage origin 0,0 was sorted out though things were fine. You can
use the England Finder to quickly re-locate specimens on different
microscopes, once you calculate the conversion factors for different
motorised/manual stages. I suppose you could make your own 0,0 reference
point using fixed features on any slide, but the entire etched grid is
really useful. Although it's easier to set the 0,0 stage origin within the
visible slide area, assuming the software allows this [ours always did], you
can use the Finder to check the reproducibility of the position of other
stage co-ordinates and that the field raster scan is moving correctly. 

Only downside is that the England Finder is fragile and costs about $250,
although both mine came free from a drawer in a lab millennia ago, so you
might find one lying about [and twenty five years on mine still aren't
chipped].

See one at:
http://www.2spi.com/catalog/magnifiers/england-finder-graticule-instructions
.html

http://www.2spi.com/catalog/magnifiers/england-finder-graticule.shtml

I used to calculate things like exact field sizes etc.. for snake and raster
scans to ensure the software gets it right. So you need the odd calibration
graticule slides as well [1mm/1,000 scale probably, with a 200um one also
useful]: 

http://www.2spi.com/catalog/ltmic/stage-micrometers-graticules.shtml

I used to stick a physical rectangular field as 'lines' [Letraset I think]
on the screen to align fields in raster scans, but I'm sure software could
do it these days. Our systems were all image analysers so checking
distances, etc. was easy [I expect your SP5 can display lengths, and it's
already been calibrated by Leica]. It's also helpful to see a bit of the
area outside the field on-screen to ensure the next field is exactly aligned
during raster scans [and to see where things in the field go]. "




Hope this is of some help [and I haven't misinterpreted your question]. Plus
I'm not familiar with the SP5 stage or its software driver options, & some
offer more control than others. But the above worked for me with our setups.
Our UCL PALM system set it's own stage origin automatically in
hardware/software, but it's motorised stage was pretty unique [and
expensive] as it can drive diagonally [for laser cutting] as well in
'straight' steps in X & Y.  

Regards

Keith

*Back in the 80s every microscope I bought for my personal research use had
a motorised stage, but collective decisions for core facilities have led to
most microscopes where I've worked since being supplied with manual XY
stages. A mistake in my opinion, particularly as they are discounted when
included in the original tender and very expensive to add afterwards on a
limited operating budget.

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  +44 (0)1865 287568
Email:  [log in to unmask]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
 
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Adrian Smith
Sent: 10 February 2009 23:01
To: [log in to unmask]
Subject: Re: Leica SP5 question - Motorised Stage

On 10/02/2009, at 4:59 PM, Cameron Nowell wrote:

>  To be able to do XYZT acquisition with multiple fields your
> scope needs to be fitted with a motorised stage. If you have that then
> you will be fine.


While we are talking SP5 and motorised stage....

I've been wondering if anyone has a come up with a good procedure for  
setting up the calibration of the stage position, ie in the Stage  
Configuration window there are settings for Tile Scan overlap, origin  
offset sizes, merge settings that need to be set up in order for tile  
scans to work.

One of the students here got them quite close through a laborious  
trial-and-error process. Unfortunately, we need to reset them because  
the stage alignment got messed up at one point (when the stage cable  
got caught in the opening into the incubation enclosure).

Before I go through the process of doing it by trial and error or  
working out a more systematic way of doing it I was wondering if  
anyone else had any suggestions as to how best approach it?

Regards,

Adrian Smith
Centenary Institute, Sydney, Australia