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Stage positions from Zeiss LSM 710, Olympus FluoView 1000, Leica SP/SP5

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Ponti, Aaron

Stage positions from Zeiss LSM 710, Olympus FluoView 1000, Leica SP/SP5

Hello

We are investigating the purchase of one of the following confocals: Zeiss LSM 710, Olympus FluoView 1000, and Leica SP/SP5. One of the criteria for the choice is the possibility to read stage positions from the files (using the loci/ome-xml tools). With loci_tools 4.1 it seems that reading the stage positions into the OME schema is supported for the Leica, but is not for the other two. Can anybody confirm this? Does anybody know if stage positions are stored at all in .lsm and .oib files? How is reading metadata information in general for these file formats (like optical parameters)?

Thanks

---------------------------------------------------------------------
| Dr. Aaron C. Ponti
| Friedrich Miescher Institute for Biomedical Research
| Facility for Advanced Microscopy and Imaging
| Software development
| Maulbeerstrasse 66 CH-4058, Basel
| WRO-1066.2.16
| Tel: +41 61 696 3513
| Fax: +41 61 697 3976
| http://www.fmi.ch/faim
----------------------------------------------------------------------

James Pawley

Re: Stage positions from Zeiss LSM 710, Olympus FluoView 1000, Leica SP/SP5

Re: Stage positions from Zeiss LSM 710, Olympus FluoView 1

Hello

We are investigating the purchase of one of the following confocals: Zeiss LSM 710, Olympus FluoView 1000, and Leica SP/SP5. One of the criteria for the choice is the possibility to read stage positions from the files (using the loci/ome-xml tools). With loci_tools 4.1 it seems that reading the stage positions into the OME schema is supported for the Leica, but is not for the other two. Can anybody confirm this? Does anybody know if stage positions are stored at all in .lsm and .oib files? How is reading metadata information in general for these file formats (like optical parameters)?

Thanks

---------------------------------------------------------------------
| Dr. Aaron C. Ponti
| Friedrich Miescher Institute for Biomedical Research
| Facility for Advanced Microscopy and Imaging
| Software development
| Maulbeerstrasse 66 CH-4058, Basel
| WRO-1066.2.16
| Tel: +41 61 696 3513
| Fax: +41 61 697 3976
| http://www.fmi.ch/faim
----------------------------------------------------------------------

Dear Aaron,

I can't answer your question directly, but I did want to point out that you are probably more interested in the position of the stage with respect to the objective lens, than in its position "on the planet," so to speak. Consequently, as the only systems that measure this parameter are those that employ some sort of constant-focus feedback system, perhaps you need to broaden your search, to include Nikon for instance.

Without this position feedback, any data from the software could only provide information about "the step number of the focus motor". Because of temperature cycling, variations in coverslips and other types of drift, over time, this will have only a coarse relationship with the position of the focus plane in the specimen.

Cheers,

Jim Pawley

--

**********************************************
Prof. James B. Pawley,

                    Ph. 608-263-3147
Room 223, Zoology Research Building,                                  FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706                                [hidden email]

3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada

Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2010

"If it ain't diffraction, it must be statistics." Anon.

Ponti, Aaron

Re: Stage positions from Zeiss LSM 710, Olympus FluoView 1000, Leica SP/SP5

In reply to this post by Ponti, Aaron

I agree with you. On the other hand, we have developed a stitching software
(http://www.xuvtools.org) that can start from the stage positions on the
planet and calculate the "correct" ones. Of course, having the correct
coordinates to begin with would be better, but I guess that the second
scenario is probably more common in real life. Or did I misunderstand you?

Jose Viña

Re: Stage positions from Zeiss LSM 710, Olympus FluoView 1000, Leica SP/SP5

Dear Aaron,

> I agree with you. On the other hand, we have developed a stitching software
> (http://www.xuvtools.org) that can start from the stage positions on the
> planet and calculate the "correct" ones.

I guess any stitching software can simply stitch images together that
have been taken under similar conditions, but unless you have some
reference 'embedded' in the image, you can't be sure that the Z
stepper was correctly calibrated and that the real distance between
planes is the reported one, for example. With this uncertainty, you
still don't know much about the real position of objects in Z.

(If the stitcher is very good, images that were acquired with
different stepping can be adapted so that they still match somehow,
but then, which one will give the correct physical dimensions?)

We've seen many cases of microscopes reporting distances between
acquired planes that were completely nonsense, and the users, normally
looking at the stack only in XY views, didn't notice. A lateral
inspection immediately reveals the problem, though: objects are
stretched or collapsed along Z. See e.g.
http://support.svi.nl/wiki/CalibrateAxialDistances.

In this sense, some kind of feedback in the microscope is probably
very very useful!

Cheers,

jose.

Tim Feinstein-2

Re: Stage positions from Zeiss LSM 710, Olympus FluoView 1000, Leica SP/SP5

If one prioritizes fine control of XYZ position then I recommend including a Nikon* on the demo list. Our lab has no problem time-lapse imaging 22 separate XY positions simultaneously for four hours or longer. We almost never notice X,Y or Z drift despite temperature swings of 5-6 degrees C when lasers are running. The software does record XYZ position, relative to the objective, for each time point. If competing brands can match that, we did not see it when demoing their scopes.

All the best,

Tim

Tim Feinstein
Postdoctoral Associate,
University of Pittsburgh Dept. of Pharmacology and Chemical Biology

(*) No commercial interest.

On Nov 16, 2009, at 10:34 AM, Jose Viña wrote:

> Dear Aaron,
>
>> I agree with you. On the other hand, we have developed a stitching software
>> (http://www.xuvtools.org) that can start from the stage positions on the
>> planet and calculate the "correct" ones.
>
> I guess any stitching software can simply stitch images together that
> have been taken under similar conditions, but unless you have some
> reference 'embedded' in the image, you can't be sure that the Z
> stepper was correctly calibrated and that the real distance between
> planes is the reported one, for example. With this uncertainty, you
> still don't know much about the real position of objects in Z.
>
> (If the stitcher is very good, images that were acquired with
> different stepping can be adapted so that they still match somehow,
> but then, which one will give the correct physical dimensions?)
>
> We've seen many cases of microscopes reporting distances between
> acquired planes that were completely nonsense, and the users, normally
> looking at the stack only in XY views, didn't notice. A lateral
> inspection immediately reveals the problem, though: objects are
> stretched or collapsed along Z. See e.g.
> http://support.svi.nl/wiki/CalibrateAxialDistances.
>
> In this sense, some kind of feedback in the microscope is probably
> very very useful!
>
> Cheers,
>
> jose.

Glen MacDonald-2

Re: Stage positions from Zeiss LSM 710, Olympus FluoView 1000, Leica SP/SP5

In reply to this post by Ponti, Aaron

Dear Aaron,
the positions are available in 2 different locations. When collecting multi-area images in the OIF format the Olympus Fluoview software creates a directory for the imaging session containing sub-folders for each stage position. Within the session folder will be a text file "MATL_Mosaic.log". This will contain the x,y coordinates of each field.

For any individual image, the XY position of the stage for each image resides the .pty parameter file associated with each TIFF. Open any .pty file (simple text file) and look for this block of text:

[Axis 7 Parameters]
AbsPositionUnitName="mm"
AbsPositionUnitNameX="nm"
AbsPositionUnitNameY="nm"
AbsPositionValue=0.0
AbsPositionValueX=2652600.0
AbsPositionValueY=-704200.0
Number=1

Note the XY positions in the mosaic.log file are in microns, the AbsPositionValueX and AbsPositionValueY are in nm.

Open an OIF image from LOCI with 'Display OME-XML metadata' and search for AbsPositionValueX and AbsPositionValueY to get the positions.
The stage positions are also in the LOCI Original Meta data window.

The meta data in the OIF is written into plain text files, the filename.oif covers the set of images, and the filename.pty is associated with each image. The .oif does not contain the stage positions, since its possible to have different stage positions within an image series. So far, I've found very optical parameter is available. I've not used the OIB format very much, but I'm sure that LOCI will read the stage positions from it.

Regards,
Glen

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923 USA
(206) 616-4156
[hidden email]

On Nov 16, 2009, at 1:32 AM, Ponti, Aaron wrote:

> Hello
>
> We are investigating the purchase of one of the following confocals: Zeiss LSM 710, Olympus FluoView 1000, and Leica SP/SP5. One of the criteria for the choice is the possibility to read stage positions from the files (using the loci/ome-xml tools). With loci_tools 4.1 it seems that reading the stage positions into the OME schema is supported for the Leica, but is not for the other two. Can anybody confirm this? Does anybody know if stage positions are stored at all in .lsm and .oib files? How is reading metadata information in general for these file formats (like optical parameters)?
>
> Thanks
>
> ---------------------------------------------------------------------
> | Dr. Aaron C. Ponti
> | Friedrich Miescher Institute for Biomedical Research
> | Facility for Advanced Microscopy and Imaging
> | Software development
> | Maulbeerstrasse 66 CH-4058, Basel
> | WRO-1066.2.16
> | Tel: +41 61 696 3513
> | Fax: +41 61 697 3976
> | http://www.fmi.ch/faim
> ----------------------------------------------------------------------

Will Moore

Re: Stage positions from Zeiss LSM 710, Olympus FluoView 1000, Leica SP/SP5

In reply to this post by Ponti, Aaron

Hi Aaron,

I've spent a bit of time looking at Stage Positions in Leica files, using the metadata exported from the Leica LAS AF viewer, as well as the "Original Metadata" and OME-XML generated in ImageJ with the LOCI BioFormats importer.

To summarise, there is nothing that looks like stage position in the SP2 (lei) files, although BioFormats is reading something else as stage position.

The SP5 (lif) files have stage position info for each image, but not each plane. Also, they have several attributes for stage position, the meaning of which I don't understand.

BioFormats is currently reading one of these attribute sets, but is not fully populating the StagePositions of every plane.

Details are below, looking at only a couple of images....

Will.

Having looked at the "stage positions" that BioFormats is reading from the Leica.lei files, I think these values are not stage positions.

E.g. Martin's Samples-SP2.lei - XYZ-Ch is a Z stack, so the Z stage position should be changing with each Z index?

However, in the OME-XML the stage position is the same for every plane

This value is being read from this attribute in the Original metadata:

XYZ-Ch Block 1 CScanActuator|Z Scan Actuator|Position|0 -2.2590361445783174E-6

I don't actually know what this attribute is, but I don't think it's the Z stage position.

The other attributes in this set of metadata look like:

XYZ-Ch Block 1 CScanActuator|X Scan Actuator|Gain|0 1.0

XYZ-Ch Block 1 CScanActuator|X Scan Actuator|Offset|0 0.0

XYZ-Ch Block 1 CScanActuator|X Scan Actuator|ScanPlane|0 Active

XYZ-Ch Block 1 CScanActuator|Y Scan Actuator|Gain|0 1.0

XYZ-Ch Block 1 CScanActuator|Y Scan Actuator|Offset|0 0.0

XYZ-Ch Block 1 CScanActuator|Y Scan Actuator|ScanPlane|0 Active

XYZ-Ch Block 1 CScanActuator|Z Scan Actuator|Position|0 -2.2590361445783174E-6

XYZ-Ch Block 1 CScanActuator|Z Scan Actuator|ScanPlane|0 Inactive

Looking at LIF files:

Martin's only image with StagePosition populated in the OME-XML is sample-files.lif - series 04 XYlamba.

The lamba scan of this image is being put in the Z dimension, but should probably but populating the C dimension (this is what we do with other formats, right)?

However, the stage positions look correct. Every plane has:

which comes from:

In a LIF file with multiple Z sections, (garcion/testOne.lif Series023) there is stage position info in the original XML file, but this is for the whole image, not each plane:

This is also what is displayed by the Leica software:

BioFormats is using this for stage-position:

OME-XML for this file has this StagePosition, but only for 4 of the 14 planes (first 4 Z planes where TheC=0).

The all are the same:

I guess the correct thing for us to do is to put this StagePosition data on every plane? or maybe just the planes with TheZ="0" ?

There's also other stage info which we can ignore for now...

Each scan has a <ATLConfocalSettingDefinition> element (within an <LDM_Block_Sequential_List> element):

The <ATLConfocalSettingDefinition> element has these attributes:

ZPosition="2.3841880645312E-10" StagePosX="328160" StagePosY="288625" StagePosZ="0"

ZPosition="2.3841880645312E-10" StagePosX="323227" StagePosY="315570" StagePosZ="0"

However, there is no info for each individual plane, equivalent to the OME model.

(There's also a <LDM_Block_Sequential_Master> <ATLConfocalSettingDefinition> element for "autofocus" with these attributes. )

ZPosition="-1.47462031805068E-06" StagePosX="312064" StagePosY="331170" StagePosZ="0"

Date: Mon, 16 Nov 2009 10:32:35 +0100
From: "Ponti, Aaron" <[hidden email]>
Subject: Stage positions from Zeiss LSM 710, Olympus FluoView 1000, Leica SP/SP5

Hello

We are investigating the purchase of one of the following confocals: =
Zeiss LSM 710, Olympus FluoView 1000, and Leica SP/SP5. One of the =
criteria for the choice is the possibility to read stage positions from =
the files (using the loci/ome-xml tools). With loci_tools 4.1 it seems =
that reading the stage positions into the OME schema is supported for =
the Leica, but is not for the other two. Can anybody confirm this? Does =
anybody know if stage positions are stored at all in .lsm and .oib =
files? How is reading metadata information in general for these file =
formats (like optical parameters)?

Thanks

---------------------------------------------------------------------
| Dr. Aaron C. Ponti
| Friedrich Miescher Institute for Biomedical Research
| Facility for Advanced Microscopy and Imaging
| Software development
| Maulbeerstrasse 66 CH-4058, Basel
| WRO-1066.2.16
| Tel: +41 61 696 3513
| Fax: +41 61 697 3976
| http://www.fmi.ch/faim
----------------------------------------------------------------------

William Moore

Division of Gene Regulation and Expression

College of Life Sciences

University of Dundee

Scotland

DD1 5PH

Tel 01382 386364

Emmanuel Gustin

Re: Stage positions from Zeiss LSM 710, Olympus FluoView 1000, Leica SP/SP5

Hi Will,

It is been a while since I studied it in detail, but with a Leica the Z position can be a combination of two different systems, the focus motor of the microscope and the Z galvo-scanner on the stage. Both are separate parameters in the system, the first grouped with the stage, the second with the X and Y galvo scanners. To get the physical Z position, you have to the take the sum (or difference) of focus motor and galvo scanner position.

When acquiring a Z-stack, at least with the SP2 software, the user has to choose which of the two systems to use to do the Z scanning.

Best Regards,

Emmanuel

--
Emmanuel Gustin, Tel. (+32) 15 46 1586, e-mail: [hidden email]

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Will Moore
Sent: donderdag 19 november 2009 16:48
To: [hidden email]
Subject: Re: Stage positions from Zeiss LSM 710, Olympus FluoView 1000, Leica SP/SP5