Pixel Size Radiance 2000 Olympus

[Eric Scarfone]

Hello List

I am having trouble with old data files, loose notes and an apparent
mismatch of pixel size if I am to believe the values that Imaris reads
from these files.

I do not have access to the system on which the data was acquired
anymore, It was a Biorad Radiance 2000MP with external detectors mounted
on an Olympus BX50 WI. Objectives were the long working distance WI 20X,
40X and 60X.

Question: does anyone has some sort of table indicating pixel size for
each of these objective with the same configuration??
I know, it is a lot to ask for....

Another point I am trying to figure out is the maximum imaging field of
this microscope/objective configuration, and how close (or much more) of
it the scanning system gets.

Thanks ahead!
Eric

Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet

Postal Address:
CFH, M1:02
Karolinska Hospital,
SE-171 76 Stockholm, Sweden

Work:  [phone],
Cell:  [phone]
Fax:   [phone]

email:  [hidden email]
http://www.ki.se/cfh/

[Glen MacDonald-2]

Eric,
Do you have the original .pic files or only exported tiff?  The .PIC  
could be opened in ImageJ.  the LOCI plugin doesn't read all of the  
metadata in the older .pic versions but Radiance files should be  
readable.  Use the Biorad Reader for older .pic versions.

Good luck.
Glen
On Aug 5, 2009, at 8:20 AM, Eric Scarfone wrote:

> Hello List
>
> I am having trouble with old data files, loose notes and an apparent
> mismatch of pixel size if I am to believe the values that Imaris reads
> from these files.
>
> I do not have access to the system on which the data was acquired
> anymore, It was a Biorad Radiance 2000MP with external detectors  
> mounted
> on an Olympus BX50 WI. Objectives were the long working distance WI  
> 20X,
> 40X and 60X.
>
> Question: does anyone has some sort of table indicating pixel size for
> each of these objective with the same configuration??
> I know, it is a lot to ask for....
>
> Another point I am trying to figure out is the maximum imaging field  
> of
> this microscope/objective configuration, and how close (or much  
> more) of
> it the scanning system gets.
>
> Thanks ahead!
> Eric
>
> Eric Scarfone, PhD, CNRS,
> Center for Hearing and communication Research
> Department of Clinical Neuroscience
> Karolinska Institutet
>
> Postal Address:
> CFH, M1:02
> Karolinska Hospital,
> SE-171 76 Stockholm, Sweden
>
> Work:  [phone],
> Cell:  [phone]
> Fax:   [phone]
>
> email:  [hidden email]
> http://www.ki.se/cfh/

[Eric Scarfone]

Hi Glen
I have the original files and I can read the header in Imaris.
The problem is that I do not know if the correct objective lens was
entered for each stack (manual objective holder on the BX50WI)..
And the time we changed objective lens/zoom settings more according to
what we could see on screen than by Nyqvist sampling....and that's one
the criteria I want to check.

Also I am not sure how to read the final magnification (including
optical system and scanning zoom) in the header, especially on older
pic.
Eric

Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet

Postal Address:
CFH, M1:02
Karolinska Hospital,
SE-171 76 Stockholm, Sweden

Work:  [phone],
Cell:  [phone]
Fax:   [phone]

email:  [hidden email]
http://www.ki.se/cfh/


----- Original Message -----
From: Glen MacDonald <[hidden email]>
Date: Wednesday, August 5, 2009 4:27 pm
Subject: Re: Pixel Size Radiance 2000 Olympus
To: [hidden email]

> Eric,
> Do you have the original .pic files or only exported tiff?  The
> .PIC  
> could be opened in ImageJ.  the LOCI plugin doesn't read all of the
>
> metadata in the older .pic versions but Radiance files should be  
> readable.  Use the Biorad Reader for older .pic versions.
>
> Good luck.
> Glen
> On Aug 5, 2009, at 8:20 AM, Eric Scarfone wrote:
>
> > Hello List
> >
> > I am having trouble with old data files, loose notes and an apparent
> > mismatch of pixel size if I am to believe the values that Imaris
> reads> from these files.
> >
> > I do not have access to the system on which the data was acquired
> > anymore, It was a Biorad Radiance 2000MP with external detectors  
> > mounted
> > on an Olympus BX50 WI. Objectives were the long working distance
> WI  
> > 20X,
> > 40X and 60X.
> >
> > Question: does anyone has some sort of table indicating pixel
> size for
> > each of these objective with the same configuration??
> > I know, it is a lot to ask for....
> >
> > Another point I am trying to figure out is the maximum imaging
> field  
> > of
> > this microscope/objective configuration, and how close (or much  
> > more) of
> > it the scanning system gets.
> >
> > Thanks ahead!
> > Eric
> >
> > Eric Scarfone, PhD, CNRS,
> > Center for Hearing and communication Research
> > Department of Clinical Neuroscience
> > Karolinska Institutet
> >
> > Postal Address:
> > CFH, M1:02
> > Karolinska Hospital,
> > SE-171 76 Stockholm, Sweden
> >
> > Work:  [phone],
> > Cell:  [phone]
> > Fax:   [phone]
> >
> > email:  [hidden email]
> > http://www.ki.se/cfh/
>

[Glen MacDonald-2]

Try the Biorad reader plugin in the Input-Output collection.
Open the image and then Image/Show Info...
It doesn't parse the metadata from the bottom of the file very well, but
The lines "Live day date time" will give the Zoom factor
"Zoom Axis_2" and "..._3" are pixel size.
Axis_4 is z step.

If you can run Object Image, I can find my old macro to extract all  
the metadata into a text file.
Hope this helps,
Glen


On Aug 5, 2009, at 10:02 AM, Eric Scarfone wrote:

> Hi Glen
> I have the original files and I can read the header in Imaris.
> The problem is that I do not know if the correct objective lens was
> entered for each stack (manual objective holder on the BX50WI)..
> And the time we changed objective lens/zoom settings more according to
> what we could see on screen than by Nyqvist sampling....and that's one
> the criteria I want to check.
>
> Also I am not sure how to read the final magnification (including
> optical system and scanning zoom) in the header, especially on older
> pic.
> Eric
>
> Eric Scarfone, PhD, CNRS,
> Center for Hearing and communication Research
> Department of Clinical Neuroscience
> Karolinska Institutet
>
> Postal Address:
> CFH, M1:02
> Karolinska Hospital,
> SE-171 76 Stockholm, Sweden
>
> Work:  [phone],
> Cell:  [phone]
> Fax:   [phone]
>
> email:  [hidden email]
> http://www.ki.se/cfh/
>
>
> ----- Original Message -----
> From: Glen MacDonald <[hidden email]>
> Date: Wednesday, August 5, 2009 4:27 pm
> Subject: Re: Pixel Size Radiance 2000 Olympus
> To: [hidden email]
>
>> Eric,
>> Do you have the original .pic files or only exported tiff?  The
>> .PIC
>> could be opened in ImageJ.  the LOCI plugin doesn't read all of the
>>
>> metadata in the older .pic versions but Radiance files should be
>> readable.  Use the Biorad Reader for older .pic versions.
>>
>> Good luck.
>> Glen
>> On Aug 5, 2009, at 8:20 AM, Eric Scarfone wrote:
>>
>>> Hello List
>>>
>>> I am having trouble with old data files, loose notes and an apparent
>>> mismatch of pixel size if I am to believe the values that Imaris
>> reads> from these files.
>>>
>>> I do not have access to the system on which the data was acquired
>>> anymore, It was a Biorad Radiance 2000MP with external detectors
>>> mounted
>>> on an Olympus BX50 WI. Objectives were the long working distance
>> WI
>>> 20X,
>>> 40X and 60X.
>>>
>>> Question: does anyone has some sort of table indicating pixel
>> size for
>>> each of these objective with the same configuration??
>>> I know, it is a lot to ask for....
>>>
>>> Another point I am trying to figure out is the maximum imaging
>> field
>>> of
>>> this microscope/objective configuration, and how close (or much
>>> more) of
>>> it the scanning system gets.
>>>
>>> Thanks ahead!
>>> Eric
>>>
>>> Eric Scarfone, PhD, CNRS,
>>> Center for Hearing and communication Research
>>> Department of Clinical Neuroscience
>>> Karolinska Institutet
>>>
>>> Postal Address:
>>> CFH, M1:02
>>> Karolinska Hospital,
>>> SE-171 76 Stockholm, Sweden
>>>
>>> Work:  [phone],
>>> Cell:  [phone]
>>> Fax:   [phone]
>>>
>>> email:  [hidden email]
>>> http://www.ki.se/cfh/
>>

[W. Chan]

Hi Eric

In addition to what Glen has suggested, you can also just open the
file in a hex editor.  The original pic file has all that info at the end
of the file.  E.g.,

INFO_OBJECTIVE_MAGNIFICATION = 60 [magnification]
INFO_OBJECTIVE_ZOOM = 2 [zoom]

The ZOOM should be what the system was used in the acquistion but the
objective might not be if you have a manual objective turret.   You should
also see that there is a field for the pic file version.  In our case it
is PIC_FF_VERSION = 5.0 written by a Radiance 2000 using lasersharp 6.  I
checked some older files and the info is the same for pic version 4.5 as
well.

On Wed, 5 Aug 2009, Eric Scarfone wrote:

> Hi Glen
> I have the original files and I can read the header in Imaris.
> The problem is that I do not know if the correct objective lens was
> entered for each stack (manual objective holder on the BX50WI)..
> And the time we changed objective lens/zoom settings more according to
> what we could see on screen than by Nyqvist sampling....and that's one
> the criteria I want to check.
>
> Also I am not sure how to read the final magnification (including
> optical system and scanning zoom) in the header, especially on older
> pic.
> Eric
>
> Eric Scarfone, PhD, CNRS,
>
> ----- Original Message -----
> From: Glen MacDonald <[hidden email]>
> Date: Wednesday, August 5, 2009 4:27 pm
> Subject: Re: Pixel Size Radiance 2000 Olympus
> To: [hidden email]
>
> > Eric,
> > Do you have the original .pic files or only exported tiff?  The
> > .PIC
> > could be opened in ImageJ.  the LOCI plugin doesn't read all of the
> >
> > metadata in the older .pic versions but Radiance files should be
> > readable.  Use the Biorad Reader for older .pic versions.
> >
> > Good luck.
> > Glen
> > On Aug 5, 2009, at 8:20 AM, Eric Scarfone wrote:
> >
> > > Hello List
> > >
> > > I am having trouble with old data files, loose notes and an apparent
> > > mismatch of pixel size if I am to believe the values that Imaris
> > reads> from these files.
> > >
> > > I do not have access to the system on which the data was acquired
> > > anymore, It was a Biorad Radiance 2000MP with external detectors
> > > mounted
> > > on an Olympus BX50 WI. Objectives were the long working distance
> > WI
> > > 20X,
> > > 40X and 60X.
> > >
> > > Question: does anyone has some sort of table indicating pixel
> > size for
> > > each of these objective with the same configuration??
> > > I know, it is a lot to ask for....
> > >
> > > Another point I am trying to figure out is the maximum imaging
> > field
> > > of
> > > this microscope/objective configuration, and how close (or much
> > > more) of
> > > it the scanning system gets.
> > >
> > > Thanks ahead!
> > > Eric
> > >

--
Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519)
The Biology Imaging Facility (http://depts.washington.edu/if/)

[Glen MacDonald-2]

Hi Pang,
I was looking at format version 4.3 from our MRC-1024. the  
Info_Objective lines don't appear with the plugin.

Glen
On Aug 5, 2009, at 11:21 AM, W. Chan wrote:

> Hi Eric
>
> In addition to what Glen has suggested, you can also just open the
> file in a hex editor.  The original pic file has all that info at  
> the end
> of the file.  E.g.,
>
> INFO_OBJECTIVE_MAGNIFICATION = 60 [magnification]
> INFO_OBJECTIVE_ZOOM = 2 [zoom]
>
> The ZOOM should be what the system was used in the acquistion but the
> objective might not be if you have a manual objective turret.   You  
> should
> also see that there is a field for the pic file version.  In our  
> case it
> is PIC_FF_VERSION = 5.0 written by a Radiance 2000 using lasersharp  
> 6.  I
> checked some older files and the info is the same for pic version  
> 4.5 as
> well.
>
> On Wed, 5 Aug 2009, Eric Scarfone wrote:
>
>> Hi Glen
>> I have the original files and I can read the header in Imaris.
>> The problem is that I do not know if the correct objective lens was
>> entered for each stack (manual objective holder on the BX50WI)..
>> And the time we changed objective lens/zoom settings more according  
>> to
>> what we could see on screen than by Nyqvist sampling....and that's  
>> one
>> the criteria I want to check.
>>
>> Also I am not sure how to read the final magnification (including
>> optical system and scanning zoom) in the header, especially on older
>> pic.
>> Eric
>>
>> Eric Scarfone, PhD, CNRS,
>>
>> ----- Original Message -----
>> From: Glen MacDonald <[hidden email]>
>> Date: Wednesday, August 5, 2009 4:27 pm
>> Subject: Re: Pixel Size Radiance 2000 Olympus
>> To: [hidden email]
>>
>>> Eric,
>>> Do you have the original .pic files or only exported tiff?  The
>>> .PIC
>>> could be opened in ImageJ.  the LOCI plugin doesn't read all of the
>>>
>>> metadata in the older .pic versions but Radiance files should be
>>> readable.  Use the Biorad Reader for older .pic versions.
>>>
>>> Good luck.
>>> Glen
>>> On Aug 5, 2009, at 8:20 AM, Eric Scarfone wrote:
>>>
>>>> Hello List
>>>>
>>>> I am having trouble with old data files, loose notes and an  
>>>> apparent
>>>> mismatch of pixel size if I am to believe the values that Imaris
>>> reads> from these files.
>>>>
>>>> I do not have access to the system on which the data was acquired
>>>> anymore, It was a Biorad Radiance 2000MP with external detectors
>>>> mounted
>>>> on an Olympus BX50 WI. Objectives were the long working distance
>>> WI
>>>> 20X,
>>>> 40X and 60X.
>>>>
>>>> Question: does anyone has some sort of table indicating pixel
>>> size for
>>>> each of these objective with the same configuration??
>>>> I know, it is a lot to ask for....
>>>>
>>>> Another point I am trying to figure out is the maximum imaging
>>> field
>>>> of
>>>> this microscope/objective configuration, and how close (or much
>>>> more) of
>>>> it the scanning system gets.
>>>>
>>>> Thanks ahead!
>>>> Eric
>>>>
>
> --
> Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519)
> The Biology Imaging Facility (http://depts.washington.edu/if/)

[Eric Scarfone]

Hi Glen, Pang,

thanks for your input. Using Imaris I can see the Biorad Notes from both version 4.5 and 4.3 of lasersharp
I can also read the info from older versions (MRC600).

However, do you know how to calculate Pixel size from these (ie using a different Objective lens than the one indicated)?
More generally isthere somewhere a description of the structure of these notes (what is the Z_correct_factor etc....)

Eric

Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet

Postal Address:
CFH, M1:02
Karolinska Hospital,
SE-171 76 Stockholm, Sweden

Work: [phone],
Cell: [phone]
Fax: [phone]

email: [hidden email]
http://www.ki.se/cfh/


----- Original Message -----
From: Glen MacDonald <[hidden email]>
Date: Wednesday, August 5, 2009 7:49 pm
Subject: Re: Pixel Size Radiance 2000 Olympus
To: [hidden email]

> Hi Pang,
> I was looking at format version 4.3 from our MRC-1024. the
> Info_Objective lines don't appear with the plugin.
>
> Glen
> On Aug 5, 2009, at 11:21 AM, W. Chan wrote:
>
> > Hi Eric
> >
> > In addition to what Glen has suggested, you can also just open the

> > file in a hex editor. The original pic file has all that info

> at
> > the end
> > of the file. E.g.,
> >
> > INFO_OBJECTIVE_MAGNIFICATION = 60 [magnification]
> > INFO_OBJECTIVE_ZOOM = 2 [zoom]
> >
> > The ZOOM should be what the system was used in the acquistion
> but the
> > objective might not be if you have a manual objective turret.
> You
> > should
> > also see that there is a field for the pic file version. In our
>
> > case it
> > is PIC_FF_VERSION = 5.0 written by a Radiance 2000 using
> lasersharp
> > 6. I
> > checked some older files and the info is the same for pic
> version
> > 4.5 as
> > well.
> >
> > On Wed, 5 Aug 2009, Eric Scarfone wrote:
> >
> >> Hi Glen

> >> I have the original files and I can read the header in Imaris.

> >> The problem is that I do not know if the correct objective lens was
> >> entered for each stack (manual objective holder on the BX50WI)..
> >> And the time we changed objective lens/zoom settings more
> according
> >> to
> >> what we could see on screen than by Nyqvist sampling....and
> that's
> >> one
> >> the criteria I want to check.
> >>
> >> Also I am not sure how to read the final magnification (including
> >> optical system and scanning zoom) in the header, especially on
> older>> pic.
> >> Eric
> >>
> >> Eric Scarfone, PhD, CNRS,
> >>
> >> ----- Original Message -----
> >> From: Glen MacDonald <[hidden email]>

> >> Date: Wednesday, August 5, 2009 4:27 pm

> >> Subject: Re: Pixel Size Radiance 2000 Olympus
> >> To: [hidden email]
> >>
> >>> Eric,
> >>> Do you have the original .pic files or only exported tiff? The
> >>> .PIC
> >>> could be opened in ImageJ. the LOCI plugin doesn't read all
> of the
> >>>
> >>> metadata in the older .pic versions but Radiance files should be
> >>> readable. Use the Biorad Reader for older .pic versions.
> >>>
> >>> Good luck.
> >>> Glen
> >>> On Aug 5, 2009, at 8:20 AM, Eric Scarfone wrote:
> >>>
> >>>> Hello List
> >>>>
> >>>> I am having trouble with old data files, loose notes and an
> >>>> apparent

> >>>> mismatch of pixel size if I am to believe the values that Imaris

> >>> reads> from these files.
> >>>>
> >>>> I do not have access to the system on which the data was acquired
> >>>> anymore, It was a Biorad Radiance 2000MP with external detectors
> >>>> mounted
> >>>> on an Olympus BX50 WI. Objectives were the long working distance
> >>> WI
> >>>> 20X,
> >>>> 40X and 60X.
> >>>>
> >>>> Question: does anyone has some sort of table indicating pixel
> >>> size for
> >>>> each of these objective with the same configuration??
> >>>> I know, it is a lot to ask for....
> >>>>
> >>>> Another point I am trying to figure out is the maximum imaging

> >>> field

> >>>> of
> >>>> this microscope/objective configuration, and how close (or much
> >>>> more) of
> >>>> it the scanning system gets.
> >>>>
> >>>> Thanks ahead!
> >>>> Eric
> >>>>
> >
> > --
> > Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-
> 1519)> The Biology Imaging Facility (http://depts.washington.edu/if/)
>

[W. Chan]

Hi Eric

Unless the setup has an ideal linear relationship for the magnification,
there is not much you can do if suspect the wrong objrctive info was
entered.  I guess you can see if there are any particular features in the
image that you know the size well and estimate the pixel size that way.
Without access to the specific setup, it is impossible to know accurately
what the spatial calibration should be.  If you can get hold of the same
sample, you can grab the same on any calibrated system and then apply to
similar images.

As for the notes, it seems to be different with different version of PIC
as Glen pointed out.  The header format is the same as far as I know.  If
you want to know more Zeiss should be able to provide a complete
description.

For a limited amount of description, if you get hold of
Biorad_Reader.java, the source file of the imagej plugin, you will see
those of the header and a few of the notes.  The header description there
is the same as presented in the CoMOS Reference of the MRC-600 manual.

Specifically:

- SCALE_FACTOR is specific for the setup

- Z_CORRECT_FACTOR is calculated with respect to the mountant:immersion
medium refractive index ratio according to White et al. 1996, J. Micro.,
181(2):99-116 (described on p.31 of the Radiance manual 9m60um04.pdf if
you can get a hold of one).  I think the first number is the RI ratio and
the 2nd number is the corrected aspect ratio.  It is used in doing 3D
reconstruction.  This information is associated with the objective used.

- AXIS_2 and AXIS_3 are the XY pixel sizes in micron

- AXIS_4 can be the Z start location and Z pixel size in a multi-slice
image, the duration if you do a time series, or represents the RGB channel
in a single-slice image

- AXIS_9 represents the RGB channel in a multi-slice image

- the INFO_* fields, method (filter, laser and detector settings) etc.,
are pretty much self explanatory.

Hope these help.

On Thu, 6 Aug 2009, Eric Scarfone wrote:

>
> Hi Glen, Pang,
>
> thanks for your input. Using Imaris I can see the Biorad Notes from both
> version 4.5 and 4.3 of lasersharp
> I can also read the info from older versions (MRC600).
>
> However, do you know how to calculate Pixel size from these (ie using a
> different Objective lens than the one indicated)?
> More generally isthere somewhere a description of the structure of these
> notes (what is the Z_correct_factor etc....)
>
> Eric
>
> ----- Original Message -----
> From: Glen MacDonald <[hidden email]>
> Date: Wednesday, August 5, 2009 7:49 pm
> Subject: Re: Pixel Size Radiance 2000 Olympus
> To: [hidden email]
>
> > Hi Pang,
> > I was looking at format version 4.3 from our MRC-1024. the
> > Info_Objective lines don't appear with the plugin.
> >
> > Glen
> > On Aug 5, 2009, at 11:21 AM, W. Chan wrote:
> >
> > > Hi Eric
> > >
> > > In addition to what Glen has suggested, you can also just open the
> > > file in a hex editor. The original pic file has all that info
> > at
> > > the end
> > > of the file. E.g.,
> > >
> > > INFO_OBJECTIVE_MAGNIFICATION = 60 [magnification]
> > > INFO_OBJECTIVE_ZOOM = 2 [zoom]
> > >
> > > The ZOOM should be what the system was used in the acquistion
> > but the
> > > objective might not be if you have a manual objective turret.
> > You
> > > should
> > > also see that there is a field for the pic file version. In our
> >
> > > case it
> > > is PIC_FF_VERSION = 5.0 written by a Radiance 2000 using
> > lasersharp
> > > 6. I
> > > checked some older files and the info is the same for pic
> > version
> > > 4.5 as
> > > well.
> > >
> > > On Wed, 5 Aug 2009, Eric Scarfone wrote:
> > >
> > >> Hi Glen
> > >> I have the original files and I can read the header in Imaris.
> > >> The problem is that I do not know if the correct objective lens was
> > >> entered for each stack (manual objective holder on the BX50WI)..
> > >> And the time we changed objective lens/zoom settings more
> > according
> > >> to
> > >> what we could see on screen than by Nyqvist sampling....and
> > that's
> > >> one
> > >> the criteria I want to check.
> > >>
> > >> Also I am not sure how to read the final magnification (including
> > >> optical system and scanning zoom) in the header, especially on
> > older>> pic.
> > >> Eric
> > >>
> > >> Eric Scarfone, PhD, CNRS,
> > >>
> > >> ----- Original Message -----
> > >> From: Glen MacDonald <[hidden email]>
> > >> Date: Wednesday, August 5, 2009 4:27 pm
> > >> Subject: Re: Pixel Size Radiance 2000 Olympus
> > >> To: [hidden email]
> > >>
> > >>> Eric,
> > >>> Do you have the original .pic files or only exported tiff? The
> > >>> .PIC
> > >>> could be opened in ImageJ. the LOCI plugin doesn't read all
> > of the
> > >>>
> > >>> metadata in the older .pic versions but Radiance files should be
> > >>> readable. Use the Biorad Reader for older .pic versions.
> > >>>
> > >>> Good luck.
> > >>> Glen
> > >>> On Aug 5, 2009, at 8:20 AM, Eric Scarfone wrote:
> > >>>
> > >>>> Hello List
> > >>>>
> > >>>> I am having trouble with old data files, loose notes and an
> > >>>> apparent
> > >>>> mismatch of pixel size if I am to believe the values that Imaris
> > >>> reads> from these files.
> > >>>>
> > >>>> I do not have access to the system on which the data was acquired
> > >>>> anymore, It was a Biorad Radiance 2000MP with external detectors
> > >>>> mounted
> > >>>> on an Olympus BX50 WI. Objectives were the long working distance
> > >>> WI
> > >>>> 20X,
> > >>>> 40X and 60X.
> > >>>>
> > >>>> Question: does anyone has some sort of table indicating pixel
> > >>> size for
> > >>>> each of these objective with the same configuration??
> > >>>> I know, it is a lot to ask for....
> > >>>>
> > >>>> Another point I am trying to figure out is the maximum imaging
> > >>> field
> > >>>> of
> > >>>> this microscope/objective configuration, and how close (or much
> > >>>> more) of
> > >>>> it the scanning system gets.
> > >>>>
> > >>>> Thanks ahead!
> > >>>> Eric

--
Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519)
The Biology Imaging Facility (http://depts.washington.edu/if/)

[Glen MacDonald-2]

Looks like Pang beat me to finding the specs for the file format.  At  
least with determining the actual  objective, if you know the zoom,  
there are only few discrete choices among the available lens  
magnifications from which to choose among.

Glen
On Aug 7, 2009, at 5:58 PM, W. Chan wrote:

> Hi Eric
>
> Unless the setup has an ideal linear relationship for the  
> magnification,
> there is not much you can do if suspect the wrong objrctive info was
> entered.  I guess you can see if there are any particular features  
> in the
> image that you know the size well and estimate the pixel size that  
> way.
> Without access to the specific setup, it is impossible to know  
> accurately
> what the spatial calibration should be.  If you can get hold of the  
> same
> sample, you can grab the same on any calibrated system and then  
> apply to
> similar images.
>
> As for the notes, it seems to be different with different version of  
> PIC
> as Glen pointed out.  The header format is the same as far as I  
> know.  If
> you want to know more Zeiss should be able to provide a complete
> description.
>
> For a limited amount of description, if you get hold of
> Biorad_Reader.java, the source file of the imagej plugin, you will see
> those of the header and a few of the notes.  The header description  
> there
> is the same as presented in the CoMOS Reference of the MRC-600 manual.
>
> Specifically:
>
> - SCALE_FACTOR is specific for the setup
>
> - Z_CORRECT_FACTOR is calculated with respect to the  
> mountant:immersion
> medium refractive index ratio according to White et al. 1996, J.  
> Micro.,
> 181(2):99-116 (described on p.31 of the Radiance manual 9m60um04.pdf  
> if
> you can get a hold of one).  I think the first number is the RI  
> ratio and
> the 2nd number is the corrected aspect ratio.  It is used in doing 3D
> reconstruction.  This information is associated with the objective  
> used.
>
> - AXIS_2 and AXIS_3 are the XY pixel sizes in micron
>
> - AXIS_4 can be the Z start location and Z pixel size in a multi-slice
> image, the duration if you do a time series, or represents the RGB  
> channel
> in a single-slice image
>
> - AXIS_9 represents the RGB channel in a multi-slice image
>
> - the INFO_* fields, method (filter, laser and detector settings)  
> etc.,
> are pretty much self explanatory.
>
> Hope these help.
>
> On Thu, 6 Aug 2009, Eric Scarfone wrote:
>
>>
>> Hi Glen, Pang,
>>
>> thanks for your input. Using Imaris I can see the Biorad Notes from  
>> both
>> version 4.5 and 4.3 of lasersharp
>> I can also read the info from older versions (MRC600).
>>
>> However, do you know how to calculate Pixel size from these (ie  
>> using a
>> different Objective lens than the one indicated)?
>> More generally isthere somewhere a description of the structure of  
>> these
>> notes (what is the Z_correct_factor etc....)
>>
>> Eric
>>
>> ----- Original Message -----
>> From: Glen MacDonald <[hidden email]>
>> Date: Wednesday, August 5, 2009 7:49 pm
>> Subject: Re: Pixel Size Radiance 2000 Olympus
>> To: [hidden email]
>>
>>> Hi Pang,
>>> I was looking at format version 4.3 from our MRC-1024. the
>>> Info_Objective lines don't appear with the plugin.
>>>
>>> Glen
>>> On Aug 5, 2009, at 11:21 AM, W. Chan wrote:
>>>
>>>> Hi Eric
>>>>
>>>> In addition to what Glen has suggested, you can also just open the
>>>> file in a hex editor. The original pic file has all that info
>>> at
>>>> the end
>>>> of the file. E.g.,
>>>>
>>>> INFO_OBJECTIVE_MAGNIFICATION = 60 [magnification]
>>>> INFO_OBJECTIVE_ZOOM = 2 [zoom]
>>>>
>>>> The ZOOM should be what the system was used in the acquistion
>>> but the
>>>> objective might not be if you have a manual objective turret.
>>> You
>>>> should
>>>> also see that there is a field for the pic file version. In our
>>>
>>>> case it
>>>> is PIC_FF_VERSION = 5.0 written by a Radiance 2000 using
>>> lasersharp
>>>> 6. I
>>>> checked some older files and the info is the same for pic
>>> version
>>>> 4.5 as
>>>> well.
>>>>
>>>> On Wed, 5 Aug 2009, Eric Scarfone wrote:
>>>>
>>>>> Hi Glen
>>>>> I have the original files and I can read the header in Imaris.
>>>>> The problem is that I do not know if the correct objective lens  
>>>>> was
>>>>> entered for each stack (manual objective holder on the BX50WI)..
>>>>> And the time we changed objective lens/zoom settings more
>>> according
>>>>> to
>>>>> what we could see on screen than by Nyqvist sampling....and
>>> that's
>>>>> one
>>>>> the criteria I want to check.
>>>>>
>>>>> Also I am not sure how to read the final magnification (including
>>>>> optical system and scanning zoom) in the header, especially on
>>> older>> pic.
>>>>> Eric
>>>>>
>>>>> Eric Scarfone, PhD, CNRS,
>>>>>
>>>>> ----- Original Message -----
>>>>> From: Glen MacDonald <[hidden email]>
>>>>> Date: Wednesday, August 5, 2009 4:27 pm
>>>>> Subject: Re: Pixel Size Radiance 2000 Olympus
>>>>> To: [hidden email]
>>>>>
>>>>>> Eric,
>>>>>> Do you have the original .pic files or only exported tiff? The
>>>>>> .PIC
>>>>>> could be opened in ImageJ. the LOCI plugin doesn't read all
>>> of the
>>>>>>
>>>>>> metadata in the older .pic versions but Radiance files should be
>>>>>> readable. Use the Biorad Reader for older .pic versions.
>>>>>>
>>>>>> Good luck.
>>>>>> Glen
>>>>>> On Aug 5, 2009, at 8:20 AM, Eric Scarfone wrote:
>>>>>>
>>>>>>> Hello List
>>>>>>>
>>>>>>> I am having trouble with old data files, loose notes and an
>>>>>>> apparent
>>>>>>> mismatch of pixel size if I am to believe the values that Imaris
>>>>>> reads> from these files.
>>>>>>>
>>>>>>> I do not have access to the system on which the data was  
>>>>>>> acquired
>>>>>>> anymore, It was a Biorad Radiance 2000MP with external detectors
>>>>>>> mounted
>>>>>>> on an Olympus BX50 WI. Objectives were the long working distance
>>>>>> WI
>>>>>>> 20X,
>>>>>>> 40X and 60X.
>>>>>>>
>>>>>>> Question: does anyone has some sort of table indicating pixel
>>>>>> size for
>>>>>>> each of these objective with the same configuration??
>>>>>>> I know, it is a lot to ask for....
>>>>>>>
>>>>>>> Another point I am trying to figure out is the maximum imaging
>>>>>> field
>>>>>>> of
>>>>>>> this microscope/objective configuration, and how close (or much
>>>>>>> more) of
>>>>>>> it the scanning system gets.
>>>>>>>
>>>>>>> Thanks ahead!
>>>>>>> Eric
>
> --
> Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519)
> The Biology Imaging Facility (http://depts.washington.edu/if/)