multichannel 3D viewing problem with Fluoview500 tiffs

[Katinka Wouters]

Hi All,

For a while, I've been using ImageJ 3D viewer or volume viewer to visualize bacterial biofilms scanned with an Olympus Fluoview 500. It works perfectly for single channel images, thanks a lot to the developers!

But, when I'm using it for multichannel images (so in my case: multispecies biofilms), my '3D' images appear 'flat' (like 2D), in both the 3D viewer and the volume viewer. When I rotate them, I get just one plane. This is particularly strange, since I can still browse through the Z-slices in the stack from which the 3D-image originate: all the (over 40) z-slices are there. So, I have a perfect 8 bit z-stack of each channel, but get a 2D image from them, both from the separate channels as from a stack in which the channels have been merged first.

Because this problem occurs in two separate plugins (both 3D Viewer as Volume Viewer), I wonder whether it's a problem with my tiff-series. An image stack originates from a Fluoview500 tiff file, which was converted for use with ImageJ using the Save_8bit_MultiCH plugin in combination with the LOCI plugin. This generates a separate tiff stack for each channel. So everything seems perfect, untill I try to render them to 3D. I used the same plugin to convert the single channel images to a tiff stack, without any 3D viewing problems.

I noticed before that I can not use the LOCI plugin as such to import my FV tiffs, because channels and z-position get mixed up: I always get a stack with repeatedly CH1; CH2; CH3; CH1; CH2; CH3... as if these are the z-slices, and this divided over 3 'channels', which are in fact not the channels. This happens with every setting I tried (e.g. changing the xyzct order). Luckily, this problem was solved when using the Save_8bit_MultiCH plugin, but now I wonder there is maybe some error left in the metadata that gets the 3D/Volume Viewer confused about the stack.

Does anyone recognize this problem? Or is there something obvious that I'm missing here? I would be very gratefull if I could find a solution for it, since I'm promoting ImageJ towards my colleagues who are now using  the very expensive program from Zurich...

Thanks a lot!

Katinka

[Benjamin Schmid-2]

Hi Katinka,

>
> For a while, I've been using ImageJ 3D viewer or volume viewer to visualize bacterial biofilms scanned with an Olympus Fluoview 500. It works perfectly for single channel images, thanks a lot to the developers!
>
> But, when I'm using it for multichannel images (so in my case: multispecies biofilms), my '3D' images appear 'flat' (like 2D), in both the 3D viewer and the volume viewer. When I rotate them, I get just one plane. This is particularly strange, since I can still browse through the Z-slices in the stack from which the 3D-image originate: all the (over 40) z-slices are there. So, I have a perfect 8 bit z-stack of each channel, but get a 2D image from them, both from the separate channels as from a stack in which the channels have been merged first.
>
> Because this problem occurs in two separate plugins (both 3D Viewer as Volume Viewer), I wonder whether it's a problem with my tiff-series. An image stack originates from a Fluoview500 tiff file, which was converted for use with ImageJ using the Save_8bit_MultiCH plugin in combination with the LOCI plugin. This generates a separate tiff stack for each channel. So everything seems perfect, untill I try to render them to 3D. I used the same plugin to convert the single channel images to a tiff stack, without any 3D viewing problems.
>
> I noticed before that I can not use the LOCI plugin as such to import my FV tiffs, because channels and z-position get mixed up: I always get a stack with repeatedly CH1; CH2; CH3; CH1; CH2; CH3... as if these are the z-slices, and this divided over 3 'channels', which are in fact not the channels. This happens with every setting I tried (e.g. changing the xyzct order). Luckily, this problem was solved when using the Save_8bit_MultiCH plugin, but now I wonder there is maybe some error left in the metadata that gets the 3D/Volume Viewer confused about the stack.
>
> Does anyone recognize this problem? Or is there something obvious that I'm missing here? I would be very gratefull if I could find a solution for it, since I'm promoting ImageJ towards my colleagues who are now using  the very expensive program from Zurich...
>

My first thought is that the calibration in the z direction may be wrong/to
small. Can you check this? (->Image->Properties: Voxel depth).

If the voxel depth seems to make sense, could you post the dimensions of
the image, i.e. what you see in the dialog when clicking
Image->Hyperstacks->Stack to Hyperstack.


Best wishes,
Bene

[Katinka Wouters]

Hi Bene,

Thanks for your very fast reply. I checked the image properties, and indeed, the voxel depth is at 0.0000000. So, problem solved! Any idea why the macro changes the voxel depths for multichannel images this dramatically?

I will now try to use a SetVoxelDepth macro, in order to keep the process automated.

Thanks a lot, you made my day!

Katinka

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Benjamin Schmid
Sent: Friday, July 23, 2010 1:09 PM
To: [hidden email]
Subject: Re: multichannel 3D viewing problem with Fluoview500 tiffs

Hi Katinka,

>
> For a while, I've been using ImageJ 3D viewer or volume viewer to visualize bacterial biofilms scanned with an Olympus Fluoview 500. It works perfectly for single channel images, thanks a lot to the developers!
>
> But, when I'm using it for multichannel images (so in my case: multispecies biofilms), my '3D' images appear 'flat' (like 2D), in both the 3D viewer and the volume viewer. When I rotate them, I get just one plane. This is particularly strange, since I can still browse through the Z-slices in the stack from which the 3D-image originate: all the (over 40) z-slices are there. So, I have a perfect 8 bit z-stack of each channel, but get a 2D image from them, both from the separate channels as from a stack in which the channels have been merged first.
>
> Because this problem occurs in two separate plugins (both 3D Viewer as Volume Viewer), I wonder whether it's a problem with my tiff-series. An image stack originates from a Fluoview500 tiff file, which was converted for use with ImageJ using the Save_8bit_MultiCH plugin in combination with the LOCI plugin. This generates a separate tiff stack for each channel. So everything seems perfect, untill I try to render them to 3D. I used the same plugin to convert the single channel images to a tiff stack, without any 3D viewing problems.
>
> I noticed before that I can not use the LOCI plugin as such to import my FV tiffs, because channels and z-position get mixed up: I always get a stack with repeatedly CH1; CH2; CH3; CH1; CH2; CH3... as if these are the z-slices, and this divided over 3 'channels', which are in fact not the channels. This happens with every setting I tried (e.g. changing the xyzct order). Luckily, this problem was solved when using the Save_8bit_MultiCH plugin, but now I wonder there is maybe some error left in the metadata that gets the 3D/Volume Viewer confused about the stack.
>
> Does anyone recognize this problem? Or is there something obvious that I'm missing here? I would be very gratefull if I could find a solution for it, since I'm promoting ImageJ towards my colleagues who are now using  the very expensive program from Zurich...
>

My first thought is that the calibration in the z direction may be wrong/to
small. Can you check this? (->Image->Properties: Voxel depth).

If the voxel depth seems to make sense, could you post the dimensions of
the image, i.e. what you see in the dialog when clicking
Image->Hyperstacks->Stack to Hyperstack.


Best wishes,
Bene

[Glen MacDonald-2]

Dear Katinka,
Scrolling through the stack is simply stepping between the images.  The voxel depth value is assigned at acquisition and written into the file metadata.  The  TIFF  does not have a native tag for storing the axial dimension.  Your macro may be converting the Fluoview file to tiff in a manner which causes that value to be lost.  ImageJ writes voxel depth to the TIFF comments tag and reads it back out, if you re-open in ImageJ. Have you tried the current stable version of LOCI, 4.2.0 to open your files?  If that does not correctly set channel order, then you can select the correct channel order by the Stack Order dropdown menu in the upper left region of the plugin window.  

Importing as Hyperstack with Composite color will maintain voxel properties and does a better job of channel color than does the Standard ImageJ Viewer option. Use Hyperstacks>Channels Tool>More to change channel colors. Use Hyperstacks>Channels Tool>More>Convert to RGB will create an 8-bit/channel RGB with voxel depth (record this once and save as a macro) for the 3D viewer.  

  Regards,Glen

On Jul 23, 2010, at 4:21 AM, Katinka Wouters wrote:

> Hi Bene,
>
> Thanks for your very fast reply. I checked the image properties, and indeed, the voxel depth is at 0.0000000. So, problem solved! Any idea why the macro changes the voxel depths for multichannel images this dramatically?
>
> I will now try to use a SetVoxelDepth macro, in order to keep the process automated.
>
> Thanks a lot, you made my day!
>
> Katinka
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Benjamin Schmid
> Sent: Friday, July 23, 2010 1:09 PM
> To: [hidden email]
> Subject: Re: multichannel 3D viewing problem with Fluoview500 tiffs
>
> Hi Katinka,
>>
>> For a while, I've been using ImageJ 3D viewer or volume viewer to visualize bacterial biofilms scanned with an Olympus Fluoview 500. It works perfectly for single channel images, thanks a lot to the developers!
>>
>> But, when I'm using it for multichannel images (so in my case: multispecies biofilms), my '3D' images appear 'flat' (like 2D), in both the 3D viewer and the volume viewer. When I rotate them, I get just one plane. This is particularly strange, since I can still browse through the Z-slices in the stack from which the 3D-image originate: all the (over 40) z-slices are there. So, I have a perfect 8 bit z-stack of each channel, but get a 2D image from them, both from the separate channels as from a stack in which the channels have been merged first.
>>
>> Because this problem occurs in two separate plugins (both 3D Viewer as Volume Viewer), I wonder whether it's a problem with my tiff-series. An image stack originates from a Fluoview500 tiff file, which was converted for use with ImageJ using the Save_8bit_MultiCH plugin in combination with the LOCI plugin. This generates a separate tiff stack for each channel. So everything seems perfect, untill I try to render them to 3D. I used the same plugin to convert the single channel images to a tiff stack, without any 3D viewing problems.
>>
>> I noticed before that I can not use the LOCI plugin as such to import my FV tiffs, because channels and z-position get mixed up: I always get a stack with repeatedly CH1; CH2; CH3; CH1; CH2; CH3... as if these are the z-slices, and this divided over 3 'channels', which are in fact not the channels. This happens with every setting I tried (e.g. changing the xyzct order). Luckily, this problem was solved when using the Save_8bit_MultiCH plugin, but now I wonder there is maybe some error left in the metadata that gets the 3D/Volume Viewer confused about the stack.
>>
>> Does anyone recognize this problem? Or is there something obvious that I'm missing here? I would be very gratefull if I could find a solution for it, since I'm promoting ImageJ towards my colleagues who are now using  the very expensive program from Zurich...
>>
>
> My first thought is that the calibration in the z direction may be wrong/to
> small. Can you check this? (->Image->Properties: Voxel depth).
>
> If the voxel depth seems to make sense, could you post the dimensions of
> the image, i.e. what you see in the dialog when clicking
> Image->Hyperstacks->Stack to Hyperstack.
>
>
> Best wishes,
> Bene

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

[Katinka Wouters]

Hi Glen,


Dear Katinka,
Scrolling through the stack is simply stepping between the images.  The voxel depth value is assigned at acquisition and written into the file metadata.  The  TIFF  does not have a native tag for storing the axial dimension.  Your macro may be converting the Fluoview file to tiff in a manner which causes that value to be lost.
-- Indeed, that's what I thought, thanks for clarifying

ImageJ writes voxel depth to the TIFF comments tag and reads it back out, if you re-open in ImageJ. Have you tried the current stable version of LOCI, 4.2.0 to open your files?  
-- yes I did

If that does not correctly set channel order, then you can select the correct channel order by the Stack Order dropdown menu in the upper left region of the plugin window.  
-- tried this, but there's no change in my outcome whether I choose xyzct, xyczt, etc... when I want to import with separating channels

Importing as Hyperstack with Composite color will maintain voxel properties and does a better job of channel color than does the Standard ImageJ Viewer option. Use Hyperstacks>Channels Tool>More to change channel colors. Use Hyperstacks>Channels Tool>More>Convert to RGB will create an 8-bit/channel RGB with voxel depth (record this once and save as a macro) for the 3D viewer.  
-- I'll try this one, thanks!

Katinka

  Regards,Glen

On Jul 23, 2010, at 4:21 AM, Katinka Wouters wrote:

> Hi Bene,
>
> Thanks for your very fast reply. I checked the image properties, and indeed, the voxel depth is at 0.0000000. So, problem solved! Any idea why the macro changes the voxel depths for multichannel images this dramatically?
>
> I will now try to use a SetVoxelDepth macro, in order to keep the process automated.
>
> Thanks a lot, you made my day!
>
> Katinka
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Benjamin Schmid
> Sent: Friday, July 23, 2010 1:09 PM
> To: [hidden email]
> Subject: Re: multichannel 3D viewing problem with Fluoview500 tiffs
>
> Hi Katinka,
>>
>> For a while, I've been using ImageJ 3D viewer or volume viewer to visualize bacterial biofilms scanned with an Olympus Fluoview 500. It works perfectly for single channel images, thanks a lot to the developers!
>>
>> But, when I'm using it for multichannel images (so in my case: multispecies biofilms), my '3D' images appear 'flat' (like 2D), in both the 3D viewer and the volume viewer. When I rotate them, I get just one plane. This is particularly strange, since I can still browse through the Z-slices in the stack from which the 3D-image originate: all the (over 40) z-slices are there. So, I have a perfect 8 bit z-stack of each channel, but get a 2D image from them, both from the separate channels as from a stack in which the channels have been merged first.
>>
>> Because this problem occurs in two separate plugins (both 3D Viewer as Volume Viewer), I wonder whether it's a problem with my tiff-series. An image stack originates from a Fluoview500 tiff file, which was converted for use with ImageJ using the Save_8bit_MultiCH plugin in combination with the LOCI plugin. This generates a separate tiff stack for each channel. So everything seems perfect, untill I try to render them to 3D. I used the same plugin to convert the single channel images to a tiff stack, without any 3D viewing problems.
>>
>> I noticed before that I can not use the LOCI plugin as such to import my FV tiffs, because channels and z-position get mixed up: I always get a stack with repeatedly CH1; CH2; CH3; CH1; CH2; CH3... as if these are the z-slices, and this divided over 3 'channels', which are in fact not the channels. This happens with every setting I tried (e.g. changing the xyzct order). Luckily, this problem was solved when using the Save_8bit_MultiCH plugin, but now I wonder there is maybe some error left in the metadata that gets the 3D/Volume Viewer confused about the stack.
>>
>> Does anyone recognize this problem? Or is there something obvious that I'm missing here? I would be very gratefull if I could find a solution for it, since I'm promoting ImageJ towards my colleagues who are now using  the very expensive program from Zurich...
>>
>
> My first thought is that the calibration in the z direction may be wrong/to
> small. Can you check this? (->Image->Properties: Voxel depth).
>
> If the voxel depth seems to make sense, could you post the dimensions of
> the image, i.e. what you see in the dialog when clicking
> Image->Hyperstacks->Stack to Hyperstack.
>
>
> Best wishes,
> Bene

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

[Katinka Wouters]

Hi again, Glen!

After reinstalling ImageJ and downloading the LOCI over again, the stack order is now OK when importing through LOCI. So, again, one problem solved (maybe I was mistaken and did not use the newest, stable, version of LOCI after all)

But, when importing as a hyperstack as you proposed, the voxel properties are still not preserved. All is set to zero immediately after importing (so not only after reopening in ImageJ). Fortunately, when adjusting the voxel settings and subsequent saving the image, the voxel settings are saved as well (which is not the case when they were adjusted in a regular z-stack). So, thanks for introducing me to 'hyperstacks'! If you would have a suggestion for another way to import, keeping the voxel settings: I'm still interested! It would save me a lot of time, since my voxel depths differ between images, so I have to look it up in my labbook for every stack...

Then, another question about orthosections, for anyone who might have some experience with it:
When looking at orthogonal views (image\stacks\orthogonal views): is there a way to save the 'entire' project as one image, so xy, flanked by xz and yz, including cross hairs? Up till now, I've only been able to save the 3 of them separately, without crosshairs. Probably it's just a way of combining the three before saving, but I can't seem to find a function for it.
The 'ortoslice' view in the 3D viewer doesn't do it for me, since I should be able to choose where to intersect. I tried VolumeJ and Volume Viewer, but couldn't really find a nice way of choosing and/or saving orthoslices.

Thanks!

Katinka

 


-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Katinka Wouters
Sent: Monday, July 26, 2010 8:40 AM
To: [hidden email]
Subject: Re: multichannel 3D viewing problem with Fluoview500 tiffs

Hi Glen,


Dear Katinka,
Scrolling through the stack is simply stepping between the images.  The voxel depth value is assigned at acquisition and written into the file metadata.  The  TIFF  does not have a native tag for storing the axial dimension.  Your macro may be converting the Fluoview file to tiff in a manner which causes that value to be lost.
-- Indeed, that's what I thought, thanks for clarifying

ImageJ writes voxel depth to the TIFF comments tag and reads it back out, if you re-open in ImageJ. Have you tried the current stable version of LOCI, 4.2.0 to open your files?  
-- yes I did

If that does not correctly set channel order, then you can select the correct channel order by the Stack Order dropdown menu in the upper left region of the plugin window.  
-- tried this, but there's no change in my outcome whether I choose xyzct, xyczt, etc... when I want to import with separating channels

Importing as Hyperstack with Composite color will maintain voxel properties and does a better job of channel color than does the Standard ImageJ Viewer option. Use Hyperstacks>Channels Tool>More to change channel colors. Use Hyperstacks>Channels Tool>More>Convert to RGB will create an 8-bit/channel RGB with voxel depth (record this once and save as a macro) for the 3D viewer.  
-- I'll try this one, thanks!

Katinka

  Regards,Glen

On Jul 23, 2010, at 4:21 AM, Katinka Wouters wrote:

> Hi Bene,
>
> Thanks for your very fast reply. I checked the image properties, and indeed, the voxel depth is at 0.0000000. So, problem solved! Any idea why the macro changes the voxel depths for multichannel images this dramatically?
>
> I will now try to use a SetVoxelDepth macro, in order to keep the process automated.
>
> Thanks a lot, you made my day!
>
> Katinka
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Benjamin Schmid
> Sent: Friday, July 23, 2010 1:09 PM
> To: [hidden email]
> Subject: Re: multichannel 3D viewing problem with Fluoview500 tiffs
>
> Hi Katinka,
>>
>> For a while, I've been using ImageJ 3D viewer or volume viewer to visualize bacterial biofilms scanned with an Olympus Fluoview 500. It works perfectly for single channel images, thanks a lot to the developers!
>>
>> But, when I'm using it for multichannel images (so in my case: multispecies biofilms), my '3D' images appear 'flat' (like 2D), in both the 3D viewer and the volume viewer. When I rotate them, I get just one plane. This is particularly strange, since I can still browse through the Z-slices in the stack from which the 3D-image originate: all the (over 40) z-slices are there. So, I have a perfect 8 bit z-stack of each channel, but get a 2D image from them, both from the separate channels as from a stack in which the channels have been merged first.
>>
>> Because this problem occurs in two separate plugins (both 3D Viewer as Volume Viewer), I wonder whether it's a problem with my tiff-series. An image stack originates from a Fluoview500 tiff file, which was converted for use with ImageJ using the Save_8bit_MultiCH plugin in combination with the LOCI plugin. This generates a separate tiff stack for each channel. So everything seems perfect, untill I try to render them to 3D. I used the same plugin to convert the single channel images to a tiff stack, without any 3D viewing problems.
>>
>> I noticed before that I can not use the LOCI plugin as such to import my FV tiffs, because channels and z-position get mixed up: I always get a stack with repeatedly CH1; CH2; CH3; CH1; CH2; CH3... as if these are the z-slices, and this divided over 3 'channels', which are in fact not the channels. This happens with every setting I tried (e.g. changing the xyzct order). Luckily, this problem was solved when using the Save_8bit_MultiCH plugin, but now I wonder there is maybe some error left in the metadata that gets the 3D/Volume Viewer confused about the stack.
>>
>> Does anyone recognize this problem? Or is there something obvious that I'm missing here? I would be very gratefull if I could find a solution for it, since I'm promoting ImageJ towards my colleagues who are now using  the very expensive program from Zurich...
>>
>
> My first thought is that the calibration in the z direction may be wrong/to
> small. Can you check this? (->Image->Properties: Voxel depth).
>
> If the voxel depth seems to make sense, could you post the dimensions of
> the image, i.e. what you see in the dialog when clicking
> Image->Hyperstacks->Stack to Hyperstack.
>
>
> Best wishes,
> Bene

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

[Glen MacDonald-2]

Dear Katinka,
Are you saving your files in the OIF or OIB format?  If using OIF, select the file "filename.oif" in LOCI open dialog.  Select OME metadata from the LOCI menu to display the metadata after the file opens. then look at the line '< Pixels' to get the PhysicalSizeX, Y, Z  dimensions and verify they were written to the metadata.  

In your second question, you have to create a new image and paste in the orthogonal views.  The sequence of operations could be recorded and turned into a macro.

regards,
Glen
On Jul 26, 2010, at 3:36 AM, Katinka Wouters wrote:

> Hi again, Glen!
>
> After reinstalling ImageJ and downloading the LOCI over again, the stack order is now OK when importing through LOCI. So, again, one problem solved (maybe I was mistaken and did not use the newest, stable, version of LOCI after all)
>
> But, when importing as a hyperstack as you proposed, the voxel properties are still not preserved. All is set to zero immediately after importing (so not only after reopening in ImageJ). Fortunately, when adjusting the voxel settings and subsequent saving the image, the voxel settings are saved as well (which is not the case when they were adjusted in a regular z-stack). So, thanks for introducing me to 'hyperstacks'! If you would have a suggestion for another way to import, keeping the voxel settings: I'm still interested! It would save me a lot of time, since my voxel depths differ between images, so I have to look it up in my labbook for every stack...
>
> Then, another question about orthosections, for anyone who might have some experience with it:
> When looking at orthogonal views (image\stacks\orthogonal views): is there a way to save the 'entire' project as one image, so xy, flanked by xz and yz, including cross hairs? Up till now, I've only been able to save the 3 of them separately, without crosshairs. Probably it's just a way of combining the three before saving, but I can't seem to find a function for it.
> The 'ortoslice' view in the 3D viewer doesn't do it for me, since I should be able to choose where to intersect. I tried VolumeJ and Volume Viewer, but couldn't really find a nice way of choosing and/or saving orthoslices.
>
> Thanks!
>
> Katinka
>
>
>
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Katinka Wouters
> Sent: Monday, July 26, 2010 8:40 AM
> To: [hidden email]
> Subject: Re: multichannel 3D viewing problem with Fluoview500 tiffs
>
> Hi Glen,
>
>
> Dear Katinka,
> Scrolling through the stack is simply stepping between the images.  The voxel depth value is assigned at acquisition and written into the file metadata.  The  TIFF  does not have a native tag for storing the axial dimension.  Your macro may be converting the Fluoview file to tiff in a manner which causes that value to be lost.
> -- Indeed, that's what I thought, thanks for clarifying
>
> ImageJ writes voxel depth to the TIFF comments tag and reads it back out, if you re-open in ImageJ. Have you tried the current stable version of LOCI, 4.2.0 to open your files?  
> -- yes I did
>
> If that does not correctly set channel order, then you can select the correct channel order by the Stack Order dropdown menu in the upper left region of the plugin window.  
> -- tried this, but there's no change in my outcome whether I choose xyzct, xyczt, etc... when I want to import with separating channels
>
> Importing as Hyperstack with Composite color will maintain voxel properties and does a better job of channel color than does the Standard ImageJ Viewer option. Use Hyperstacks>Channels Tool>More to change channel colors. Use Hyperstacks>Channels Tool>More>Convert to RGB will create an 8-bit/channel RGB with voxel depth (record this once and save as a macro) for the 3D viewer.  
> -- I'll try this one, thanks!
>
> Katinka
>
>  Regards,Glen
>
> On Jul 23, 2010, at 4:21 AM, Katinka Wouters wrote:
>
>> Hi Bene,
>>
>> Thanks for your very fast reply. I checked the image properties, and indeed, the voxel depth is at 0.0000000. So, problem solved! Any idea why the macro changes the voxel depths for multichannel images this dramatically?
>>
>> I will now try to use a SetVoxelDepth macro, in order to keep the process automated.
>>
>> Thanks a lot, you made my day!
>>
>> Katinka
>>
>> -----Original Message-----
>> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Benjamin Schmid
>> Sent: Friday, July 23, 2010 1:09 PM
>> To: [hidden email]
>> Subject: Re: multichannel 3D viewing problem with Fluoview500 tiffs
>>
>> Hi Katinka,
>>>
>>> For a while, I've been using ImageJ 3D viewer or volume viewer to visualize bacterial biofilms scanned with an Olympus Fluoview 500. It works perfectly for single channel images, thanks a lot to the developers!
>>>
>>> But, when I'm using it for multichannel images (so in my case: multispecies biofilms), my '3D' images appear 'flat' (like 2D), in both the 3D viewer and the volume viewer. When I rotate them, I get just one plane. This is particularly strange, since I can still browse through the Z-slices in the stack from which the 3D-image originate: all the (over 40) z-slices are there. So, I have a perfect 8 bit z-stack of each channel, but get a 2D image from them, both from the separate channels as from a stack in which the channels have been merged first.
>>>
>>> Because this problem occurs in two separate plugins (both 3D Viewer as Volume Viewer), I wonder whether it's a problem with my tiff-series. An image stack originates from a Fluoview500 tiff file, which was converted for use with ImageJ using the Save_8bit_MultiCH plugin in combination with the LOCI plugin. This generates a separate tiff stack for each channel. So everything seems perfect, untill I try to render them to 3D. I used the same plugin to convert the single channel images to a tiff stack, without any 3D viewing problems.
>>>
>>> I noticed before that I can not use the LOCI plugin as such to import my FV tiffs, because channels and z-position get mixed up: I always get a stack with repeatedly CH1; CH2; CH3; CH1; CH2; CH3... as if these are the z-slices, and this divided over 3 'channels', which are in fact not the channels. This happens with every setting I tried (e.g. changing the xyzct order). Luckily, this problem was solved when using the Save_8bit_MultiCH plugin, but now I wonder there is maybe some error left in the metadata that gets the 3D/Volume Viewer confused about the stack.
>>>
>>> Does anyone recognize this problem? Or is there something obvious that I'm missing here? I would be very gratefull if I could find a solution for it, since I'm promoting ImageJ towards my colleagues who are now using  the very expensive program from Zurich...
>>>
>>
>> My first thought is that the calibration in the z direction may be wrong/to
>> small. Can you check this? (->Image->Properties: Voxel depth).
>>
>> If the voxel depth seems to make sense, could you post the dimensions of
>> the image, i.e. what you see in the dialog when clicking
>> Image->Hyperstacks->Stack to Hyperstack.
>>
>>
>> Best wishes,
>> Bene
>
>
>
> Glen MacDonald
> Core for Communication Research
> Virginia Merrill Bloedel Hearing Research Center
> Box 357923
> University of Washington
> Seattle, WA 98195-7923  USA
> [phone]
> [hidden email]

[Felipe Franco]

          Guys,

          I m working in a molecular imaging project and we need to measure the fluorescence of organs in diferents intervals, not in a specific time. I just cannot find any software that is able to take an video stream ( working in set intervals ) and transforme it in a stack .tiff. Do you guys have some sugestion? Do you guys  think that if i convert my .vob file ( dvd video ) in quick time and then into a stack will lose some quality?

           I need to point out that this video has about 1 hour in high quality and I intent to take some intervals between 5 and 15 minutes.

           Thanks
     

[dscho]

On Mon, 2 Aug 2010, Felipe Franco wrote:

>           Guys,

            Girl,

>           I m working in a molecular imaging project and we need to
>           measure the fluorescence of organs in diferents intervals, not
>           in a specific time. I just cannot find any software that is
>           able to take an video stream ( working in set intervals ) and
>           transforme it in a stack .tiff. Do you guys have some
>           sugestion? Do you guys think that if i convert my .vob file (
>           dvd video ) in quick time and then into a stack will lose some
>           quality?

There are a number of tools, the easiest method on Linux is to use mplayer
with the "-vo png" option to populate a directory of .png files which you
then read back via File>Import>Image Sequence...

However, guessing (I cannot know because you neglected to let us know that
pretty important piece of information) that you are rather a Windows guy,
you could use VirtualDub:

http://www.virtualdub.org/blog/pivot/entry.php?id=34

By the way, I fail to see the reason why your mail is part of the
"multichannel 3D viewing problem with Fluoview500 tiffs + orthosections"
thread:

http://thread.gmane.org/gmane.comp.java.imagej/18923/focus=19032

Hth,
Johannes