What has happened to "extended focus" in Leica Software?

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Torsten.Fregin Torsten.Fregin
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What has happened to "extended focus" in Leica Software?

Hey,

a "long" time ago I used an Leica TCS NT confocal, and I remember that I was able to view
the 3D stacks with two differend methods in 2D: "Extended Focus" and "Projection",
respectively.

With our new Leica Confocal with the new LAS AF software, I have just one way to view the
stacks: "Maximum projection", which gives me results same as with the old "Projection". (If I
open old .tiff files and process them)...

The old "ExtFoc" worked for me quite well sometimes, the new "Maximum Projection" very
often needs deconvolution to get a clear, that is, crisp image...

Does anyone remember what the difference was between the two modes in the old software?

:-) Torsten

 
Jonathan Boyd Jonathan Boyd
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Re: What has happened to "extended focus" in Leica Software?

Hi Torsten,

Under the Processing tab go to 3D projection.  Look under the dropdown tab. It defaults to Max projection.  You will see there are other methods for rendering your volumes in this dropdown.

Hope this helps,

Jonathan
 
Jonathan Boyd
 Confocal Application Specialist - Life Science Division
 Leica Microsystems, Inc.
 Living up to Life

 (415) 515-9964

 [hidden email]
 http://www.leica-microsystems.com/

 Click Below to view our current promotions!
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----- Original Message -----
From: Torsten.Fregin
Sent: 01/21/2010 04:38 PM CET
To: [hidden email]
Subject: What has happened to "extended focus" in Leica Software?



Hey,

a "long" time ago I used an Leica TCS NT confocal, and I remember that I was able to view
the 3D stacks with two differend methods in 2D: "Extended Focus" and "Projection",
respectively.

With our new Leica Confocal with the new LAS AF software, I have just one way to view the
stacks: "Maximum projection", which gives me results same as with the old "Projection". (If I
open old .tiff files and process them)...

The old "ExtFoc" worked for me quite well sometimes, the new "Maximum Projection" very
often needs deconvolution to get a clear, that is, crisp image...

Does anyone remember what the difference was between the two modes in the old software?

:-) Torsten

 

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Torsten.Fregin Torsten.Fregin
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Re: What has happened to "extended focus" in Leica Software?

Hi Jonathan,

I cannot check it right now, b/c the PC I am working with has only the LAS AF Light version installed, and it is a "NO LICENCE!" feature, so I cannot go to the dropdown tab... :-(

But I think to remember that there are "Maximum" and "Average", and my impression was that "average" does not exactly do the same as the old "Extended Focus" - is it the same algorithm??

Thanks so far!

:-) Torsten


On 20 Jan 2010 at 10:07, Jonathan Boyd wrote:

> Hi Torsten,
>
> Under the Processing tab go to 3D projection.  Look under the dropdown tab. It defaults to Max projection.  You will see there are other methods for rendering your volumes in this dropdown.
>
> Hope this helps,
>
> Jonathan
  
rjpalmer rjpalmer
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Re: What has happened to "extended focus" in Leica Software?

Re: What has happened to "extended focus" in Leica Softwar
Have you contacted your Leica applications person about this?  Doesn't seem to be a topic that could take more than 10 min on the phone...... just a thought ;-)
 
Hi Jonathan,

I cannot check it right now, b/c the PC I am working with has only the LAS AF Light version installed, and it is a "NO LICENCE!" feature, so I cannot go to the dropdown tab... :-(

But I think to remember that there are "Maximum" and "Average", and my impression was that "average" does not exactly do the same as the old "Extended Focus" - is it the same algorithm??

Thanks so far!

:-) Torsten


On 20 Jan 2010 at 10:07, Jonathan Boyd wrote:

> Hi Torsten,
>
> Under the Processing tab go to 3D projection.  Look under the dropdown tab. It defaults to Max projection.  You will see there are other methods for rendering your volumes in this dropdown.
>
> Hope this helps,
>
> Jonathan
 


-- 
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396
Glen MacDonald-2 Glen MacDonald-2
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Re: What has happened to "extended focus" in Leica Software?

In reply to this post by Torsten.Fregin
The Extended Focus plugin for ImageJ can give good results,.  We found identical results compared with the Image Pro function.  
No, an average projection is not the same, but may be of benefit when working with features displaying a wide range of intensities.

Regards,
Glen
On Jan 21, 2010, at 11:16 AM, [hidden email] wrote:

> Hi Jonathan,
>
> I cannot check it right now, b/c the PC I am working with has only the LAS AF Light version installed, and it is a "NO LICENCE!" feature, so I cannot go to the dropdown tab... :-(
>
> But I think to remember that there are "Maximum" and "Average", and my impression was that "average" does not exactly do the same as the old "Extended Focus" - is it the same algorithm??
>
> Thanks so far!
>
> :-) Torsten
>
>
> On 20 Jan 2010 at 10:07, Jonathan Boyd wrote:
>
> > Hi Torsten,
> >
> > Under the Processing tab go to 3D projection.  Look under the dropdown tab. It defaults to Max projection.  You will see there are other methods for rendering your volumes in this dropdown.
> >
> > Hope this helps,
> >
> > Jonathan
>  
Guy Cox Guy Cox
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Re: What has happened to "extended focus" in Leica Software?

In reply to this post by Torsten.Fregin
There are two simple ways of projecting a stack -

1. maximum brightness: you take the brightest pixel in each vertical
column and put that in the output image
2. average: you take the average of all the pixels in each column, and
put that in the output image.  It will usually need to be normalized to
make it bright enough so you really need to work in 16 bits.

Various names have been applied to these - in his early papers Colin
Sheppard called one extended focus and the other autofocus (I think in
that order, but I'm not sure).  Usually maximum brightness will give the
clearer image whereas average will show more subtle detail - there is a
figure in my book (below) showing both.

If you are not getting a clear crisp image from a maximum brightness
projection there must be something wrong with your original image stack
- most likely saturation somewhere which is obviously going to be fatal.
(You should never have saturation .... but if you do, an average
projection will make a much better job of it).  False-colour palettes
can also sometimes be a bit funny with maximum projections since there
will be so many bright pixels - check it in grey-scale if it looks
funny.

The thought of applying deconvolution to a maximum projection makes me
want to pour myself a stiff drink - but I'd better not since it's still
mid-morning.  If ever there was an example of illegitimate image
processing that would be it!

                                          Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006

Phone +61 2 9351 3176     Fax +61 2 9351 7682
             Mobile 0413 281 861
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of [hidden email]
Sent: Friday, 22 January 2010 2:39 AM
To: [hidden email]
Subject: What has happened to "extended focus" in Leica Software?

Hey,

a "long" time ago I used an Leica TCS NT confocal, and I remember that I
was able to view
the 3D stacks with two differend methods in 2D: "Extended Focus" and
"Projection",
respectively.

With our new Leica Confocal with the new LAS AF software, I have just
one way to view the
stacks: "Maximum projection", which gives me results same as with the
old "Projection". (If I
open old .tiff files and process them)...

The old "ExtFoc" worked for me quite well sometimes, the new "Maximum
Projection" very
often needs deconvolution to get a clear, that is, crisp image...

Does anyone remember what the difference was between the two modes in
the old software?

:-) Torsten

 

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.730 / Virus Database: 270.14.122/2590 - Release Date:
01/20/10 04:49:00
Torsten.Fregin Torsten.Fregin
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Re: What has happened to "extended focus" in Leica Software?

In reply to this post by rjpalmer
Thanks everyone for the replies!

Guy - I did the deconvolution on the whole stack, not the projection... ;-)

But I still don't know what kind of algorithm Leica used for the "Extended Focus" function in the old software package. Today I took my old .tif stacks and produced new projections (maximum and average) with the Leica LAS package, but they don't look as the old pictures I still have on my hard disk (from the old equipment)... So I get three different projection versions from a single stack.

Robert - I contacted Leica to get another licence key, as it is depends on a hardware key the setup program of the TCS NT PowerScan software produces...  (I try to install the old package on a modern PC; Win NT works fine so far). Actually, I send the first email to the list b/c I am almost always surprised about the responses... Over the years I learned a lot from the list...:-)

Glen - Thanks for this hint, I will give the ImageJ plugin a try asap!

:-)  Torsten





  
rjpalmer rjpalmer
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Re: What has happened to "extended focus" in Leica Software?

Re: What has happened to "extended focus" in Leica Softwar
Torsten - I meant ask Leica about the algorithm, which does not belong to the two "simple" ways to do a projection (maximum or average).

Thanks everyone for the replies!

Guy - I did the deconvolution on the whole stack, not the projection... ;-)

But I still don't know what kind of algorithm Leica used for the "Extended Focus" function in the old software package. Today I took my old .tif stacks and produced new projections (maximum and average) with the Leica LAS package, but they don't look as the old pictures I still have on my hard disk (from the old equipment)... So I get three different projection versions from a single stack.

Robert - I contacted Leica to get another licence key, as it is depends on a hardware key the setup program of the TCS NT PowerScan software produces...  (I try to install the old package on a modern PC; Win NT works fine so far). Actually, I send the first email to the list b/c I am almost always surprised about the responses... Over the years I learned a lot from the list...:-)

Glen - Thanks for this hint, I will give the ImageJ plugin a try asap!

:-)  Torsten





 


-- 
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396
DrSmithMartin@gmail.com DrSmithMartin@gmail.com
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Re: What has happened to "extended focus" in Leica Software?

In reply to this post by Torsten.Fregin
Assuming you do a simple top down projection then a maximum or average project will just consider a single pixel/voxel per Z section in the calculation. You would use them as Guy Cox suggests, maximum is the default and easy 1 click mode as it usually looks fine. If you took your Z-stack ok then max brightness is idiot proof. Average is better for almost saturated images.

The name extended focus implies a slightly more complex algorithm than just analysing a single voxel per plane. These algorithms will look for which Z plane has the ‘best’ information, meaning most in focus information. This is usually determined by looking at regions within each plane and looking for the local energy. The section with the highest energy for that region is selected. Unlike average and maximum brightness projections extended depth tend to vary in implementation between software programs I have seen as there are different algorithms like High-pass for picking what is in focus.

Extended focus algorithms are traditionally best for transmitted light images where the inverted contrast makes techniques like max brightness and average not suitable.

I would just ask what the replacement for this previous Extended focus is and what they recommend for the transmission source is.

Regards
 - Martin


On Thu, Jan 21, 2010 at 2:39 PM, Robert J. Palmer Jr. <[hidden email]> wrote:
Torsten - I meant ask Leica about the algorithm, which does not belong to the two "simple" ways to do a projection (maximum or average).

Thanks everyone for the replies!

Guy - I did the deconvolution on the whole stack, not the projection... ;-)

But I still don't know what kind of algorithm Leica used for the "Extended Focus" function in the old software package. Today I took my old .tif stacks and produced new projections (maximum and average) with the Leica LAS package, but they don't look as the old pictures I still have on my hard disk (from the old equipment)... So I get three different projection versions from a single stack.

Robert - I contacted Leica to get another licence key, as it is depends on a hardware key the setup program of the TCS NT PowerScan software produces...  (I try to install the old package on a modern PC; Win NT works fine so far). Actually, I send the first email to the list b/c I am almost always surprised about the responses... Over the years I learned a lot from the list...:-)

Glen - Thanks for this hint, I will give the ImageJ plugin a try asap!

:-)  Torsten





 


-- 
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396

Boswell, Carl A - (cboswell) Boswell, Carl A - (cboswell)
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Re: What has happened to "extended focus" in Leica Software?

In reply to this post by Guy Cox
My interpretation of extended focus is the ability to take only the in-focus
(high contrast) portion of images from an image stack and make a composite,
effectively increasing depth of field well beyond the original objective's
specs.  This would be in lieu of a confocal image, and is quite useful for
non-flourescent specimens acquired from bright field microscopes.  ImageJ
has an algoritm for this, and the free software, Helicon Focus
http://HeliconFocus.com, does it pretty well.

Maybe this is a new use of an older term.

Cheers,
C

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX 520-621-3709
----- Original Message -----
From: "Guy Cox" <[hidden email]>
To: <[hidden email]>
Sent: Wednesday, January 20, 2010 5:00 PM
Subject: Re: What has happened to "extended focus" in Leica Software?


There are two simple ways of projecting a stack -

1. maximum brightness: you take the brightest pixel in each vertical
column and put that in the output image
2. average: you take the average of all the pixels in each column, and
put that in the output image.  It will usually need to be normalized to
make it bright enough so you really need to work in 16 bits.

Various names have been applied to these - in his early papers Colin
Sheppard called one extended focus and the other autofocus (I think in
that order, but I'm not sure).  Usually maximum brightness will give the
clearer image whereas average will show more subtle detail - there is a
figure in my book (below) showing both.

If you are not getting a clear crisp image from a maximum brightness
projection there must be something wrong with your original image stack
- most likely saturation somewhere which is obviously going to be fatal.
(You should never have saturation .... but if you do, an average
projection will make a much better job of it).  False-colour palettes
can also sometimes be a bit funny with maximum projections since there
will be so many bright pixels - check it in grey-scale if it looks
funny.

The thought of applying deconvolution to a maximum projection makes me
want to pour myself a stiff drink - but I'd better not since it's still
mid-morning.  If ever there was an example of illegitimate image
processing that would be it!

                                          Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006

Phone +61 2 9351 3176     Fax +61 2 9351 7682
             Mobile 0413 281 861
______________________________________________
      http://www.guycox.net



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of [hidden email]
Sent: Friday, 22 January 2010 2:39 AM
To: [hidden email]
Subject: What has happened to "extended focus" in Leica Software?

Hey,

a "long" time ago I used an Leica TCS NT confocal, and I remember that I
was able to view
the 3D stacks with two differend methods in 2D: "Extended Focus" and
"Projection",
respectively.

With our new Leica Confocal with the new LAS AF software, I have just
one way to view the
stacks: "Maximum projection", which gives me results same as with the
old "Projection". (If I
open old .tiff files and process them)...

The old "ExtFoc" worked for me quite well sometimes, the new "Maximum
Projection" very
often needs deconvolution to get a clear, that is, crisp image...

Does anyone remember what the difference was between the two modes in
the old software?

:-) Torsten



No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.730 / Virus Database: 270.14.122/2590 - Release Date:
01/20/10 04:49:00
Lloyd Donaldson Lloyd Donaldson
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Re: What has happened to "extended focus" in Leica Software?

In reply to this post by Torsten.Fregin

Torsten

 

The TCS NT implementation of extended focus is a direct average of each column with the result resampled back to 8-bit. In the newer LAS implementation Leica have added a threshold feature so you can calculate an average projection without including the dark/black pixels which tend to dilute detail in parts of the sample surrounded above and below by dark pixels/voxels. The end result is a projection intermediate between average intensity and maximum intensity which is why your new images look different. I usually simplistically explain to users that maximum intensity tends to find surfaces while extended focus gives a more transparent rendering.

If you are using LAS you should be able to get a projection the same as the old software if you set the threshold to 0.

 

Dr Lloyd Donaldson

Senior Scientist

Scion - Next Generation Biomaterials

49 Sala St. Rotorua

Private Bag 3020, Rotorua 3046

NEW ZEALAND

Ph: 64 7 343 5581

Fx: 64 7 343 5507

www.scionresearch.com

 

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Saturday, 23 January 2010 1:59 a.m.
To: [hidden email]
Subject: Re: What has happened to "extended focus" in Leica Software?

 

Thanks everyone for the replies!

 

Guy - I did the deconvolution on the whole stack, not the projection... ;-)

 

But I still don't know what kind of algorithm Leica used for the "Extended Focus" function in the old software package. Today I took my old .tif stacks and produced new projections (maximum and average) with the Leica LAS package, but they don't look as the old pictures I still have on my hard disk (from the old equipment)... So I get three different projection versions from a single stack.

 

Robert - I contacted Leica to get another licence key, as it is depends on a hardware key the setup program of the TCS NT PowerScan software produces...  (I try to install the old package on a modern PC; Win NT works fine so far). Actually, I send the first email to the list b/c I am almost always surprised about the responses... Over the years I learned a lot from the list...:-)

 

Glen - Thanks for this hint, I will give the ImageJ plugin a try asap!

 

:-)  Torsten

 

 

 

 

 

  



Disclaimer: This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the course of Scion’s usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.
Glen MacDonald-2 Glen MacDonald-2
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Re: What has happened to "extended focus" in Leica Software?

So Carl's concern was correct, this is redefining the term.  If one does not have access to the Leica software, ImageJ can do this through the 3D Project... command (requires 8-bit or RGB).  select projection method and choose Z-Axis for axis of rotation.  Set Total Rotation = 0 and enter your desired Lower and Upper Transparency Bounds to set the threshold.  Look at the ImageJ  documentation for using the depth cueing and opacity settings.

Regards,
Glen

On Jan 21, 2010, at 11:16 AM, Lloyd Donaldson wrote:

> Torsten
>  
> The TCS NT implementation of extended focus is a direct average of each column with the result resampled back to 8-bit. In the newer LAS implementation Leica have added a threshold feature so you can calculate an average projection without including the dark/black pixels which tend to dilute detail in parts of the sample surrounded above and below by dark pixels/voxels. The end result is a projection intermediate between average intensity and maximum intensity which is why your new images look different. I usually simplistically explain to users that maximum intensity tends to find surfaces while extended focus gives a more transparent rendering.
> If you are using LAS you should be able to get a projection the same as the old software if you set the threshold to 0.
>  
> Dr Lloyd Donaldson
> Senior Scientist
> Scion - Next Generation Biomaterials
> 49 Sala St. Rotorua
> Private Bag 3020, Rotorua 3046
> NEW ZEALAND
> Ph: 64 7 343 5581
> Fx: 64 7 343 5507
> www.scionresearch.com
>  
>  
>  
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
> Sent: Saturday, 23 January 2010 1:59 a.m.
> To: [hidden email]
> Subject: Re: What has happened to "extended focus" in Leica Software?
>  
> Thanks everyone for the replies!
>  
> Guy - I did the deconvolution on the whole stack, not the projection... ;-)
>  
> But I still don't know what kind of algorithm Leica used for the "Extended Focus" function in the old software package. Today I took my old .tif stacks and produced new projections (maximum and average) with the Leica LAS package, but they don't look as the old pictures I still have on my hard disk (from the old equipment)... So I get three different projection versions from a single stack.
>  
> Robert - I contacted Leica to get another licence key, as it is depends on a hardware key the setup program of the TCS NT PowerScan software produces...  (I try to install the old package on a modern PC; Win NT works fine so far). Actually, I send the first email to the list b/c I am almost always surprised about the responses... Over the years I learned a lot from the list...:-)
>  
> Glen - Thanks for this hint, I will give the ImageJ plugin a try asap!
>  
> :-)  Torsten
>  
>  
>  
>  
>  
>  
>
> Disclaimer: This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
> Scion does not accept responsibility for anything in this e-mail which is not provided in the course of Scion’s usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.



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]
Guy Cox Guy Cox
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Re: What has happened to "extended focus" in Leica Software?

In reply to this post by Torsten.Fregin

>Guy - I did the deconvolution on the whole stack, not the projection... ;-)

 

Phew, that’s a relief!

 

>But I still don't know what kind of algorithm Leica used for the "Extended Focus" function in the old software package. Today I took my old .tif stacks and produced new projections (maximum and average) with the Leica LAS package, but they don't look as the old pictures I still have on my hard disk (from the old equipment)... So I get three different projection versions from a single stack.

 

As others have suggested, try using some other package such as Image J where the functions are well defined and see what they do.

 

Essentially there are three ways of projecting a series – average and maximum as I mentioned before, and ‘alpha-blending’ or ‘rendering’ where you assign more prominence to the voxels at the top of the stack – ie those ‘closest’ to you.  Once you get into this there are several algorithms and endless possible variations of settings and no two packages will give the same result.  Maybe Leica are doing something like this now? 

 

The only other thing I can think of is that in the old NT software Leica  were doing an average with insufficient bit depth so that there were rounding errors – it seems unlikely that they would do that now. 

 

                                                                                             Guy

 

 

Guy Cox Guy Cox
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Re: What has happened to "extended focus" in Leica Software?

In reply to this post by DrSmithMartin@gmail.com

The sort of algorithm that  Martin mentions is a very clever way for getting, electronically, an extended depth of field in a wide-field microscope.  As he says, there are several ways to do it – one is to blur the image at each plane and subtract it from  the original image.  Out of focus stuff won’t change much so will be removed – in focus stuff will change a lot and will stay (in fact be enhanced).  But this doesn’t really apply to confocal imaging since (in principle, anyway) there shouldn’t be any out of focus information.  Therefore it seems unlikely Leica were doing anything like that. 

 

One slightly ‘off the wall’ idea I just had was that maybe they were doing BOTH max and average projections than taking the mean of the two.  In principle that could give quite a nice result – has anyone tried it?

 

                                                                                                                Guy

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Electron Microscope Unit, Madsen Building F09,

University of Sydney, NSW 2006

 

Phone +61 2 9351 3176     Fax +61 2 9351 7682

             Mobile 0413 281 861

______________________________________________

      http://www.guycox.net

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Friday, 22 January 2010 12:44 AM
To: [hidden email]
Subject: Re: What has happened to "extended focus" in Leica Software?

 

Assuming you do a simple top down projection then a maximum or average project will just consider a single pixel/voxel per Z section in the calculation. You would use them as Guy Cox suggests, maximum is the default and easy 1 click mode as it usually looks fine. If you took your Z-stack ok then max brightness is idiot proof. Average is better for almost saturated images.

The name extended focus implies a slightly more complex algorithm than just analysing a single voxel per plane. These algorithms will look for which Z plane has the ‘best’ information, meaning most in focus information. This is usually determined by looking at regions within each plane and looking for the local energy. The section with the highest energy for that region is selected. Unlike average and maximum brightness projections extended depth tend to vary in implementation between software programs I have seen as there are different algorithms like High-pass for picking what is in focus.

Extended focus algorithms are traditionally best for transmitted light images where the inverted contrast makes techniques like max brightness and average not suitable.

I would just ask what the replacement for this previous Extended focus is and what they recommend for the transmission source is.

Regards
 - Martin

On Thu, Jan 21, 2010 at 2:39 PM, Robert J. Palmer Jr. <[hidden email]> wrote:

Torsten - I meant ask Leica about the algorithm, which does not belong to the two "simple" ways to do a projection (maximum or average).

 

Thanks everyone for the replies!

 

Guy - I did the deconvolution on the whole stack, not the projection... ;-)

 

But I still don't know what kind of algorithm Leica used for the "Extended Focus" function in the old software package. Today I took my old .tif stacks and produced new projections (maximum and average) with the Leica LAS package, but they don't look as the old pictures I still have on my hard disk (from the old equipment)... So I get three different projection versions from a single stack.

 

Robert - I contacted Leica to get another licence key, as it is depends on a hardware key the setup program of the TCS NT PowerScan software produces...  (I try to install the old package on a modern PC; Win NT works fine so far). Actually, I send the first email to the list b/c I am almost always surprised about the responses... Over the years I learned a lot from the list...:-)

 

Glen - Thanks for this hint, I will give the ImageJ plugin a try asap!

 

:-)  Torsten

 

 

 

 

 

 

 

 

-- 

Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396

 

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.730 / Virus Database: 270.14.122/2590 - Release Date: 01/21/10 06:18:00

Lloyd Donaldson Lloyd Donaldson
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Re: What has happened to "extended focus" in Leica Software?

I tried Guy's suggestion. The average of a maximum and an average projection looks like a maximum projection with reduced noise. In other words it looks better.

If anyone is interested a standard deviation projection also looks better than the maximum projection.

Thanks for the suggestion Guy.

 

 

Dr Lloyd Donaldson

Senior Scientist

Scion - Next Generation Biomaterials

49 Sala St. Rotorua

Private Bag 3020, Rotorua 3046

NEW ZEALAND

Ph: 64 7 343 5581

Fx: 64 7 343 5507

www.scionresearch.com

 

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Friday, 22 January 2010 1:32 p.m.
To: [hidden email]
Subject: Re: What has happened to "extended focus" in Leica Software?

 

The sort of algorithm that  Martin mentions is a very clever way for getting, electronically, an extended depth of field in a wide-field microscope.  As he says, there are several ways to do it – one is to blur the image at each plane and subtract it from  the original image.  Out of focus stuff won’t change much so will be removed – in focus stuff will change a lot and will stay (in fact be enhanced).  But this doesn’t really apply to confocal imaging since (in principle, anyway) there shouldn’t be any out of focus information.  Therefore it seems unlikely Leica were doing anything like that. 

 

One slightly ‘off the wall’ idea I just had was that maybe they were doing BOTH max and average projections than taking the mean of the two.  In principle that could give quite a nice result – has anyone tried it?

 

                                                                                                                Guy

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Electron Microscope Unit, Madsen Building F09,

University of Sydney, NSW 2006

 

Phone +61 2 9351 3176     Fax +61 2 9351 7682

             Mobile 0413 281 861

______________________________________________

      http://www.guycox.net

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Friday, 22 January 2010 12:44 AM
To: [hidden email]
Subject: Re: What has happened to "extended focus" in Leica Software?

 

Assuming you do a simple top down projection then a maximum or average project will just consider a single pixel/voxel per Z section in the calculation. You would use them as Guy Cox suggests, maximum is the default and easy 1 click mode as it usually looks fine. If you took your Z-stack ok then max brightness is idiot proof. Average is better for almost saturated images.

The name extended focus implies a slightly more complex algorithm than just analysing a single voxel per plane. These algorithms will look for which Z plane has the ‘best’ information, meaning most in focus information. This is usually determined by looking at regions within each plane and looking for the local energy. The section with the highest energy for that region is selected. Unlike average and maximum brightness projections extended depth tend to vary in implementation between software programs I have seen as there are different algorithms like High-pass for picking what is in focus.

Extended focus algorithms are traditionally best for transmitted light images where the inverted contrast makes techniques like max brightness and average not suitable.

I would just ask what the replacement for this previous Extended focus is and what they recommend for the transmission source is.

Regards
 - Martin

On Thu, Jan 21, 2010 at 2:39 PM, Robert J. Palmer Jr. <[hidden email]> wrote:

Torsten - I meant ask Leica about the algorithm, which does not belong to the two "simple" ways to do a projection (maximum or average).

 

Thanks everyone for the replies!

 

Guy - I did the deconvolution on the whole stack, not the projection... ;-)

 

But I still don't know what kind of algorithm Leica used for the "Extended Focus" function in the old software package. Today I took my old .tif stacks and produced new projections (maximum and average) with the Leica LAS package, but they don't look as the old pictures I still have on my hard disk (from the old equipment)... So I get three different projection versions from a single stack.

 

Robert - I contacted Leica to get another licence key, as it is depends on a hardware key the setup program of the TCS NT PowerScan software produces...  (I try to install the old package on a modern PC; Win NT works fine so far). Actually, I send the first email to the list b/c I am almost always surprised about the responses... Over the years I learned a lot from the list...:-)

 

Glen - Thanks for this hint, I will give the ImageJ plugin a try asap!

 

:-)  Torsten

 

 

 

 

 

 

 

 

-- 

Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396

 

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.730 / Virus Database: 270.14.122/2590 - Release Date: 01/21/10 06:18:00



Disclaimer: This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the course of Scion’s usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.
Anda Cornea Anda Cornea
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Re: What has happened to "extended focus" in Leica Software?

I wonder for what type of images, the standard deviation projection may look better than the Max.  Does not seem intuitive…

 

Anda Cornea, Ph.D.

Director of the Imaging Core

Oregon National Primate Research Center

Oregon Heath & Science University

503-690-5293

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Lloyd Donaldson
Sent: Thursday, January 21, 2010 5:04 PM
To: [hidden email]
Subject: Re: What has happened to "extended focus" in Leica Software?

 

I tried Guy's suggestion. The average of a maximum and an average projection looks like a maximum projection with reduced noise. In other words it looks better.

If anyone is interested a standard deviation projection also looks better than the maximum projection.

Thanks for the suggestion Guy.

 

 

Dr Lloyd Donaldson

Senior Scientist

Scion - Next Generation Biomaterials

49 Sala St. Rotorua

Private Bag 3020, Rotorua 3046

NEW ZEALAND

Ph: 64 7 343 5581

Fx: 64 7 343 5507

www.scionresearch.com

 

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Friday, 22 January 2010 1:32 p.m.
To: [hidden email]
Subject: Re: What has happened to "extended focus" in Leica Software?

 

The sort of algorithm that  Martin mentions is a very clever way for getting, electronically, an extended depth of field in a wide-field microscope.  As he says, there are several ways to do it – one is to blur the image at each plane and subtract it from  the original image.  Out of focus stuff won’t change much so will be removed – in focus stuff will change a lot and will stay (in fact be enhanced).  But this doesn’t really apply to confocal imaging since (in principle, anyway) there shouldn’t be any out of focus information.  Therefore it seems unlikely Leica were doing anything like that. 

 

One slightly ‘off the wall’ idea I just had was that maybe they were doing BOTH max and average projections than taking the mean of the two.  In principle that could give quite a nice result – has anyone tried it?

 

                                                                                                                Guy

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Electron Microscope Unit, Madsen Building F09,

University of Sydney, NSW 2006

 

Phone +61 2 9351 3176     Fax +61 2 9351 7682

             Mobile 0413 281 861

______________________________________________

      http://www.guycox.net

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Friday, 22 January 2010 12:44 AM
To: [hidden email]
Subject: Re: What has happened to "extended focus" in Leica Software?

 

Assuming you do a simple top down projection then a maximum or average project will just consider a single pixel/voxel per Z section in the calculation. You would use them as Guy Cox suggests, maximum is the default and easy 1 click mode as it usually looks fine. If you took your Z-stack ok then max brightness is idiot proof. Average is better for almost saturated images.

The name extended focus implies a slightly more complex algorithm than just analysing a single voxel per plane. These algorithms will look for which Z plane has the ‘best’ information, meaning most in focus information. This is usually determined by looking at regions within each plane and looking for the local energy. The section with the highest energy for that region is selected. Unlike average and maximum brightness projections extended depth tend to vary in implementation between software programs I have seen as there are different algorithms like High-pass for picking what is in focus.

Extended focus algorithms are traditionally best for transmitted light images where the inverted contrast makes techniques like max brightness and average not suitable.

I would just ask what the replacement for this previous Extended focus is and what they recommend for the transmission source is.

Regards
 - Martin

On Thu, Jan 21, 2010 at 2:39 PM, Robert J. Palmer Jr. <[hidden email]> wrote:

Torsten - I meant ask Leica about the algorithm, which does not belong to the two "simple" ways to do a projection (maximum or average).

 

Thanks everyone for the replies!

 

Guy - I did the deconvolution on the whole stack, not the projection... ;-)

 

But I still don't know what kind of algorithm Leica used for the "Extended Focus" function in the old software package. Today I took my old .tif stacks and produced new projections (maximum and average) with the Leica LAS package, but they don't look as the old pictures I still have on my hard disk (from the old equipment)... So I get three different projection versions from a single stack.

 

Robert - I contacted Leica to get another licence key, as it is depends on a hardware key the setup program of the TCS NT PowerScan software produces...  (I try to install the old package on a modern PC; Win NT works fine so far). Actually, I send the first email to the list b/c I am almost always surprised about the responses... Over the years I learned a lot from the list...:-)

 

Glen - Thanks for this hint, I will give the ImageJ plugin a try asap!

 

:-)  Torsten

 

 

 

 

 

 

 

 

-- 

Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396

 

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.730 / Virus Database: 270.14.122/2590 - Release Date: 01/21/10 06:18:00

 


Disclaimer: This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the course of Scion’s usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.

Cameron Nowell Cameron Nowell
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Re: What has happened to "extended focus" in Leica Software?

Standard deviation projections work really well at turning a DIC/Transmitted stack into something that can be used for image segmentation. The result will be white/higher grey for the cells and black for the background. This is due to large fluctuations in intensity in the cell that lead to a higher standard deviation when compared to the uniform background.
 
See http://www.ncbi.nlm.nih.gov/pubmed/19847301 for a nice paper about using brightfiled images for segmentation.
 
 
Cheers
 
Cam
 
 
 
Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
 
Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104
 
http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
 

________________________________

From: Confocal Microscopy List on behalf of Anda Cornea
Sent: Fri 22/01/2010 12:09 PM
To: [hidden email]
Subject: Re: What has happened to "extended focus" in Leica Software?



I wonder for what type of images, the standard deviation projection may look better than the Max.  Does not seem intuitive...

 

Anda Cornea, Ph.D.

Director of the Imaging Core

Oregon National Primate Research Center

Oregon Heath & Science University

503-690-5293

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Lloyd Donaldson
Sent: Thursday, January 21, 2010 5:04 PM
To: [hidden email]
Subject: Re: What has happened to "extended focus" in Leica Software?

 

I tried Guy's suggestion. The average of a maximum and an average projection looks like a maximum projection with reduced noise. In other words it looks better.

If anyone is interested a standard deviation projection also looks better than the maximum projection.

Thanks for the suggestion Guy.

 

 

Dr Lloyd Donaldson

Senior Scientist

Scion - Next Generation Biomaterials

49 Sala St. Rotorua

Private Bag 3020, Rotorua 3046

NEW ZEALAND

Ph: 64 7 343 5581

Fx: 64 7 343 5507

www.scionresearch.com

 

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Friday, 22 January 2010 1:32 p.m.
To: [hidden email]
Subject: Re: What has happened to "extended focus" in Leica Software?

 

The sort of algorithm that  Martin mentions is a very clever way for getting, electronically, an extended depth of field in a wide-field microscope.  As he says, there are several ways to do it - one is to blur the image at each plane and subtract it from  the original image.  Out of focus stuff won't change much so will be removed - in focus stuff will change a lot and will stay (in fact be enhanced).  But this doesn't really apply to confocal imaging since (in principle, anyway) there shouldn't be any out of focus information.  Therefore it seems unlikely Leica were doing anything like that.  

 

One slightly 'off the wall' idea I just had was that maybe they were doing BOTH max and average projections than taking the mean of the two.  In principle that could give quite a nice result - has anyone tried it?

 

                                                                                                                Guy

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Electron Microscope Unit, Madsen Building F09,

University of Sydney, NSW 2006

 

Phone +61 2 9351 3176     Fax +61 2 9351 7682

             Mobile 0413 281 861

______________________________________________

      http://www.guycox.net <http://www.guycox.net/>

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Friday, 22 January 2010 12:44 AM
To: [hidden email]
Subject: Re: What has happened to "extended focus" in Leica Software?

 

Assuming you do a simple top down projection then a maximum or average project will just consider a single pixel/voxel per Z section in the calculation. You would use them as Guy Cox suggests, maximum is the default and easy 1 click mode as it usually looks fine. If you took your Z-stack ok then max brightness is idiot proof. Average is better for almost saturated images.

The name extended focus implies a slightly more complex algorithm than just analysing a single voxel per plane. These algorithms will look for which Z plane has the 'best' information, meaning most in focus information. This is usually determined by looking at regions within each plane and looking for the local energy. The section with the highest energy for that region is selected. Unlike average and maximum brightness projections extended depth tend to vary in implementation between software programs I have seen as there are different algorithms like High-pass for picking what is in focus.

Extended focus algorithms are traditionally best for transmitted light images where the inverted contrast makes techniques like max brightness and average not suitable.

I would just ask what the replacement for this previous Extended focus is and what they recommend for the transmission source is.

Regards
 - Martin

On Thu, Jan 21, 2010 at 2:39 PM, Robert J. Palmer Jr. <[hidden email]> wrote:

Torsten - I meant ask Leica about the algorithm, which does not belong to the two "simple" ways to do a projection (maximum or average).

 

        Thanks everyone for the replies!

         

        Guy - I did the deconvolution on the whole stack, not the projection... ;-)

         

        But I still don't know what kind of algorithm Leica used for the "Extended Focus" function in the old software package. Today I took my old .tif stacks and produced new projections (maximum and average) with the Leica LAS package, but they don't look as the old pictures I still have on my hard disk (from the old equipment)... So I get three different projection versions from a single stack.

         

        Robert - I contacted Leica to get another licence key, as it is depends on a hardware key the setup program of the TCS NT PowerScan software produces...  (I try to install the old package on a modern PC; Win NT works fine so far). Actually, I send the first email to the list b/c I am almost always surprised about the responses... Over the years I learned a lot from the list...:-)

         

        Glen - Thanks for this hint, I will give the ImageJ plugin a try asap!

         

        :-)  Torsten

         

         

         

         

         

         

 

 

--

Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396

 

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.730 / Virus Database: 270.14.122/2590 - Release Date: 01/21/10 06:18:00

 

________________________________

Disclaimer: This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the course of Scion's usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.