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
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From: Confocal Microscopy List on behalf of Anda Cornea
Sent: Fri 22/01/2010 12:09 PM
To: [log in to unmask]
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:[log in to unmask]] On Behalf Of Lloyd Donaldson
Sent: Thursday, January 21, 2010 5:04 PM
To: [log in to unmask]
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
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Scion - Next Generation Biomaterials
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From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Guy Cox
Sent: Friday, 22 January 2010 1:32 p.m.
To: [log in to unmask]
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
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http://www.guycox.net <http://www.guycox.net/>
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of [log in to unmask]
Sent: Friday, 22 January 2010 12:44 AM
To: [log in to unmask]
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. <[log in to unmask]> 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
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