Posted by
Boswell, Carl A - (cboswell) on
URL: http://confocal-microscopy-list.275.s1.nabble.com/What-has-happened-to-extended-focus-in-Leica-Software-tp4427300p4434956.html
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
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