Well, I can't make much sense out of these. The Zeiss one has a pinhole diameter in µm but no magnification figure, so I can't see how that computes. The Olympus one doesn't say what the pinhole size is measured in, but it also contains m and M which are not explained, but I assume one of these is the mag at the pinhole.
The formula I have (from Colin Sheppard) dz = 0.4 lambda / n.sin^2 (alpha/2)
where dz is FWHM in Z, lambda is the wavelength, n is the refractive index and alpha is the half-angle of the objective.
It assumes the pinhole is open no wider than the Airy disk, and works out to 390nm for lambda 500, alpha 72 and n 1.5. This seems to be achievable in practice. Opening the pinhole wider is also opening a can of worms - you are getting some mix of confocal and widefield which will be a bit of a dog's breakfast. (How's that for a glorious mixed metaphor!)
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)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006
Phone +61 2 9351 3176 Fax +61 2 9351 7682
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______________________________________________
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Cameron Nowell
Sent: Thursday, 3 November 2011 11:10 AM
To: [log in to unmask]
Subject: Re: Deconvolution of Confocal Images? Volocity Course London
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Hi All,
There is an "optical slice thickness" calculation in each confocal system isn't there?
Ziess use this
www.fileden.com/files/2011/10/11/3207952/Zeiss%20Optical%20Thickness.png
and Olympus use this
www.fileden.com/files/2011/10/11/3207952/Olympus%20Optical%20Thickness.png
I assume the other companies use some variation of them as well.
But of course as Guy points out you should sample based on the measured (or theoretical) resolution of your system
Cheers
Cam
Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research Melbourne - Parkville Branch
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Guy Cox
Sent: Thursday, 3 November 2011 9:27 AM
To: [log in to unmask]
Subject: Re: Deconvolution of Confocal Images? Volocity Course London
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I'm a bit puzzled by this "0.9µm optical slice". If this is an NA 1.4 lens Z resolution should be 400-500nm, I've measured this FWHM on 100nm beads, so this is practical not theoretical. So you should be sampling at 200nm or less in Z to meet Nyquist. This is before thinking about oversampling for decon.
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) Australian Centre for Microscopy & Microanalysis, 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:[log in to unmask]] On Behalf Of Keith Morris
Sent: Thursday, 3 November 2011 12:59 AM
To: [log in to unmask]
Subject: Re: Deconvolution of Confocal Images? Volocity Course London
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On a practical note regarding whether to bother with deconvolving a confocal z stack using our Zeiss 510 Metahead confocal and our Volocity 3D deconvolution [Restoration] software:
The advice from one of our Perkin Elmer's Volocity software engineers was that in practice you don't see much improvement, if any, using Volocity's Restoration module to deconvolve a standard confocal z-stack. Traditionally using our Zeiss 510 we double over-sample a z-stack, i.e. for a 0.9um optical slice [63x objective, Airy=1] we take a z-slice every 0.45um in the z direction. In order to get a significant improvement over that with Volocity's Restoration module, Perkin Elmer support recommended oversampling by at least 10 [perhaps even up to 100] times rather than just 2. In practice the confocal is limited to a minimum focus z step of around 0.05um, giving about 20 Z scans when oversampling through each '0.9um optical slice'
[although you can push up the numbers, and scan time, by applying image averaging as well]. This increases the scan time by about 10x for each z stack, to typically over twenty minutes with 1024x1024 image sizes. Plus you have to make time to apply Volocity's Fast Restoration or the slower Iterative Restoration to the image z-stack.
Although time consuming, z-stack oversampling with deconvolution does get a little bit more detail from our 3D z-stacks - it's just whether you want to take another half hour to go though the acquisition and deconvolution process for every z-stack, as the extra detail may not provide anything more informative. Volocity Support added that Volocity's Restoration deconvolution module predictably looks it's best when dealing with a brighter and fuzzier z-stack from a normal wide-field microscope - where the out of focus light can be put to use rather than be simply excluded by the confocal iris. There is talk on the listserver of confocal deconvolution also producing superior 3D spatial information for quantification of sub-micron structures and well as improving resolution. Without something like serial TEM section corroboration of the structure you can't really allay the fear that the extra detail might include processing artefacts, although most of this extra detail can be seen in the original fuzzier confocal z-stack once the deconvolution has highlighted it. These days though I expect you'd also look towards a colleagues super-resolution STED/PALM system.
For those in the UK with an interest in using Volocity 3D software, next week Perkin Elmer are running a two day Volocity user training course in London on the 8th and 9th November 2011 [price £300 a day, £550 both days],
see:
http://now.eloqua.com/e/es.aspx?s=643&e=123818&elq=9c93259d524640798ee7b233e
bf0246c
Day 1: Volocity Essentials, Day 2: Volocity Quantitation
With our Volocity 3D software it tends to be the Quantitation module we use the most, with standard Zeiss [non-de]convolved 2x over-sampled confocal z-stacks, as it can measure intracellular structure volume and track objects. We have the old Volocity v4.2 [latest is v6.0], although it's used less frequently here than our 2D image analysis options, MetaMorph v7.7 and ImageJ.
Regards
Keith
I have placed a deconvolved confocal ~20x oversampled 45 slice z-stack 3D image at http://www.well.ox.ac.uk/cytogenetics/deconvolved.jpg
It's a slice from the top of a couple of Invitrogen FocalCheck fluorescent microspheres [Volocity Fast Restoration, see right image], where the diameter of the slice shown is about 10 um and the depth of the ring about 2 um.
---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core, Laboratory 00/069 and 00/070, The Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.
Telephone: +44 (0)1865 287568
Email: [log in to unmask]
Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of John Oreopoulos
Sent: 30 October 2011 20:42
To: [log in to unmask]
Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
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Peter, there are some advantages to deconvolving even confocal imaging data.
Until I took Pawley's course, I too had always seen these two imaging techniques as mutually exclusive, but that's not the case.
Here is a passage from Chapter 2 of Pawley's Handbook:
"Although deconvolution and confocal imaging are often seen as competing methods aimed at the same goal, namely producing images of 3D stain distributions, in fact, they are not exclusive, and there is much to be said for combining them. No only does deconvolution suppress the "single-pixel"
features created by Poisson noise, it also effectively averages the signal in the Nyquist-sampled image of a point object. In other words, it has the same effect of reducing the uncertainty in the estimate of the brightness in individual voxels as Kalman averaging for 64 to 125 frames. This point is so important that the present edition of this volume devotes an entire chapter to it: Chapter 25."
Now, having repeated that passage, can I say that I deconvolve my confocal data all the time? No, mainly due to lack of access to deconvolution software, and partly due to time constraints usually. But I can assure you that your images will look a bit better after deconvolving them. Apparently all the best confocalists do this, but I seldom see it practiced in the literature.
Have a look at Chapter 25 of his book. It's quite interesting.
John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca
On 2011-10-30, at 4:09 PM, Peter Werner wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> An interesting point was made here by Jim Pawley:
>
>> I agree that sampling a bit higher than Nyquist never hurts,
>> especially
if you deconvolve (as you always should), but I think that it is a mistake to think that one can "separate" out the noise by decon. I think that noise is pretty fundamental.
>
> I had always heard that if you're doing confocal microscopy, at least
point-scanning confocal with a pinhole size of 1AU or smaller, that deconvolution was superfluous, because you shouldn't be getting out of focus light. So what is gained by deconvolution when one is sampling voxel by voxel?
>
> Peter G. Werner
> Merritt College Microscopy Program
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