Deconvolved Widefield versus Confocal

[Ellen T. Arena]

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Hello Everyone…

Perhaps my question is a bit naive… But I am hoping you can help me.  Our lab is purchasing a new widefield microscope with a deconvolution package - and I need to convince our users to move some of their imaging onto this machine - away from overly-booked confocal.  So - my question…  Are there circumstances I can present to our users in which wide-field deconvolved images can match/surpass confocal images?  (tissue versus cell culture, etc.?)  Most people in the lab are not doing any quantitative data.  I realize it depends on the experiment and the question at hand.  However, if I can give them a few some-what concrete examples - would be most helpful.

Thank you!

Ellen


------------------------------------------------------
Ellen T. Arena, PhD
Pasteur Foundation Postdoctoral Fellow
Unité de Pathogénie Microbienne Moléculaire
INSERM U786
Institut Pasteur
28 rue du Dr Roux
F - 75724 PARIS Cédex 15
France
Tel: [phone]
Fax: [phone]
[hidden email]
------------------------------------------------------

[Arne Seitz]

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Dear Ellen,

I suggest reading chapter 23 "Comparison of Widefield/Deconvolution and Confocal Microscopy for Three-Dimensional Imaging" form Peter J. Shaw in the "Handbook of Biological Confocal Microscopy", 3rd. Edition J. P. Pawley.

From my personal experience: it really depends on the application and although I have some experience with both techniques I'm unable to predict in which cases deconvolution is giving similar or even better results. Thus one would have to try it out. Nevertheless I'm in favour of deconvolution in the following cases:

- rather thin specimen < 10 um
- low to moderate amount of out of focus light
- High signal to noise ratio

Whenever temporal resolution is becoming an issue and/or photodamage of the specimen is critical, deconvolution is also worth giving a try.

Please do not forget that confocal images can be deconvolved as well in order to obtain even "better" images.  

Just my 2c.

Best regards
Arne


---------------------------------------------------------------
Dr. Arne Seitz
Head of Bioimaging and Optics Platform (PT-BIOP)
Ecole Polytechnique Fédérale de Lausanne (EPFL)
Faculty of Life Sciences
Station 15, AI 0241
CH-1015 Lausanne

Phone: [phone]
Fax:      [phone]
http://biop.epfl.ch/
---------------------------------------------------------------

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Ellen T.
> Arena
> Sent: mardi 5 février 2013 10:24
> To: [hidden email]
> Subject: Deconvolved Widefield versus Confocal
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello Everyone.
>
> Perhaps my question is a bit naive. But I am hoping you can help me.  Our
> lab is purchasing a new widefield microscope with a deconvolution package -
> and I need to convince our users to move some of their imaging onto this
> machine - away from overly-booked confocal.  So - my question.  Are there
> circumstances I can present to our users in which wide-field deconvolved
> images can match/surpass confocal images?  (tissue versus cell culture, etc.?)
> Most people in the lab are not doing any quantitative data.  I realize it
> depends on the experiment and the question at hand.  However, if I can give
> them a few some-what concrete examples - would be most helpful.
>
> Thank you!
>
> Ellen
>
>
> ------------------------------------------------------
> Ellen T. Arena, PhD
> Pasteur Foundation Postdoctoral Fellow
> Unité de Pathogénie Microbienne Moléculaire INSERM U786 Institut Pasteur
> 28 rue du Dr Roux
> F - 75724 PARIS Cédex 15
> France
> Tel: [phone]
> Fax: [phone]
> [hidden email]
> ------------------------------------------------------

[Guy Cox-2]

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To join, leave or search the confocal microscopy listserv, go to:
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Well, as you implicitly suggest, deconvolution is not (IMHO) quantitative (and I know I'll get some flak for this).  But it has a hugely better photon budget, so the users you should seek to shift are those with dim signals who are struggling on the confocal.  

                                                      Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ellen T. Arena
Sent: Tuesday, 5 February 2013 8:24 PM
To: [hidden email]
Subject: Deconvolved Widefield versus Confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello Everyone.

Perhaps my question is a bit naive. But I am hoping you can help me.  Our lab is purchasing a new widefield microscope with a deconvolution package - and I need to convince our users to move some of their imaging onto this machine - away from overly-booked confocal.  So - my question.  Are there circumstances I can present to our users in which wide-field deconvolved images can match/surpass confocal images?  (tissue versus cell culture, etc.?)  Most people in the lab are not doing any quantitative data.  I realize it depends on the experiment and the question at hand.  However, if I can give them a few some-what concrete examples - would be most helpful.

Thank you!

Ellen


------------------------------------------------------
Ellen T. Arena, PhD
Pasteur Foundation Postdoctoral Fellow
Unité de Pathogénie Microbienne Moléculaire INSERM U786 Institut Pasteur
28 rue du Dr Roux
F - 75724 PARIS Cédex 15
France
Tel: [phone]
Fax: [phone]
[hidden email]
------------------------------------------------------

[Chris Tully]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello,

Based on my experience, Confocal excels at thicker specimens, especially if
you have multiphonton capabilities. Also, confocal is absolutely necessary
for some whole mount specimens like C. Elegans, which since it is mostly
clear and round tends to act like a lens, making PSF determination highly
impractical (you have to determine a new PSF for every worm!), and throwing
blind deconvolution methods into non-converging states, because of the
unaccounted for "lens" otherwise known as the sample.

With that said I would strongly suggest looking at you costs for both
systems and charging usage fees accordingly. My guess is that the wide
field system will cost out enough cheaper that some people will naturally
shift to it just to stretch their research budgets.

I would also ask for a justification from the confocal users of why they
need the confocal - if it has been the only available scope then some may
have been using it by default and be happy to switch to the wide field
system.


Chris Tully
Microscopy and Image Analysis Expert
[hidden email]
240-475-9753 (c)

[image: View my profile on LinkedIn]<http://www.linkedin.com/in/christully/>


On Tue, Feb 5, 2013 at 6:43 AM, Guy Cox <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Well, as you implicitly suggest, deconvolution is not (IMHO) quantitative
> (and I know I'll get some flak for this).  But it has a hugely better
> photon budget, so the users you should seek to shift are those with dim
> signals who are struggling on the confocal.
>
>                                                       Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Ellen T. Arena
> Sent: Tuesday, 5 February 2013 8:24 PM
> To: [hidden email]
> Subject: Deconvolved Widefield versus Confocal
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello Everyone.
>
> Perhaps my question is a bit naive. But I am hoping you can help me.  Our
> lab is purchasing a new widefield microscope with a deconvolution package -
> and I need to convince our users to move some of their imaging onto this
> machine - away from overly-booked confocal.  So - my question.  Are there
> circumstances I can present to our users in which wide-field deconvolved
> images can match/surpass confocal images?  (tissue versus cell culture,
> etc.?)  Most people in the lab are not doing any quantitative data.  I
> realize it depends on the experiment and the question at hand.  However, if
> I can give them a few some-what concrete examples - would be most helpful.
>
> Thank you!
>
> Ellen
>
>
> ------------------------------------------------------
> Ellen T. Arena, PhD
> Pasteur Foundation Postdoctoral Fellow
> Unité de Pathogénie Microbienne Moléculaire INSERM U786 Institut Pasteur
> 28 rue du Dr Roux
> F - 75724 PARIS Cédex 15
> France
> Tel: [phone]
> Fax: [phone]
> [hidden email]
> ------------------------------------------------------
>

[Kevin Ryan]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Disclosure - Media Cybernetics makes AutoQuant deconvolution software.
 

Confocal and deconvolution techniques both have their pluses and minuses.

Neither, in the case of single channel fluorescence, are "quantitative": given all the variables in staining, probe penetration, total light exposure of the sample to date, oxidation micro-environment, quenching, etc., what you get is a measure of _relative_ fluorescence/staining, not dye molecule absolute counts. Absolute quantitation requires internal standards, as in ratio imaging and FRET, or in single molecule counting (as you can't get less than one!).

Resolution is roughly equivalent between good confocal and good deconvolved data, albeit with some structural dependencies (see below).


Deconvolution pluses:
- Low photobleaching: in vivo, low stability dyes, samples that will be revisited.
- Fast acquisition: again, in vivo samples, live cell cultures, time resolution.
- Very good with small point-like or linear structures; sample presents a PSF from a point, noise does not - and gets removed.
- Lower system cost, can be used with existing widefield scopes.

Minuses:
- Large changes in refractive index or strong absorption (deep, lensing samples) can distort reconstruction.
- Requires some above/below sample acquisition to capture the PSF.
- Difficulty resolving flat uniform sheets, due to low Z contrast.
- Processing time, although that can take place offline (on other computers) from your acquisition system.


Confocal pluses:
- Little/no post-processing required, less time to finished data (although can benefit from additional deconvolution).
- Can go very deep in absorbing/distorting media: for example a slice deep in the sample. While sample absorption/refractive lensing still distorts the data, those distortions don't propagate through slices. Multiphoton confocal is particularly good at deep observations.
- Single or thin slices don't need supporting data above/below.

Minuses:
- Photobleaching: high fluorescent decay, hard on the sample, makes it difficult to compare intensities over time or Z.
- Generally slower acquisition speeds due to scanning.
- Higher noise levels: Poisson noise and point-like structures can be difficult to distinguish.


Overall, given the samples you described, I would consider deconvolution preferable for cell cultures, particularly live cultures, while confocal may do better for thick tissue samples, as long as they're photostable. But as in all things, your mileage may vary - I would suggest trying samples on both systems. While there are strengths and weaknesses, I suspect your users will find both confocal and deconvolution useful and acceptable.


Kevin Ryan
Media Cybernetics, Inc.



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ellen T. Arena
Sent: Tuesday, February 05, 2013 4:24 AM
To: [hidden email]
Subject: Deconvolved Widefield versus Confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello Everyone.

Perhaps my question is a bit naive. But I am hoping you can help me.  Our lab is purchasing a new widefield microscope with a deconvolution package - and I need to convince our users to move some of their imaging onto this machine - away from overly-booked confocal.  So - my question.  Are there circumstances I can present to our users in which wide-field deconvolved images can match/surpass confocal images?  (tissue versus cell culture, etc.?)  Most people in the lab are not doing any quantitative data.  I realize it depends on the experiment and the question at hand.  However, if I can give them a few some-what concrete examples - would be most helpful.

Thank you!

Ellen


------------------------------------------------------
Ellen T. Arena, PhD
Pasteur Foundation Postdoctoral Fellow
Unité de Pathogénie Microbienne Moléculaire INSERM U786 Institut Pasteur
28 rue du Dr Roux
F - 75724 PARIS Cédex 15
France
Tel: [phone]
Fax: [phone]
[hidden email]
------------------------------------------------------

[Jason Swedlow-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi-

Disclaimer: Shameless self-promotion:

Live Cell Imaging (
http://www.amazon.com/Live-Cell-Imaging-Laboratory-Manual/dp/0879698934)
has a great chapter by John Murray that summarises all the advantages and
compromises of each method.

Two direct comparisons:

http://www.ncbi.nlm.nih.gov/pubmed/11830634

http://www.ncbi.nlm.nih.gov/pubmed/18045334

(Note that many of the platforms that were tested in these studies are now
obsolete, so that absolute measures of performance observed will have
changed, but the general trends are probably the same; anybody up for doing
it again on the latest generation of platforms? Definitely should include
SPIM/LSM....)

Cheers,

Jason


On Tue, Feb 5, 2013 at 1:19 PM, Chris Tully <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> Based on my experience, Confocal excels at thicker specimens, especially if
> you have multiphonton capabilities. Also, confocal is absolutely necessary
> for some whole mount specimens like C. Elegans, which since it is mostly
> clear and round tends to act like a lens, making PSF determination highly
> impractical (you have to determine a new PSF for every worm!), and throwing
> blind deconvolution methods into non-converging states, because of the
> unaccounted for "lens" otherwise known as the sample.
>
> With that said I would strongly suggest looking at you costs for both
> systems and charging usage fees accordingly. My guess is that the wide
> field system will cost out enough cheaper that some people will naturally
> shift to it just to stretch their research budgets.
>
> I would also ask for a justification from the confocal users of why they
> need the confocal - if it has been the only available scope then some may
> have been using it by default and be happy to switch to the wide field
> system.
>
>
> Chris Tully
> Microscopy and Image Analysis Expert
> [hidden email]
> 240-475-9753 (c)
>
> [image: View my profile on LinkedIn]<
> http://www.linkedin.com/in/christully/>
>
>
> On Tue, Feb 5, 2013 at 6:43 AM, Guy Cox <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Well, as you implicitly suggest, deconvolution is not (IMHO) quantitative
> > (and I know I'll get some flak for this).  But it has a hugely better
> > photon budget, so the users you should seek to shift are those with dim
> > signals who are struggling on the confocal.
> >
> >                                                       Guy
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of Ellen T. Arena
> > Sent: Tuesday, 5 February 2013 8:24 PM
> > To: [hidden email]
> > Subject: Deconvolved Widefield versus Confocal
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hello Everyone.
> >
> > Perhaps my question is a bit naive. But I am hoping you can help me.  Our
> > lab is purchasing a new widefield microscope with a deconvolution
> package -
> > and I need to convince our users to move some of their imaging onto this
> > machine - away from overly-booked confocal.  So - my question.  Are there
> > circumstances I can present to our users in which wide-field deconvolved
> > images can match/surpass confocal images?  (tissue versus cell culture,
> > etc.?)  Most people in the lab are not doing any quantitative data.  I
> > realize it depends on the experiment and the question at hand.  However,
> if
> > I can give them a few some-what concrete examples - would be most
> helpful.
> >
> > Thank you!
> >
> > Ellen
> >
> >
> > ------------------------------------------------------
> > Ellen T. Arena, PhD
> > Pasteur Foundation Postdoctoral Fellow
> > Unité de Pathogénie Microbienne Moléculaire INSERM U786 Institut Pasteur
> > 28 rue du Dr Roux
> > F - 75724 PARIS Cédex 15
> > France
> > Tel: [phone]
> > Fax: [phone]
> > [hidden email]
> > ------------------------------------------------------
> >
>

--
**************************
Wellcome Trust Centre for Gene Regulation & Expression
College of Life Sciences
MSI/WTB/JBC Complex
University of Dundee
Dow Street
Dundee  DD1 5EH
United Kingdom

phone [phone]
Intl phone:  [phone]
FAX   [phone]
email: [hidden email]

Lab Page: http://www.lifesci.dundee.ac.uk/gre/staff/jason-swedlow
Open Microscopy Environment: http://openmicroscopy.org
**************************

[Vitaly Boyko]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear All,

I have one very simple question.

Does anyone have Leica SP5 or SP8 with 440 nm laser or both 405 nm and 440 nm laser lines.

Best,

Vitaly

[Gary G. Li-2]

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To join, leave or search the confocal microscopy listserv, go to:
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Hi Vitaly,

The 405 nm and 440 nm laser lines have different applications.  The
440 nm is a blue line in the visible.  Although the violet line 405 nm
is still in the visible range, it is a cheaper solution for exciting
UV dyes (DAPI), than a real UV laser (355nm).

Regards

Gary G Li, PhD

>
>
> On Fri, Feb 8, 2013 at 11:01 AM, Vitaly Boyko <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear All,
>>
>> I have one very simple question.
>>
>> Does anyone have Leica SP5 or SP8 with 440 nm laser or both 405 nm and 440 nm laser lines.
>>
>> Best,
>>
>> Vitaly

[Eleanor Kable]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Great.  Can you please put it in your calendar.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jason Swedlow
Sent: Friday, 8 February 2013 7:37 AM
To: [hidden email]
Subject: Re: Deconvolved Widefield versus Confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi-

Disclaimer: Shameless self-promotion:

Live Cell Imaging (
http://www.amazon.com/Live-Cell-Imaging-Laboratory-Manual/dp/0879698934)
has a great chapter by John Murray that summarises all the advantages and compromises of each method.

Two direct comparisons:

http://www.ncbi.nlm.nih.gov/pubmed/11830634

http://www.ncbi.nlm.nih.gov/pubmed/18045334

(Note that many of the platforms that were tested in these studies are now obsolete, so that absolute measures of performance observed will have changed, but the general trends are probably the same; anybody up for doing it again on the latest generation of platforms? Definitely should include
SPIM/LSM....)

Cheers,

Jason


On Tue, Feb 5, 2013 at 1:19 PM, Chris Tully <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> Based on my experience, Confocal excels at thicker specimens,
> especially if you have multiphonton capabilities. Also, confocal is
> absolutely necessary for some whole mount specimens like C. Elegans,
> which since it is mostly clear and round tends to act like a lens,
> making PSF determination highly impractical (you have to determine a
> new PSF for every worm!), and throwing blind deconvolution methods
> into non-converging states, because of the unaccounted for "lens" otherwise known as the sample.
>
> With that said I would strongly suggest looking at you costs for both
> systems and charging usage fees accordingly. My guess is that the wide
> field system will cost out enough cheaper that some people will
> naturally shift to it just to stretch their research budgets.
>
> I would also ask for a justification from the confocal users of why
> they need the confocal - if it has been the only available scope then
> some may have been using it by default and be happy to switch to the
> wide field system.
>
>
> Chris Tully
> Microscopy and Image Analysis Expert
> [hidden email]
> 240-475-9753 (c)
>
> [image: View my profile on LinkedIn]<
> http://www.linkedin.com/in/christully/>
>
>
> On Tue, Feb 5, 2013 at 6:43 AM, Guy Cox <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Well, as you implicitly suggest, deconvolution is not (IMHO)
> > quantitative (and I know I'll get some flak for this).  But it has a
> > hugely better photon budget, so the users you should seek to shift
> > are those with dim signals who are struggling on the confocal.
> >
> >                                                       Guy
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> > [mailto:[hidden email]]
> > On Behalf Of Ellen T. Arena
> > Sent: Tuesday, 5 February 2013 8:24 PM
> > To: [hidden email]
> > Subject: Deconvolved Widefield versus Confocal
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hello Everyone.
> >
> > Perhaps my question is a bit naive. But I am hoping you can help me.  
> > Our lab is purchasing a new widefield microscope with a
> > deconvolution
> package -
> > and I need to convince our users to move some of their imaging onto
> > this machine - away from overly-booked confocal.  So - my question.  
> > Are there circumstances I can present to our users in which
> > wide-field deconvolved images can match/surpass confocal images?  
> > (tissue versus cell culture,
> > etc.?)  Most people in the lab are not doing any quantitative data.  
> > I realize it depends on the experiment and the question at hand.  
> > However,
> if
> > I can give them a few some-what concrete examples - would be most
> helpful.
> >
> > Thank you!
> >
> > Ellen
> >
> >
> > ------------------------------------------------------
> > Ellen T. Arena, PhD
> > Pasteur Foundation Postdoctoral Fellow Unité de Pathogénie
> > Microbienne Moléculaire INSERM U786 Institut Pasteur
> > 28 rue du Dr Roux
> > F - 75724 PARIS Cédex 15
> > France
> > Tel: [phone]
> > Fax: [phone]
> > [hidden email]
> > ------------------------------------------------------
> >
>

--
**************************
Wellcome Trust Centre for Gene Regulation & Expression College of Life Sciences MSI/WTB/JBC Complex University of Dundee Dow Street Dundee  DD1 5EH United Kingdom

phone [phone]
Intl phone:  [phone]
FAX   [phone]
email: [hidden email]

Lab Page: http://www.lifesci.dundee.ac.uk/gre/staff/jason-swedlow
Open Microscopy Environment: http://openmicroscopy.org
**************************