"Out of Office autoreply" courtesy

[Brian Northan]

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Jim

Right.  Deconvolution does not provide a complete separation between
noise and signal.  As you mention it is a noise reduction.

Brian






On Thu, Oct 27, 2011 at 11:35 AM, James Pawley <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>>
>> This is why it is good to sample a little higher than the Nyqyist
>> rate.  Airy discs can be separated from Poisson noise by deconvolution
>> or by other noise reduction algorithms.
>>
>> Brian
>
> Hi Brian,
>
> 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.
>
> Would you not agree that the best you can do is effectively average the
> statistical noise over all the measurements needed to define a PSF at the
> sampling you are using? If we assume that from 64 to 125 pixels are needed
> to define a PSF, this produces a significant noise reduction. But not an
> infinite one.
>
> Best
>
> Jim Pawley
> --
> ***************************************************************************
> Prof. James B. Pawley,                                      Ph. 608-238-3953
>                           21. N. Prospect Ave. Madison, WI 53726 USA
> [hidden email]
> 3D Microscopy of Living Cells Course, June 10-22, 2012, UBC, Vancouver
> Canada
> Info: http://www.3dcourse.ubc.ca/       Applications accepted after 11/15/12
>               "If it ain't diffraction, it must be statistics." Anon.
>

[Guy Cox-2]

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That paper says it all perfectly - and even deals with different mapping
algorithms.  The link Christophe gave is just to a site that links to it
- the direct link is http://alvyray.com/Memos/CG/Microsoft/6_pixel.pdf .
Thanks a lot, Christophe!

                                                        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 [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Christophe Leterrier
Sent: Friday, 28 October 2011 12:32 AM
To: [hidden email]
Subject: Re: Airy Units -

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On Thu, Oct 27, 2011 at 15:16, Martin Wessendorf <[hidden email]> wrote

>
> On 10/26/2011 11:41 PM, Guy Cox wrote:
>
>  As to Jim's point about digital images, this brings me to another of
my
>> 'hobby horses'.  We should NOT just look at our images as collected,
we
>> should map them into a larger space using sine-wave fitting.  Only
then
>> does Nyquist mean anything.
>>
>
> Can you go into how this is be done, or suggest a reference?
>
> Thanks!
>
> Martin
>

I guess Guy is talking about image reconstruction as in the famous "A
pixel
is not a little square" paper:
http://pentileblog.com/uncategorized/a-pixel-is-not-a-little-square-a-pi
xel-is-not-a-little-square-a-pixel-is-not-a-little-square-and-a-voxel-is
-not-a-little-cube/

Or am I confused ?

Christophe

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1411 / Virus Database: 2092/3977 - Release Date: 10/26/11

[Guy Cox-2]

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In stochastic imaging techniques such as PALM and STORM the optical
resolution does not come into the calculation.  The limit to resolution
is the precision with which you can locate the centre of the Airy disk.
This in turn depends on the number of photons you have collected at that
point - more photons = better resolution.  It is essentially
localization rather than resolution.  You can locate the position of a
point with the same precision in conventional microscopy - so long as it
is isolated.  By imaging molecules 'one at a time' you can extend the
principle to points that are close together - PALM, STORM etc.  So when
people claim 20nm resolution they mean that that is their expected
precision given the number of photons they have recorded for each
molecule.  I'd like to see a clear demonstration - the idea that comes
to mind is finding a couple of microtubules that are diverging at a
small angle.  

                                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 [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Armstrong, Brian
Sent: Friday, 28 October 2011 2:44 AM
To: [hidden email]
Subject: Re: Airy Units

*****
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The thought I had was that if when people are using algorithms to refine
the images collected using a super-resolution technique, such PALM,
STORM, etc.. and they use 0.51 rather than 0.61 are they accurately
representing the true resolution of the technique; 488/1.4NA * 0.61 =
212 whereas 488/1.4NA * 0.51 = 177.
When they report resolution of 20nm is this a true value?  

Brian D Armstrong PhD
Assistant Research Professor
Light Microscopy Core
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Cammer, Michael
Sent: Thursday, October 27, 2011 8:21 AM
To: [hidden email]
Subject: Re: Airy Units - getting off topic

*****
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Actually, I'm interested in the cilia on gut bacteria which look like
hairy discs inside the mice.  If we sample small enough we'll find the
fundamental particle.  Which I bet is a pixel.


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of simon walker (BI)
Sent: Thursday, October 27, 2011 11:12 AM
To: [hidden email]
Subject: Re: Airy Units - getting off topic

Do mice appear as Hairy discs?

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Brian Northan
Sent: 27 October 2011 15:56
To: [hidden email]
Subject: Re: Airy Units - getting off topic

*****
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Michael

Coming from a deconvolution background I've thought about this issue
quite a bit.  If "people" were the smallest thing that could be counted
than it wouldn't make sense to go any smaller (assuming you didn't care
what they looked like).

But sampling an area smaller than the size of a person would be useful
if there was some other smaller creature that could be confused with a
person.  Maybe there are mice running around randomly.   If we sample
at the size of a person, both people and mice appear as points.  If we
sample just a little finer, then people appear as a larger correlated
cluster of pixels and mice appear as points.  We can then separate
people from mice.  We don't even have to get a good picture of the
people just sample a little finer so we can tell the different between
people and mice.

This is why it is good to sample a little higher than the Nyqyist rate.
Airy discs can be separated from Poisson noise by deconvolution or by
other noise reduction algorithms.

Brian

On Thu, Oct 27, 2011 at 10:18 AM, Cammer, Michael
<[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> A pixel is not a little square.  It is a discrete sampling of some
measurement, such as light or temperature or radioactivity or events in
time or people who live in a given area. Let's look at the last example.
Sampling an area smaller than the size of a person really isn't useful.
And it leads to the ridiculous measurement of fractional people.
Sampling a square km can't resolve finer the locations of people if
there is more than one living there. I think this is the original
question.

>
> (I know, what about sampling differently, such as overlapping circular
> areas or some stochastic sampling method, but we have to store the
> data somehow and converting matrices, little squares, is the current
> method.  Is there anyone out there storing confocal sampled images or
> displaying them with a non 2D matrix display?)
>
> -Michael C.
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Christophe
> Leterrier
> Sent: Thursday, October 27, 2011 9:32 AM
> To: [hidden email]
> Subject: Re: Airy Units -
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> On Thu, Oct 27, 2011 at 15:16, Martin Wessendorf <[hidden email]>
> wrote
>
>>
>> On 10/26/2011 11:41 PM, Guy Cox wrote:
>>
>>  As to Jim's point about digital images, this brings me to another of
>> my
>>> 'hobby horses'.  We should NOT just look at our images as collected,
>>> we should map them into a larger space using sine-wave fitting.
>>> Only then does Nyquist mean anything.
>>>
>>
>> Can you go into how this is be done, or suggest a reference?
>>
>> Thanks!
>>
>> Martin
>>
>
> I guess Guy is talking about image reconstruction as in the famous "A
> pixel is not a little square" paper:
> http://pentileblog.com/uncategorized/a-pixel-is-not-a-little-square-a-
> pixel-is-not-a-little-square-a-pixel-is-not-a-little-square-and-a-voxe
> l-is-not-a-little-cube/
>
> Or am I confused ?
>
> Christophe
>
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[Andreas Bruckbauer]

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In super resolution microscopy you also have a point spread function, just that this is the PSF of the whole process including image analysis. The position markers (e.g. gold particles) provide a stable signal so that you get thousands of localisations during one aquisition series (thousands of frames) and in the image reconstruction you get a nice PSF with the claimed resolution of e.g. 20 nm.
If the signal to noise ratio of the fluorophores used to label the biological features is similar to the gold particles you would expect the same PSF for the features of interest. The Nature methods paper by Andrew York (2011, 8(4) 327) contains some information in the supplemetary material.

best wishes

Andreas




 

 

 

-----Original Message-----
From: Armstrong, Brian <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Thu, 27 Oct 2011 17:29
Subject: Re: Airy Units


*****





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





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





*****











The thought I had was that if when people are using algorithms to refine the





images collected using a super-resolution technique, such PALM, STORM, etc.. and





they use 0.51 rather than 0.61 are they accurately representing the true





resolution of the technique; 488/1.4NA * 0.61 = 212 whereas 488/1.4NA * 0.51 =





177.





When they report resolution of 20nm is this a true value?  











Brian D Armstrong PhD





Assistant Research Professor





Light Microscopy Core





Beckman Research Institute





City of Hope





Dept of Neuroscience





1450 E Duarte Rd





Duarte, CA 91010





626-256-4673 x62872











http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx

















-----Original Message-----





From: Confocal Microscopy List [mailto:[hidden email]] On





Behalf Of Cammer, Michael





Sent: Thursday, October 27, 2011 8:21 AM





To: [hidden email]





Subject: Re: Airy Units - getting off topic











*****





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





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





*****











Actually, I'm interested in the cilia on gut bacteria which look like hairy





discs inside the mice.  If we sample small enough we'll find the fundamental





particle.  Which I bet is a pixel.

















-----Original Message-----





From: Confocal Microscopy List [mailto:[hidden email]] On





Behalf Of simon walker (BI)





Sent: Thursday, October 27, 2011 11:12 AM





To: [hidden email]





Subject: Re: Airy Units - getting off topic











Do mice appear as Hairy discs?











-----Original Message-----





From: Confocal Microscopy List [mailto:[hidden email]] On





Behalf Of Brian Northan





Sent: 27 October 2011 15:56





To: [hidden email]





Subject: Re: Airy Units - getting off topic











*****





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





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





*****











Michael











Coming from a deconvolution background I've thought about this issue quite a





bit.  If "people" were the smallest thing that could be counted than it wouldn't





make sense to go any smaller (assuming you didn't care what they looked like).











But sampling an area smaller than the size of a person would be useful if there





was some other smaller creature that could be confused with a





person.  Maybe there are mice running around randomly.   If we sample





at the size of a person, both people and mice appear as points.  If we sample





just a little finer, then people appear as a larger correlated cluster of pixels





and mice appear as points.  We can then separate people from mice.  We don't





even have to get a good picture of the people just sample a little finer so we





can tell the different between people and mice.











This is why it is good to sample a little higher than the Nyqyist rate.  Airy





discs can be separated from Poisson noise by deconvolution or by other noise





reduction algorithms.











Brian











On Thu, Oct 27, 2011 at 10:18 AM, Cammer, Michael <[hidden email]>





wrote:





> *****





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





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





> *****





>





> A pixel is not a little square.  It is a discrete sampling of some





measurement, such as light or temperature or radioactivity or events in time or





people who live in a given area. Let's look at the last example.  Sampling an





area smaller than the size of a person really isn't useful.  And it leads to the





ridiculous measurement of fractional people.  Sampling a square km can't resolve





finer the locations of people if there is more than one living there. I think





this is the original question.





>





> (I know, what about sampling differently, such as overlapping circular





> areas or some stochastic sampling method, but we have to store the





> data somehow and converting matrices, little squares, is the current





> method.  Is there anyone out there storing confocal sampled images or





> displaying them with a non 2D matrix display?)





>





> -Michael C.





>





>





> -----Original Message-----





> From: Confocal Microscopy List





> [mailto:[hidden email]] On Behalf Of Christophe





> Leterrier





> Sent: Thursday, October 27, 2011 9:32 AM





> To: [hidden email]





> Subject: Re: Airy Units -





>





> *****





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





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





> *****





>





> On Thu, Oct 27, 2011 at 15:16, Martin Wessendorf <[hidden email]>





> wrote





>





>>





>> On 10/26/2011 11:41 PM, Guy Cox wrote:





>>





>>  As to Jim's point about digital images, this brings me to another of





>> my





>>> 'hobby horses'.  We should NOT just look at our images as collected,





>>> we should map them into a larger space using sine-wave fitting.





>>> Only then does Nyquist mean anything.





>>>





>>





>> Can you go into how this is be done, or suggest a reference?





>>





>> Thanks!





>>





>> Martin





>>





>





> I guess Guy is talking about image reconstruction as in the famous "A





> pixel is not a little square" paper:





> http://pentileblog.com/uncategorized/a-pixel-is-not-a-little-square-a-





> pixel-is-not-a-little-square-a-pixel-is-not-a-little-square-and-a-voxe





> l-is-not-a-little-cube/





>





> Or am I confused ?





>





> Christophe





>





> ------------------------------------------------------------





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this email in error please notify the sender by return email and delete the





original message. Please note, the recipient should check this email and any





attachments for the presence of viruses. The organization accepts no liability





for any damage caused by any virus transmitted by this email.





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>





The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT Registered





Charity No. 1053902.





The information transmitted in this email is directed only to the addressee. If





you received this in error, please contact the sender and delete this email from





your system. The contents of this e-mail are the views of the sender and do not





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[Christian Soeller]

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The localisation precision is only one part of the story, in practice we find that sampling is much more limiting. By building the image from points the imaging process is taken into the digital domain and spatial Nyquist sampling considerations apply. This is discussed in various papers, e.g. Shroff et al.  Nat Meth (2008) vol. 5 (5) pp. 417-23; our own Baddeley et al. Visualization of localization microscopy data. Microsc Microanal (2010) vol. 16 (1) pp. 64-72 and probably many others.

Demonstrations of the resolution exist in various papers, some are simulated as in the two above, some simulated and experimental demos are in van de Linde et al. Journal of biotechnology (2010) vol. 149 (4) pp. 260-6. Various additional examples are in the other early PALM/STORM/etc papers (and many more by now). There is no paradox here, as usual the diffraction limit is not broken but circumvented, etc....

Christian
 
On 28/10/2011, at 7:54 PM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> In stochastic imaging techniques such as PALM and STORM the optical
> resolution does not come into the calculation.  The limit to resolution
> is the precision with which you can locate the centre of the Airy disk.
> This in turn depends on the number of photons you have collected at that
> point - more photons = better resolution.  It is essentially
> localization rather than resolution.  You can locate the position of a
> point with the same precision in conventional microscopy - so long as it
> is isolated.  By imaging molecules 'one at a time' you can extend the
> principle to points that are close together - PALM, STORM etc.  So when
> people claim 20nm resolution they mean that that is their expected
> precision given the number of photons they have recorded for each
> molecule.  I'd like to see a clear demonstration - the idea that comes
> to mind is finding a couple of microtubules that are diverging at a
> small angle.  
>
>                                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 [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Armstrong, Brian
> Sent: Friday, 28 October 2011 2:44 AM
> To: [hidden email]
> Subject: Re: Airy Units
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The thought I had was that if when people are using algorithms to refine
> the images collected using a super-resolution technique, such PALM,
> STORM, etc.. and they use 0.51 rather than 0.61 are they accurately
> representing the true resolution of the technique; 488/1.4NA * 0.61 =
> 212 whereas 488/1.4NA * 0.51 = 177.
> When they report resolution of 20nm is this a true value?  
>
> Brian D Armstrong PhD
> Assistant Research Professor
> Light Microscopy Core
> Beckman Research Institute
> City of Hope
> Dept of Neuroscience
> 1450 E Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
>
> http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
> ing/Pages/default.aspx
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Cammer, Michael
> Sent: Thursday, October 27, 2011 8:21 AM
> To: [hidden email]
> Subject: Re: Airy Units - getting off topic
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Actually, I'm interested in the cilia on gut bacteria which look like
> hairy discs inside the mice.  If we sample small enough we'll find the
> fundamental particle.  Which I bet is a pixel.
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of simon walker (BI)
> Sent: Thursday, October 27, 2011 11:12 AM
> To: [hidden email]
> Subject: Re: Airy Units - getting off topic
>
> Do mice appear as Hairy discs?
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Brian Northan
> Sent: 27 October 2011 15:56
> To: [hidden email]
> Subject: Re: Airy Units - getting off topic
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Michael
>
> Coming from a deconvolution background I've thought about this issue
> quite a bit.  If "people" were the smallest thing that could be counted
> than it wouldn't make sense to go any smaller (assuming you didn't care
> what they looked like).
>
> But sampling an area smaller than the size of a person would be useful
> if there was some other smaller creature that could be confused with a
> person.  Maybe there are mice running around randomly.   If we sample
> at the size of a person, both people and mice appear as points.  If we
> sample just a little finer, then people appear as a larger correlated
> cluster of pixels and mice appear as points.  We can then separate
> people from mice.  We don't even have to get a good picture of the
> people just sample a little finer so we can tell the different between
> people and mice.
>
> This is why it is good to sample a little higher than the Nyqyist rate.
> Airy discs can be separated from Poisson noise by deconvolution or by
> other noise reduction algorithms.
>
> Brian
>
> On Thu, Oct 27, 2011 at 10:18 AM, Cammer, Michael
> <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> A pixel is not a little square.  It is a discrete sampling of some
> measurement, such as light or temperature or radioactivity or events in
> time or people who live in a given area. Let's look at the last example.
> Sampling an area smaller than the size of a person really isn't useful.
> And it leads to the ridiculous measurement of fractional people.
> Sampling a square km can't resolve finer the locations of people if
> there is more than one living there. I think this is the original
> question.
>>
>> (I know, what about sampling differently, such as overlapping circular
>> areas or some stochastic sampling method, but we have to store the
>> data somehow and converting matrices, little squares, is the current
>> method.  Is there anyone out there storing confocal sampled images or
>> displaying them with a non 2D matrix display?)
>>
>> -Michael C.
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Christophe
>> Leterrier
>> Sent: Thursday, October 27, 2011 9:32 AM
>> To: [hidden email]
>> Subject: Re: Airy Units -
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> On Thu, Oct 27, 2011 at 15:16, Martin Wessendorf <[hidden email]>
>> wrote
>>
>>>
>>> On 10/26/2011 11:41 PM, Guy Cox wrote:
>>>
>>> As to Jim's point about digital images, this brings me to another of
>>> my
>>>> 'hobby horses'.  We should NOT just look at our images as collected,
>>>> we should map them into a larger space using sine-wave fitting.
>>>> Only then does Nyquist mean anything.
>>>>
>>>
>>> Can you go into how this is be done, or suggest a reference?
>>>
>>> Thanks!
>>>
>>> Martin
>>>
>>
>> I guess Guy is talking about image reconstruction as in the famous "A
>> pixel is not a little square" paper:
>> http://pentileblog.com/uncategorized/a-pixel-is-not-a-little-square-a-
>> pixel-is-not-a-little-square-a-pixel-is-not-a-little-square-and-a-voxe
>> l-is-not-a-little-cube/
>>
>> Or am I confused ?
>>
>> Christophe
>>
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[Michelle Peckham]

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*****

Having just done some overnight filming from cells infected with a
adenovirus (eGFP-tagged construct), I need to know how long it takes for
the eGFP to become fluorescent..

Does anyone know?

Thanks

Michelle

[Zaal, Kristina (NIH/NIAMS)]

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We have seen expression 6 h after a fugene transfections of plasmid encoding a eGFP labeled nuclear membrane protein in COS7 cells. I think the cell type and the patterns of the protein expression makes a difference: diffuse proteins are harder to pick up than those concentrated in structures.
 
Kristien J.M. Zaal, Ph.D.
Staff Scientist, Facility Manager
Light Imaging Section
NIAMS, NIH
Bldg 50, Rm 1533
Bethesda, MD 20892
Phone [phone]; FAX [phone]
________________________________________
From: Michelle Peckham [[hidden email]]
Sent: Friday, October 28, 2011 12:37 PM
To: [hidden email]
Subject: how long does it take eGFP to fold and become fluorescent in mammalian cells?

*****
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Having just done some overnight filming from cells infected with a
adenovirus (eGFP-tagged construct), I need to know how long it takes for
the eGFP to become fluorescent..

Does anyone know?

Thanks

Michelle

[Michelle Peckham]

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Yes, that's about the length of time before I see fluorescence too - and
it's organised into structures.  But if it takes 6 hours for GFP to
fold...then is this GFP folding time, or does GFP fold very quickly and I
don't have to worry about that?

M

On 28/10/2011 20:47, "Zaal, Kristina (NIH/NIAMS) [E]" <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>We have seen expression 6 h after a fugene transfections of plasmid
>encoding a eGFP labeled nuclear membrane protein in COS7 cells. I think
>the cell type and the patterns of the protein expression makes a
>difference: diffuse proteins are harder to pick up than those
>concentrated in structures.
>
>Kristien J.M. Zaal, Ph.D.
>Staff Scientist, Facility Manager
>Light Imaging Section
>NIAMS, NIH
>Bldg 50, Rm 1533
>Bethesda, MD 20892
>Phone [phone]; FAX [phone]
>________________________________________
>From: Michelle Peckham [[hidden email]]
>Sent: Friday, October 28, 2011 12:37 PM
>To: [hidden email]
>Subject: how long does it take eGFP to fold and become fluorescent in
>mammalian cells?
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Having just done some overnight filming from cells infected with a
>adenovirus (eGFP-tagged construct), I need to know how long it takes for
>the eGFP to become fluorescent..
>
>Does anyone know?
>
>Thanks
>
>Michelle

[Kurt Thorn]

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There are a number of papers characterizing the folding and maturation
rate of EGFP, and they usually find rates of around 30 minutes.  Here is
one with some detailed characterization:
http://pubs.acs.org/doi/full/10.1021/ja0580439

Whether or not this depends on the fusion context of the GFP, I don't know.

Kurt

On 10/28/2011 1:09 PM, Michelle Peckham wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Yes, that's about the length of time before I see fluorescence too - and
> it's organised into structures.  But if it takes 6 hours for GFP to
> fold...then is this GFP folding time, or does GFP fold very quickly and I
> don't have to worry about that?
>
> M
>
> On 28/10/2011 20:47, "Zaal, Kristina (NIH/NIAMS) [E]"<[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> We have seen expression 6 h after a fugene transfections of plasmid
>> encoding a eGFP labeled nuclear membrane protein in COS7 cells. I think
>> the cell type and the patterns of the protein expression makes a
>> difference: diffuse proteins are harder to pick up than those
>> concentrated in structures.
>>
>> Kristien J.M. Zaal, Ph.D.
>> Staff Scientist, Facility Manager
>> Light Imaging Section
>> NIAMS, NIH
>> Bldg 50, Rm 1533
>> Bethesda, MD 20892
>> Phone [phone]; FAX [phone]
>> ________________________________________
>> From: Michelle Peckham [[hidden email]]
>> Sent: Friday, October 28, 2011 12:37 PM
>> To: [hidden email]
>> Subject: how long does it take eGFP to fold and become fluorescent in
>> mammalian cells?
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Having just done some overnight filming from cells infected with a
>> adenovirus (eGFP-tagged construct), I need to know how long it takes for
>> the eGFP to become fluorescent..
>>
>> Does anyone know?
>>
>> Thanks
>>
>> Michelle

[Michelle Peckham]

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

That's great, just what I wanted to know but couldn't find the right reference
Thanks



On 28 Oct 2011, at 22:49, "Kurt Thorn" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> There are a number of papers characterizing the folding and maturation
> rate of EGFP, and they usually find rates of around 30 minutes.  Here is
> one with some detailed characterization:
> http://pubs.acs.org/doi/full/10.1021/ja0580439
>
> Whether or not this depends on the fusion context of the GFP, I don't know.
>
> Kurt
>
> On 10/28/2011 1:09 PM, Michelle Peckham wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Yes, that's about the length of time before I see fluorescence too - and
>> it's organised into structures.  But if it takes 6 hours for GFP to
>> fold...then is this GFP folding time, or does GFP fold very quickly and I
>> don't have to worry about that?
>>
>> M
>>
>> On 28/10/2011 20:47, "Zaal, Kristina (NIH/NIAMS) [E]"<[hidden email]>
>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> We have seen expression 6 h after a fugene transfections of plasmid
>>> encoding a eGFP labeled nuclear membrane protein in COS7 cells. I think
>>> the cell type and the patterns of the protein expression makes a
>>> difference: diffuse proteins are harder to pick up than those
>>> concentrated in structures.
>>>
>>> Kristien J.M. Zaal, Ph.D.
>>> Staff Scientist, Facility Manager
>>> Light Imaging Section
>>> NIAMS, NIH
>>> Bldg 50, Rm 1533
>>> Bethesda, MD 20892
>>> Phone [phone]; FAX [phone]
>>> ________________________________________
>>> From: Michelle Peckham [[hidden email]]
>>> Sent: Friday, October 28, 2011 12:37 PM
>>> To: [hidden email]
>>> Subject: how long does it take eGFP to fold and become fluorescent in
>>> mammalian cells?
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Having just done some overnight filming from cells infected with a
>>> adenovirus (eGFP-tagged construct), I need to know how long it takes for
>>> the eGFP to become fluorescent..
>>>
>>> Does anyone know?
>>>
>>> Thanks
>>>
>>> Michelle

[Guy Cox-2]

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And, of course, that's why there are birds sitting on the backs of cows
- the cows have fleas and the birds are there to Peckham! :)

(Sorry, the coincidence was too good to miss!)

                                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 [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Michelle Peckham
Sent: Friday, 28 October 2011 3:00 AM
To: [hidden email]
Subject: Re: Airy Units -even more off topic

*****
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A colleague suggested this:

Great fleas have little fleas upon their backs to bite 'em,
And little fleas have lesser fleas, and so ad infinitum.



(quotation from augustus de Morgan)

On 27/10/2011 12:37, "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
>*****
>
>As I recall, depth (Z) resolution is said to be at a maximum at that
>value.  Lateral (XY) resolution is at a maximum when the pinhole is
>infinitely small ... which is not terribly useful.  By 0.7 Airy any
gain
>is minimal, but it's not too bad down at 0.3 Airy.  Again, see the Cox
&

>Sheppard paper I mentioned earlier (MICROSCOPY RESEARCH AND TECHNIQUE
>63:18-22 (2004)) which gives both theoretical and experimental
>measurements.
>
>                                          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 [phone]     Fax [phone]
>             Mobile [phone]
>______________________________________________
>      http://www.guycox.net
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List

[mailto:[hidden email]]

>On Behalf Of Mark Cannell
>Sent: Thursday, 27 October 2011 6:49 PM
>To: [hidden email]
>Subject: Re: Airy Units -
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>In addition to Guy's point, I believe the confocal pinhole setting of 1
>Airy unit represents some sort of compromise between resolution and

S/N.

>Resolution will increase if you close the pinhole further albeit at the
>expense of signal. I think the resolution is maximal at 0.7 Airy units
>but I don't have the Handbook of BCM with me to check that value.
>
>Cheers Mark
>
>Mark  B. Cannell Ph.D. FRSNZ
>Professor of Cardiac Cell Biology
>School of Physiology&  Pharmacology
>Medical Sciences Building
>University of Bristol
>Bristol
>BS8 1TD UK
>
>[hidden email]
>
>
>On 27/10/2011, at 12:40 AM, Guy Cox wrote:
>>
>> Brian,
>>
>>       0.5 lambda is the Abbe resolution.  That criterion is based on
>> diffraction at the specimen so it requires partially coherent light
>and
>> does not apply in fluorescence.  But Abbe's criterion is absolute -

it
>> describes the limiting frequency which the lens can pass.  Rayleigh's
>is
>> not - at the Rayleigh minimum resolved distance there Is till an
>> intensity minimum (about .75) between the points.  In other terms,
the
>> intensity modulation is reduced to about 1/4 at that frequency.  The
>> limiting cutoff frequency is given by FWHM - full width half maximum
-

>> so that is a fairer comparison with Abbe for the incoherent case.
>This
>> is probably what was being used by your speakers.
>>
>>       The Airy unit as used for confocal pinhole size is not affected
>> by this, of course, it is the diameter of the disk, 1.22 lambda
>> (multiplied by the magnification, of course).
>>
>>                                        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 [phone]     Fax [phone]
>>             Mobile [phone]
>> ______________________________________________
>>      http://www.guycox.net
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>[mailto:[hidden email]]
>> On Behalf Of Armstrong, Brian
>> Sent: Thursday, 27 October 2011 2:25 AM
>> To: [hidden email]
>> Subject: Re: Airy Units -
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Guy (and list), in a couple of super-resolution talks I've attended
>they
>> were using 0.51 instead of 0.61 for the constant. Do you know the
>> rationale behind this?
>> Thanks, Brian
>>
>> Brian Armstrong PhD
>> Light Microscopy Core
>> Beckman Research Institute
>> 1450 East Duarte Rd
>> Duarte, CA 91010
>> 626-256-4673 x62872
>>
>http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHom

e

>> .htm
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>[mailto:[hidden email]]
>> On Behalf Of Guy Cox
>> Sent: Wednesday, October 26, 2011 2:46 AM
>> To: [hidden email]
>> Subject: Airy Units - was: RE: "Out of Office autoreply" courtesy
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> You are right, it has been totally drowned out!
>>
>> The Airy unit is defined by the size of the Airy disk, that is the
>size
>> of the disk representing the image of a point object.  Airy was an
>> astronomer and thus derived it by reference to stars (which, though
>> huge, are so far away that they appear as point objects).  John
>Strutt,
>> Lord Rayleigh, proposed a general resolution criterion that two
>objects
>> can be considered resolved if the maximum of one Airy disk lies on

the
>> first minimum of the other.  This criterion, the radius of the
central

>> disk (ignoring surrounding haloes) is given by  r   =  0.61 lambda  /
>> NA, where lambda is the wavelength of the light being used.
>>
>>                                         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 [phone]     Fax [phone]
>>             Mobile [phone]
>> ______________________________________________
>>      http://www.guycox.net
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>[mailto:[hidden email]]
>> On Behalf Of Peter G. Werner
>> Sent: Sunday, 23 October 2011 4:55 AM
>> To: [hidden email]
>> Subject: Re: "Out of Office autoreply" courtesy
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> And I hate to point this out, but the question I originally asked
>> (concerning the definition of Airy Units) has been drowned out by all
>> the
>> commentary about the "Out of Office autoreply" that my initial email
>> generated.
>>
>> If nobody has an answer to the question, no worries, but I'd hate to
>see
>> the
>> topic get lost under the weight of discussion of listserv function.
>>
>> -----
>> No virus found in this message.
>> Checked by AVG - www.avg.com
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>>
>> ---------------------------------------------------------------------
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>> or entity to which they are addressed. This communication may contain
>> information that is privileged, confidential, or exempt from
>disclosure
>> under applicable law (e.g., personal health information, research
>data,
>> financial information). Because this e-mail has been sent without
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>to
>> view the information, forward it to others or tamper with the
>> information without the knowledge or consent of the sender. If you

are

>> not the intended recipient, or the employee or person responsible for
>> delivering the message to the intended recipient, any dissemination,
>> distribution or copying of the communication is strictly prohibited.
>If
>> you received the communication in error, please notify the sender
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[Zac Arrac Atelaz]

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LOL!!! Science & humor I really really really love this list!!!
Gabriel OHPD: Ben are you ok? we were all really worried :D
 > Date: Sun, 30 Oct 2011 00:07:02 +1100

> From: [hidden email]
> Subject: Re: Airy Units -even more off topic
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> And, of course, that's why there are birds sitting on the backs of cows
> - the cows have fleas and the birds are there to Peckham! :)
>
> (Sorry, the coincidence was too good to miss!)
>
>                                 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 [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net
>  
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Michelle Peckham
> Sent: Friday, 28 October 2011 3:00 AM
> To: [hidden email]
> Subject: Re: Airy Units -even more off topic
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> A colleague suggested this:
>
> Great fleas have little fleas upon their backs to bite 'em,
> And little fleas have lesser fleas, and so ad infinitum.
>
>
>
> (quotation from augustus de Morgan)
>
> On 27/10/2011 12:37, "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
> >*****
> >
> >As I recall, depth (Z) resolution is said to be at a maximum at that
> >value.  Lateral (XY) resolution is at a maximum when the pinhole is
> >infinitely small ... which is not terribly useful.  By 0.7 Airy any
> gain
> >is minimal, but it's not too bad down at 0.3 Airy.  Again, see the Cox
> &
> >Sheppard paper I mentioned earlier (MICROSCOPY RESEARCH AND TECHNIQUE
> >63:18-22 (2004)) which gives both theoretical and experimental
> >measurements.
> >
> >                                          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 [phone]     Fax [phone]
> >             Mobile [phone]
> >______________________________________________
> >      http://www.guycox.net
> >
> >
> >
> >-----Original Message-----
> >From: Confocal Microscopy List
> [mailto:[hidden email]]
> >On Behalf Of Mark Cannell
> >Sent: Thursday, 27 October 2011 6:49 PM
> >To: [hidden email]
> >Subject: Re: Airy Units -
> >
> >*****
> >To join, leave or search the confocal microscopy listserv, go to:
> >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >*****
> >
> >In addition to Guy's point, I believe the confocal pinhole setting of 1
> >Airy unit represents some sort of compromise between resolution and
> S/N.
> >Resolution will increase if you close the pinhole further albeit at the
> >expense of signal. I think the resolution is maximal at 0.7 Airy units
> >but I don't have the Handbook of BCM with me to check that value.
> >
> >Cheers Mark
> >
> >Mark  B. Cannell Ph.D. FRSNZ
> >Professor of Cardiac Cell Biology
> >School of Physiology&  Pharmacology
> >Medical Sciences Building
> >University of Bristol
> >Bristol
> >BS8 1TD UK
> >
> >[hidden email]
> >
> >
> >On 27/10/2011, at 12:40 AM, Guy Cox wrote:
> >>
> >> Brian,
> >>
> >>       0.5 lambda is the Abbe resolution.  That criterion is based on
> >> diffraction at the specimen so it requires partially coherent light
> >and
> >> does not apply in fluorescence.  But Abbe's criterion is absolute -
> it
> >> describes the limiting frequency which the lens can pass.  Rayleigh's
> >is
> >> not - at the Rayleigh minimum resolved distance there Is till an
> >> intensity minimum (about .75) between the points.  In other terms,
> the
> >> intensity modulation is reduced to about 1/4 at that frequency.  The
> >> limiting cutoff frequency is given by FWHM - full width half maximum
> -
> >> so that is a fairer comparison with Abbe for the incoherent case.
> >This
> >> is probably what was being used by your speakers.
> >>
> >>       The Airy unit as used for confocal pinhole size is not affected
> >> by this, of course, it is the diameter of the disk, 1.22 lambda
> >> (multiplied by the magnification, of course).
> >>
> >>                                        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 [phone]     Fax [phone]
> >>             Mobile [phone]
> >> ______________________________________________
> >>      http://www.guycox.net
> >>
> >>
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List
> >[mailto:[hidden email]]
> >> On Behalf Of Armstrong, Brian
> >> Sent: Thursday, 27 October 2011 2:25 AM
> >> To: [hidden email]
> >> Subject: Re: Airy Units -
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Guy (and list), in a couple of super-resolution talks I've attended
> >they
> >> were using 0.51 instead of 0.61 for the constant. Do you know the
> >> rationale behind this?
> >> Thanks, Brian
> >>
> >> Brian Armstrong PhD
> >> Light Microscopy Core
> >> Beckman Research Institute
> >> 1450 East Duarte Rd
> >> Duarte, CA 91010
> >> 626-256-4673 x62872
> >>
> >http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHom
> e
> >> .htm
> >>
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List
> >[mailto:[hidden email]]
> >> On Behalf Of Guy Cox
> >> Sent: Wednesday, October 26, 2011 2:46 AM
> >> To: [hidden email]
> >> Subject: Airy Units - was: RE: "Out of Office autoreply" courtesy
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> You are right, it has been totally drowned out!
> >>
> >> The Airy unit is defined by the size of the Airy disk, that is the
> >size
> >> of the disk representing the image of a point object.  Airy was an
> >> astronomer and thus derived it by reference to stars (which, though
> >> huge, are so far away that they appear as point objects).  John
> >Strutt,
> >> Lord Rayleigh, proposed a general resolution criterion that two
> >objects
> >> can be considered resolved if the maximum of one Airy disk lies on
> the
> >> first minimum of the other.  This criterion, the radius of the
> central
> >> disk (ignoring surrounding haloes) is given by  r   =  0.61 lambda  /
> >> NA, where lambda is the wavelength of the light being used.
> >>
> >>                                         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 [phone]     Fax [phone]
> >>             Mobile [phone]
> >> ______________________________________________
> >>      http://www.guycox.net
> >>
> >>
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List
> >[mailto:[hidden email]]
> >> On Behalf Of Peter G. Werner
> >> Sent: Sunday, 23 October 2011 4:55 AM
> >> To: [hidden email]
> >> Subject: Re: "Out of Office autoreply" courtesy
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> And I hate to point this out, but the question I originally asked
> >> (concerning the definition of Airy Units) has been drowned out by all
> >> the
> >> commentary about the "Out of Office autoreply" that my initial email
> >> generated.
> >>
> >> If nobody has an answer to the question, no worries, but I'd hate to
> >see
> >> the
> >> topic get lost under the weight of discussion of listserv function.
> >>
> >> -----
> >> No virus found in this message.
> >> Checked by AVG - www.avg.com
> >> Version: 10.0.1411 / Virus Database: 1522/3970 - Release Date:
> >10/23/11
> >>
> >>
> >> ---------------------------------------------------------------------
> >> *SECURITY/CONFIDENTIALITY WARNING:
> >> This message and any attachments are intended solely for the
> >individual
> >> or entity to which they are addressed. This communication may contain
> >> information that is privileged, confidential, or exempt from
> >disclosure
> >> under applicable law (e.g., personal health information, research
> >data,
> >> financial information). Because this e-mail has been sent without
> >> encryption, individuals other than the intended recipient may be able
> >to
> >> view the information, forward it to others or tamper with the
> >> information without the knowledge or consent of the sender. If you
> are
> >> not the intended recipient, or the employee or person responsible for
> >> delivering the message to the intended recipient, any dissemination,
> >> distribution or copying of the communication is strictly prohibited.
> >If
> >> you received the communication in error, please notify the sender
> >> immediately by replying to this message and deleting the message and
> >any
> >> accompanying files from your system. If, due to the security risks,
> >you
> >> do not wish to receive further communications via e-mail, please
> reply
> >> to this message and inform the sender that you do not wish to receive
> >> further e-mail from the sender.
> >>
> >> ---------------------------------------------------------------------
> >>
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> >> No virus found in this message.
> >> Checked by AVG - www.avg.com
> >> Version: 2012.0.1834 / Virus Database: 2092/4574 - Release Date:
> >> 10/25/11
> >
> >-----
> >No virus found in this message.
> >Checked by AVG - www.avg.com
> >Version: 2012.0.1834 / Virus Database: 2092/4577 - Release Date:
> >10/26/11
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1411 / Virus Database: 2092/3977 - Release Date: 10/26/11

     

[Rosemary.White]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Dear all,

While we're on the topic of GFP, what would be considered a "standard" GFP
protein turnover time?  I've seen only a couple of papers on this, both
from transfected animal (i.e. not plant!) cells in culture, with
approximate turnover of 24 h. Mainly interested in free, cytoplasmic GFP,
any further information would be very useful.

thanks,
Rosemary White

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601

T 02 6246 5475
F 02 6246 5334
E [hidden email]

[Vitaly Boyko]

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Sorry for the late posting - now confirmation required within a CERTAIN TIME FRAME - What an INNOVATION!!!???
Hi Michelle,

it depends. 3-10 hours. 
3+ hours when expressed in neurons (axons which are 5um thick with low autofluorescence) and 
up to 8 hours in Cos-7 cells, and 
up to 10 hours in 293T or Hela. 
It also depends on your construct and promoter.

Cos-7 and 293T lines carry large T-antigen of SV-40 (transcriptional activator), autofluorescence of 293T cells is a bit high.

Under TIR illumination you would see the signal above background a couple of hours earlier.
 
I suggest to use YFP instead of GFP (a bit better Signal to Noise ratio) - you may detect YFP signal up to one hour earlier. Also, mCherry could be a good choice, though it is more sticky to membranes, but it should not be an issue at early time points.

Good luck,

Vitaly   



________________________________
From: Michelle Peckham <[hidden email]>
To: [hidden email]
Sent: Friday, October 28, 2011 7:38 PM
Subject: Re: how long does it take eGFP to fold and become fluorescent in mammalian cells?

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

That's great, just what I wanted to know but couldn't find the right reference
Thanks



On 28 Oct 2011, at 22:49, "Kurt Thorn" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> There are a number of papers characterizing the folding and maturation
> rate of EGFP, and they usually find rates of around 30 minutes.  Here is
> one with some detailed characterization:
> http://pubs.acs.org/doi/full/10.1021/ja0580439
>
> Whether or not this depends on the fusion context of the GFP, I don't know.
>
> Kurt
>
> On 10/28/2011 1:09 PM, Michelle Peckham wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Yes, that's about the length of time before I see fluorescence too - and
>> it's organised into structures.  But if it takes 6 hours for GFP to
>> fold...then is this GFP folding time, or does GFP fold very quickly and I
>> don't have to worry about that?
>>
>> M
>>
>> On 28/10/2011 20:47, "Zaal, Kristina (NIH/NIAMS) [E]"<[hidden email]>
>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> We have seen expression 6 h after a fugene transfections of plasmid
>>> encoding a eGFP labeled nuclear membrane protein in COS7 cells. I think
>>> the cell type and the patterns of the protein expression makes a
>>> difference: diffuse proteins are harder to pick up than those
>>> concentrated in structures.
>>>
>>> Kristien J.M. Zaal, Ph.D.
>>> Staff Scientist, Facility Manager
>>> Light Imaging Section
>>> NIAMS, NIH
>>> Bldg 50, Rm 1533
>>> Bethesda, MD 20892
>>> Phone [phone]; FAX [phone]
>>> ________________________________________
>>> From: Michelle Peckham [[hidden email]]
>>> Sent: Friday, October 28, 2011 12:37 PM
>>> To: [hidden email]
>>> Subject: how long does it take eGFP to fold and become fluorescent in
>>> mammalian cells?
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Having just done some overnight filming from cells infected with a
>>> adenovirus (eGFP-tagged construct), I need to know how long it takes for
>>> the eGFP to become fluorescent..
>>>
>>> Does anyone know?
>>>
>>> Thanks
>>>
>>> Michelle

[Andreas Bruckbauer]

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 Back to the original question, and Craig's reply about using the 1/e parameter. There are even more measurements for PSF, laser beam or focal spot size. In Fluorescence correlation spectroscopy the 1/e^2 diameter is important and some people give the (half)width they get from a Gaussian fit. The last one is quite interesting as it is fairly easy to do the fit. However using a normal Gaussian fit e.g. in imagej

y = a + (b-a)*exp(-(x-c)*(x-c)/(2*d*d))

the width parameter d is the standard deviation of the Gaussian distribution, which would be the radius where the curves drops to sqrt(1/e) = 0.61. If you think about each photon beeing a position measurement, this also would be the root mean square error (RMSE) of the measurement. Obmitting the factor 2 in the fitting equation would give you the 1/e radius and to go from the standard deviation to the full width at half maximum (fwhm) one would need to multiply with 2*sqrt(2*ln(2)) = 2.35, to go from the 1/e radius to the fwhm one multiplies with 2*sqrt(ln(2)) = 1.67. Please check the math ...

Just wanted to point this out as i always found this quite confusing.

best wishes

Andreas




 

 

-----Original Message-----
From: Craig Brideau <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Wed, 26 Oct 2011 19:25
Subject: Re: Airy Units -


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There are two brightness criteria for measuring width.  There's FWHM as

mentioned by Christophe, but there is also 1/e (where e is euler's number).

 Basically when the power level drops to 1/e on both sides of the curve is

the point where the width measurement is taken.  This is also frequently

used to describe beam diameters in lasers.  For example a laser described as

having a 2mm beam means that the points on opposite sides of the beam at

which intensity drops to 1/e of the maximum is 2mm apart.



Craig





On Wed, Oct 26, 2011 at 9:43 AM, Christophe Leterrier <

[hidden email]> wrote:



> *****

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

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

> *****

>

> I think the formula with 0.51 is the full width at half maximum (FWHM),

> i.e.

> the distance between the two points where the intensity is half the

> maximum,

> whereas the one with 0.61 is the radius of the Airy disk, i.e. the distance

> between the maximum and the first minimum.

>

> Christophe

>

> On Wed, Oct 26, 2011 at 17:25, Armstrong, Brian <[hidden email]>

> wrote:

>

> > *****

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

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

> > *****

> >

> > Guy (and list), in a couple of super-resolution talks I've attended they

> > were using 0.51 instead of 0.61 for the constant. Do you know the

> rationale

> > behind this?

> > Thanks, Brian

> >

> > Brian Armstrong PhD

> > Light Microscopy Core

> > Beckman Research Institute

> > 1450 East Duarte Rd

> > Duarte, CA 91010

> > 626-256-4673 x62872

> >

> >

> http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm

> >

> >

> > -----Original Message-----

> > From: Confocal Microscopy List [mailto:[hidden email]]

> > On Behalf Of Guy Cox

> > Sent: Wednesday, October 26, 2011 2:46 AM

> > To: [hidden email]

> > Subject: Airy Units - was: RE: "Out of Office autoreply" courtesy

> >

> > *****

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

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

> > *****

> >

> > You are right, it has been totally drowned out!

> >

> > The Airy unit is defined by the size of the Airy disk, that is the size

> > of the disk representing the image of a point object.  Airy was an

> > astronomer and thus derived it by reference to stars (which, though

> > huge, are so far away that they appear as point objects).  John Strutt,

> > Lord Rayleigh, proposed a general resolution criterion that two objects

> > can be considered resolved if the maximum of one Airy disk lies on the

> > first minimum of the other.  This criterion, the radius of the central

> > disk (ignoring surrounding haloes) is given by  r   =  0.61 lambda  /

> > NA, where lambda is the wavelength of the light being used.

> >

> >                                         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 [phone]     Fax [phone]

> >             Mobile [phone]

> > ______________________________________________

> >      http://www.guycox.net

> >

> >

> >

> > -----Original Message-----

> > From: Confocal Microscopy List [mailto:[hidden email]]

> > On Behalf Of Peter G. Werner

> > Sent: Sunday, 23 October 2011 4:55 AM

> > To: [hidden email]

> > Subject: Re: "Out of Office autoreply" courtesy

> >

> > *****

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

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

> > *****

> >

> > And I hate to point this out, but the question I originally asked

> > (concerning the definition of Airy Units) has been drowned out by all

> > the

> > commentary about the "Out of Office autoreply" that my initial email

> > generated.

> >

> > If nobody has an answer to the question, no worries, but I'd hate to see

> > the

> > topic get lost under the weight of discussion of listserv function.

> >

> > -----

> > No virus found in this message.

> > Checked by AVG - www.avg.com

> > Version: 10.0.1411 / Virus Database: 1522/3970 - Release Date: 10/23/11

> >

> >

> > ---------------------------------------------------------------------

> > *SECURITY/CONFIDENTIALITY WARNING:

> > This message and any attachments are intended solely for the individual

> or

> > entity to which they are addressed. This communication may contain

> > information that is privileged, confidential, or exempt from disclosure

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>