what fluorescent proteins are working best for precision localization microscopy (nanoscopy) - in your and your users hands?

[George McNamara]

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Dear confocal listserv,

what fluorescent proteins are working best for precision localization
microscopy (nanoscopy) - in your and your users hands?

I know the literature pretty well - what I am looking for is YOUR
experiences, ideally good, but if you've been burned, what turned out to
be bad.


thanks in advance,

George
p.s. and yes, I will email Michael Davidson directly for his preferences.

[John Oreopoulos]

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tdEos

John Oreopoulos

On 2013-04-09, at 8:04 PM, George McNamara <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear confocal listserv,
>
> what fluorescent proteins are working best for precision localization microscopy (nanoscopy) - in your and your users hands?
>
> I know the literature pretty well - what I am looking for is YOUR experiences, ideally good, but if you've been burned, what turned out to be bad.
>
>
> thanks in advance,
>
> George
> p.s. and yes, I will email Michael Davidson directly for his preferences.

[Roger Phillips]

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John,
Have you tried 2P activation of tdEOS?
Roger

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: 10 April 2013 02:10
To: [hidden email]
Subject: Re: what fluorescent proteins are working best for precision localization microscopy (nanoscopy) - in your and your users hands?

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tdEos

John Oreopoulos

On 2013-04-09, at 8:04 PM, George McNamara <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear confocal listserv,
>
> what fluorescent proteins are working best for precision localization microscopy (nanoscopy) - in your and your users hands?
>
> I know the literature pretty well - what I am looking for is YOUR experiences, ideally good, but if you've been burned, what turned out to be bad.
>
>
> thanks in advance,
>
> George
> p.s. and yes, I will email Michael Davidson directly for his preferences.

[Guy Cox-2]

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Well, I have and the answer is that it just doesn't work.

                                                                      Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Roger Phillips
Sent: Wednesday, 10 April 2013 6:16 PM
To: [hidden email]
Subject: Re: what fluorescent proteins are working best for precision localization microscopy (nanoscopy) - in your and your users hands?

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John,
Have you tried 2P activation of tdEOS?
Roger

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: 10 April 2013 02:10
To: [hidden email]
Subject: Re: what fluorescent proteins are working best for precision localization microscopy (nanoscopy) - in your and your users hands?

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tdEos

John Oreopoulos

On 2013-04-09, at 8:04 PM, George McNamara <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear confocal listserv,
>
> what fluorescent proteins are working best for precision localization microscopy (nanoscopy) - in your and your users hands?
>
> I know the literature pretty well - what I am looking for is YOUR experiences, ideally good, but if you've been burned, what turned out to be bad.
>
>
> thanks in advance,
>
> George
> p.s. and yes, I will email Michael Davidson directly for his preferences.

[Rickman, Colin]

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Hi

We have used photoactivatable mCherry successfully both for PALM and sptPALM in a couple of published projects.. We started off using photoactivatable GFP which in hindsight is less than optimal due to spontaneous activation and activation induced by the excitation source.

Colin
Dr Colin Rickman
Institute of Biological Chemistry, Biophysics and Bioengineering
School of Engineering and Physical Sciences
Heriot-Watt University
Edinburgh
EH14 4AS

Tel: [phone] (Office)

http://www.ib3.eps.hw.ac.uk
http://www.esric.org


On 10 Apr 2013, at 01:04, George McNamara <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear confocal listserv,
>
> what fluorescent proteins are working best for precision localization microscopy (nanoscopy) - in your and your users hands?
>
> I know the literature pretty well - what I am looking for is YOUR experiences, ideally good, but if you've been burned, what turned out to be bad.
>
>
> thanks in advance,
>
> George
> p.s. and yes, I will email Michael Davidson directly for his preferences.

[Tom Blanpied]

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Hi,

We use mEos whenever possible. mEos2 seemed great to us, as we never saw
any effects of the small dimerization tendency. mEos3 does look to be at least
as good, and it does seem a bit brighter; it is our go-to probe now. We avoid
the bulk of the tdEos, but did think it was a good molecule.

PA-mCherry we thought was pretty terrific at first, but we no longer use it
unless we really need the separate GFP channel for something. It is
considerably dimmer in our hands than mEos  (photons/frame and number of
frames till bleaching), and also seems to have a tendency to produce a smaller
population of photoactivated molecules. We've not tried to determine whether
this was due to expression level or folding or a non-activatable fraction, but it
does not seem to be due simply to the diminished ability to localize dim or
quickly bleached molecules.  

PA-GFP and Dronpa we did a little bit with, but in general we have so much
autofluorescent green stuff in our relatively old cultured neurons that anything
in that spectral range will be unsatisfying. We finally managed to obtain some
PS-CFP2, but haven't yet measured much about it; we're hopeful it will be the
best of that range.  

I'll be curious to hear comparisons of dendra2-type molecules.

Tom

[Tom Blanpied]

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Hi,
Sorry, I conflated two molecules in my previous post.
In our experience, PA-mCherry provides adequate numbers of molecules, though
isn't as bright or long-lived as mEos2/3.
PA-TagRFP is considerably brighter than PA-mCherry. However, on two or three
different proteins with side-by-side comparisons, it has provided a smaller number
of localized molecules than Eos or Cherry. Thus, longer tracks than PA-mCherry,
but fewer.
Tom

[Tim Feinstein-2]

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I wonder whether anyone has experience with the inducible dark states approach reported by Christian Soeller in New Zealand.  In theory one could do super-resolution TIRF with any fluorophore you want.    

http://www.sciencedirect.com/science/article/pii/S0006349508000763

Markus Sauer followed up with a protocol:

http://www.nature.com/nprot/journal/v6/n7/abs/nprot.2011.336.html   

Has anyone tried this?  

cheers,


TF

Timothy Feinstein, PhD
Visiting Research Associate
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Apr 11, 2013, at 8:35 AM, Tom Blanpied wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> We use mEos whenever possible. mEos2 seemed great to us, as we never saw
> any effects of the small dimerization tendency. mEos3 does look to be at least
> as good, and it does seem a bit brighter; it is our go-to probe now. We avoid
> the bulk of the tdEos, but did think it was a good molecule.
>
> PA-mCherry we thought was pretty terrific at first, but we no longer use it
> unless we really need the separate GFP channel for something. It is
> considerably dimmer in our hands than mEos  (photons/frame and number of
> frames till bleaching), and also seems to have a tendency to produce a smaller
> population of photoactivated molecules. We've not tried to determine whether
> this was due to expression level or folding or a non-activatable fraction, but it
> does not seem to be due simply to the diminished ability to localize dim or
> quickly bleached molecules.  
>
> PA-GFP and Dronpa we did a little bit with, but in general we have so much
> autofluorescent green stuff in our relatively old cultured neurons that anything
> in that spectral range will be unsatisfying. We finally managed to obtain some
> PS-CFP2, but haven't yet measured much about it; we're hopeful it will be the
> best of that range.  
>
> I'll be curious to hear comparisons of dendra2-type molecules.
>
> Tom

[Douglas Richardson]

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This is STORM.  It doesn't work with every dye, but many.  See here:

Evaluation of fluorophores for optimal performance in localization-based
super-resolution
imaging<http://www.nature.com/nmeth/journal/v8/n12/full/nmeth.1768.html>

   - Graham T Dempsey<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-1>
   ,
   - Joshua C Vaughan<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-2>
   ,
   - Kok Hao Chen<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-3>
   ,
   - Mark Bates<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-4>
   - & Xiaowei Zhuang<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-5>

Nature Methods 8,1027–1036(2011)doi:10.1038/nmeth.1768

-Doug



On Thu, Apr 11, 2013 at 10:20 AM, Tim Feinstein <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I wonder whether anyone has experience with the inducible dark states
> approach reported by Christian Soeller in New Zealand.  In theory one could
> do super-resolution TIRF with any fluorophore you want.
>
> http://www.sciencedirect.com/science/article/pii/S0006349508000763
>
> Markus Sauer followed up with a protocol:
>
> http://www.nature.com/nprot/journal/v6/n7/abs/nprot.2011.336.html
>
> Has anyone tried this?
>
> cheers,
>
>
> TF
>
> Timothy Feinstein, PhD
> Visiting Research Associate
> Laboratory for GPCR Biology
> Dept. of Pharmacology & Chemical Biology
> University of Pittsburgh, School of Medicine
> BST W1301, 200 Lothrop St.
> Pittsburgh, PA  15261
>
> On Apr 11, 2013, at 8:35 AM, Tom Blanpied wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi,
> >
> > We use mEos whenever possible. mEos2 seemed great to us, as we never saw
> > any effects of the small dimerization tendency. mEos3 does look to be at
> least
> > as good, and it does seem a bit brighter; it is our go-to probe now. We
> avoid
> > the bulk of the tdEos, but did think it was a good molecule.
> >
> > PA-mCherry we thought was pretty terrific at first, but we no longer use
> it
> > unless we really need the separate GFP channel for something. It is
> > considerably dimmer in our hands than mEos  (photons/frame and number of
> > frames till bleaching), and also seems to have a tendency to produce a
> smaller
> > population of photoactivated molecules. We've not tried to determine
> whether
> > this was due to expression level or folding or a non-activatable
> fraction, but it
> > does not seem to be due simply to the diminished ability to localize dim
> or
> > quickly bleached molecules.
> >
> > PA-GFP and Dronpa we did a little bit with, but in general we have so
> much
> > autofluorescent green stuff in our relatively old cultured neurons that
> anything
> > in that spectral range will be unsatisfying. We finally managed to
> obtain some
> > PS-CFP2, but haven't yet measured much about it; we're hopeful it will
> be the
> > best of that range.
> >
> > I'll be curious to hear comparisons of dendra2-type molecules.
> >
> > Tom
>

[Tim Feinstein-2]

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Thanks, Doug.  For some reason I thought STORM had a more limited dye selection.  


TF

Timothy Feinstein, PhD
Visiting Research Associate
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Apr 11, 2013, at 1:30 PM, Douglas Richardson wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> This is STORM.  It doesn't work with every dye, but many.  See here:
>
> Evaluation of fluorophores for optimal performance in localization-based
> super-resolution
> imaging<http://www.nature.com/nmeth/journal/v8/n12/full/nmeth.1768.html>
>
>   - Graham T Dempsey<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-1>
>   ,
>   - Joshua C Vaughan<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-2>
>   ,
>   - Kok Hao Chen<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-3>
>   ,
>   - Mark Bates<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-4>
>   - & Xiaowei Zhuang<http://www.nature.com/nmeth/journal/v8/n12/fig_tab/nmeth.1768_ft.html#auth-5>
>
> Nature Methods 8,1027–1036(2011)doi:10.1038/nmeth.1768
>
> -Doug
>
>
>
> On Thu, Apr 11, 2013 at 10:20 AM, Tim Feinstein <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I wonder whether anyone has experience with the inducible dark states
>> approach reported by Christian Soeller in New Zealand.  In theory one could
>> do super-resolution TIRF with any fluorophore you want.
>>
>> http://www.sciencedirect.com/science/article/pii/S0006349508000763
>>
>> Markus Sauer followed up with a protocol:
>>
>> http://www.nature.com/nprot/journal/v6/n7/abs/nprot.2011.336.html
>>
>> Has anyone tried this?
>>
>> cheers,
>>
>>
>> TF
>>
>> Timothy Feinstein, PhD
>> Visiting Research Associate
>> Laboratory for GPCR Biology
>> Dept. of Pharmacology & Chemical Biology
>> University of Pittsburgh, School of Medicine
>> BST W1301, 200 Lothrop St.
>> Pittsburgh, PA  15261
>>
>> On Apr 11, 2013, at 8:35 AM, Tom Blanpied wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi,
>>>
>>> We use mEos whenever possible. mEos2 seemed great to us, as we never saw
>>> any effects of the small dimerization tendency. mEos3 does look to be at
>> least
>>> as good, and it does seem a bit brighter; it is our go-to probe now. We
>> avoid
>>> the bulk of the tdEos, but did think it was a good molecule.
>>>
>>> PA-mCherry we thought was pretty terrific at first, but we no longer use
>> it
>>> unless we really need the separate GFP channel for something. It is
>>> considerably dimmer in our hands than mEos  (photons/frame and number of
>>> frames till bleaching), and also seems to have a tendency to produce a
>> smaller
>>> population of photoactivated molecules. We've not tried to determine
>> whether
>>> this was due to expression level or folding or a non-activatable
>> fraction, but it
>>> does not seem to be due simply to the diminished ability to localize dim
>> or
>>> quickly bleached molecules.
>>>
>>> PA-GFP and Dronpa we did a little bit with, but in general we have so
>> much
>>> autofluorescent green stuff in our relatively old cultured neurons that
>> anything
>>> in that spectral range will be unsatisfying. We finally managed to
>> obtain some
>>> PS-CFP2, but haven't yet measured much about it; we're hopeful it will
>> be the
>>> best of that range.
>>>
>>> I'll be curious to hear comparisons of dendra2-type molecules.
>>>
>>> Tom
>>

[George McNamara]

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Hi Tom,

thanks! ... Michael Davidson, who told me: "The best photoswitchable
proteins are tdEos, mEos4, PS-CFP2, PA-mCherry1, and Dendra2."

I anticipate getting all of these from Michael (collaboration with our
lab - no redistribution from us, so anyone interested will need to
contact Michael directly for plasmids). Looks like I will start with mEos4.

Sincerely,

George
p.s. I had training on M.D. Anderson Cancer Center's OMX yesterday. A
couple of observations (which may not be new to the listserv):
* refractive index of the immersion oil matters a lot. We (Tomasz and
Anna Zal were the trainers) had best results with one of Michael
Davidson's triple immunofluorescence slides mounted in CytoSeal. Olympus
100x/1.4 NA lens. The best oil was RI=1.512. The RI=1.510 and 1.514 oils
were noticably worse in the reconstructions. The RI=1.518 was obviously
a wrong choice from the acquisition screen (no or low contrast fringes).
* Michael's DAPI in CytoSeal photoconverted to green fluorescence after
prolonged excitation with 405 nm laser (several of us have seen, and
mentioned on listserv, this with Hoechst ... I suppose it is possible
the slide was mislabeled and was actually Hoechst???).
* Prolong Gold (on Zeiss 18x18 mm coverglass) ... looks like the R.I.
changes from the edge to the middle of the coverglass. This makes sense
in that the stuff solidifies by outgasssing a [relatively low refractive
index] solvent. Since 0.002 steps in oil matter, it appears that the
Prlong Gold R.I. changes by at least 0.002 (maybe a lot more) from edge
to middle. This suggests that instead of spending time to change the oil
for different areas (distances), that it may be more efficient to select
an oil and then search the coverglass for optimum R.I. match (best fring
contrast and then final check on the reconstructed image). ... I may
decide to use Prolong Gold with "open face" imaging dishes, or buy and
test CytoSeal etc.
* The Applied Precision reconstruction software is called "SoftWoRx" but
a much more accurate name is "painfully tedious manual steps to do
everything anti-productivity WoRx".
* Plan V: I am scheduled for training on M.D. Anderson's Vutara SR-200
on Monday (Tomasz and Anna doing the training again). Maybe I'll end up
optimizing sample preps for this instead of the OMX.


On 4/11/2013 9:14 AM, Tom Blanpied wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
> Sorry, I conflated two molecules in my previous post.
> In our experience, PA-mCherry provides adequate numbers of molecules, though
> isn't as bright or long-lived as mEos2/3.
> PA-TagRFP is considerably brighter than PA-mCherry. However, on two or three
> different proteins with side-by-side comparisons, it has provided a smaller number
> of localized molecules than Eos or Cherry. Thus, longer tracks than PA-mCherry,
> but fewer.
> Tom
>
>    

Hi,

We use mEos whenever possible. mEos2 seemed great to us, as we never saw
any effects of the small dimerization tendency. mEos3 does look to be at least
as good, and it does seem a bit brighter; it is our go-to probe now. We avoid
the bulk of the tdEos, but did think it was a good molecule.

PA-mCherry we thought was pretty terrific at first, but we no longer use it
unless we really need the separate GFP channel for something. It is
considerably dimmer in our hands than mEos  (photons/frame and number of
frames till bleaching), and also seems to have a tendency to produce a smaller
population of photoactivated molecules. We've not tried to determine whether
this was due to expression level or folding or a non-activatable fraction, but it
does not seem to be due simply to the diminished ability to localize dim or
quickly bleached molecules.

PA-GFP and Dronpa we did a little bit with, but in general we have so much
autofluorescent green stuff in our relatively old cultured neurons that anything
in that spectral range will be unsatisfying. We finally managed to obtain some
PS-CFP2, but haven't yet measured much about it; we're hopeful it will be the
best of that range.

I'll be curious to hear comparisons of dendra2-type molecules.

Tom

[Alison J. North]

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Dear George,

You are absolutely correct that the refractive index of the immersion
oil is critical for 3D-SIM, which is why I feel I have to respond to
your posting and expand it a bit, in case any new or potential OMX users
out there get confused.  You say that the 1.512 oil was "the best oil" -
I think it needs to be clarified that you mean this was the best oil for
that particular sample, looking at the particular wavelength
fluorochromes you were imaging, and working at the exact temperature of
that room.   In picking the oil for any OMX experiment you need to first
check the room temp (if only ours were perfectly stable that wouldn't be
an issue, but sadly we do get 1 degree fluctuations from day to day
depending on the weather etc., despite having stipulated that our
engineers needed to give me the most stable room environment
possible!).  Then you need to consider the sample prep - by encouraging
people to use the high performance coverslips and to try to stick to
ProLong Gold (talking about fixed samples only of course) we aim to
minimize the amount of time we need to optimize the oil r.i. each time.  
But you will still need to decide which is the most critical channel in
your experiment - if you are most interested in the red signal then you
will pick a different oil than if the blue or green signals are the most
critical, as you won't get a perfectly optimal signal in every channel
with just one oil.

I also think you are being a tad unfair on SoftWorx - I can't say I find
it at all painful to use.  Every software has its glitches and annoying
features but of the many different softwares we have in our facility we
actually find SoftWorx to be one of the most user-friendly - and it
hardly ever crashes, which is a big plus in my book.  I don't know which
tedious manual steps you are referring to, but the more I have worked
with the system, the more I want to slow the users down and force them
to look at each log file while the reconstruction is going along.  
Checking numbers in the logs like the line spacing, the amplitude, and
the ko angles gives a good initial indication of whether you have a
decent data set or not.  If our users just ran everything as automatic
batch files I fear that the number of artifacts published would shoot up.

Anyway, that's just my two cents and I expect you will disagree
vehemently! :-)
All the best,
Alison

>
> George
> p.s. I had training on M.D. Anderson Cancer Center's OMX yesterday. A
> couple of observations (which may not be new to the listserv):
> * refractive index of the immersion oil matters a lot. We (Tomasz and
> Anna Zal were the trainers) had best results with one of Michael
> Davidson's triple immunofluorescence slides mounted in CytoSeal.
> Olympus 100x/1.4 NA lens. The best oil was RI=1.512. The RI=1.510 and
> 1.514 oils were noticably worse in the reconstructions. The RI=1.518
> was obviously a wrong choice from the acquisition screen (no or low
> contrast fringes).
> * Michael's DAPI in CytoSeal photoconverted to green fluorescence
> after prolonged excitation with 405 nm laser (several of us have seen,
> and mentioned on listserv, this with Hoechst ... I suppose it is
> possible the slide was mislabeled and was actually Hoechst???).
> * Prolong Gold (on Zeiss 18x18 mm coverglass) ... looks like the R.I.
> changes from the edge to the middle of the coverglass. This makes
> sense in that the stuff solidifies by outgasssing a [relatively low
> refractive index] solvent. Since 0.002 steps in oil matter, it appears
> that the Prlong Gold R.I. changes by at least 0.002 (maybe a lot more)
> from edge to middle. This suggests that instead of spending time to
> change the oil for different areas (distances), that it may be more
> efficient to select an oil and then search the coverglass for optimum
> R.I. match (best fring contrast and then final check on the
> reconstructed image). ... I may decide to use Prolong Gold with "open
> face" imaging dishes, or buy and test CytoSeal etc.
> * The Applied Precision reconstruction software is called "SoftWoRx"
> but a much more accurate name is "painfully tedious manual steps to do
> everything anti-productivity WoRx".
> * Plan V: I am scheduled for training on M.D. Anderson's Vutara SR-200
> on Monday (Tomasz and Anna doing the training again). Maybe I'll end
> up optimizing sample preps for this instead of the OMX.
>
>

--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office ++ 212 327 7488
Tel: lab     ++ 212 327 7486
Fax:         ++ 212 327 7489

[George McNamara]

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

Thanks for clarifying for me about refractive index per specimen (I did
mention that even across a specimen the mountant R.I. can change) - and
for pointing out the room temperature and critical fluorescent channel
are both critical issues as well.

SoftWoRx - as far as I can see, Applied Precision / General Electric,
has failed to "nail the landing" on the
acquisition/recontruction/registration. Every step that I have to do
manually, they could have automated years ago. probably the only way
they will go to automation is when they start to lose sales because
someone has a much more efficient workflow.

I do agree with you that critical evaluation of the data is even more
important for structured illumination (and the other nanoscopes) than it
has been for confocal, deconvolution, and even plain old widefield.

Thanks for the tip that I also need to learn to look at - and understand
- the log files.

Sincerely,

George


On 4/12/2013 9:51 AM, Alison North wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear George,
>
> You are absolutely correct that the refractive index of the immersion
> oil is critical for 3D-SIM, which is why I feel I have to respond to
> your posting and expand it a bit, in case any new or potential OMX
> users out there get confused.  You say that the 1.512 oil was "the
> best oil" - I think it needs to be clarified that you mean this was
> the best oil for that particular sample, looking at the particular
> wavelength fluorochromes you were imaging, and working at the exact
> temperature of that room.   In picking the oil for any OMX experiment
> you need to first check the room temp (if only ours were perfectly
> stable that wouldn't be an issue, but sadly we do get 1 degree
> fluctuations from day to day depending on the weather etc., despite
> having stipulated that our engineers needed to give me the most stable
> room environment possible!).  Then you need to consider the sample
> prep - by encouraging people to use the high performance coverslips
> and to try to stick to ProLong Gold (talking about fixed samples only
> of course) we aim to minimize the amount of time we need to optimize
> the oil r.i. each time.  But you will still need to decide which is
> the most critical channel in your experiment - if you are most
> interested in the red signal then you will pick a different oil than
> if the blue or green signals are the most critical, as you won't get a
> perfectly optimal signal in every channel with just one oil.
>
> I also think you are being a tad unfair on SoftWorx - I can't say I
> find it at all painful to use.  Every software has its glitches and
> annoying features but of the many different softwares we have in our
> facility we actually find SoftWorx to be one of the most user-friendly
> - and it hardly ever crashes, which is a big plus in my book.  I don't
> know which tedious manual steps you are referring to, but the more I
> have worked with the system, the more I want to slow the users down
> and force them to look at each log file while the reconstruction is
> going along.  Checking numbers in the logs like the line spacing, the
> amplitude, and the ko angles gives a good initial indication of
> whether you have a decent data set or not.  If our users just ran
> everything as automatic batch files I fear that the number of
> artifacts published would shoot up.
>
> Anyway, that's just my two cents and I expect you will disagree
> vehemently! :-)
> All the best,
> Alison
>
>
>>
>> George
>> p.s. I had training on M.D. Anderson Cancer Center's OMX yesterday. A
>> couple of observations (which may not be new to the listserv):
>> * refractive index of the immersion oil matters a lot. We (Tomasz and
>> Anna Zal were the trainers) had best results with one of Michael
>> Davidson's triple immunofluorescence slides mounted in CytoSeal.
>> Olympus 100x/1.4 NA lens. The best oil was RI=1.512. The RI=1.510 and
>> 1.514 oils were noticably worse in the reconstructions. The RI=1.518
>> was obviously a wrong choice from the acquisition screen (no or low
>> contrast fringes).
>> * Michael's DAPI in CytoSeal photoconverted to green fluorescence
>> after prolonged excitation with 405 nm laser (several of us have
>> seen, and mentioned on listserv, this with Hoechst ... I suppose it
>> is possible the slide was mislabeled and was actually Hoechst???).
>> * Prolong Gold (on Zeiss 18x18 mm coverglass) ... looks like the R.I.
>> changes from the edge to the middle of the coverglass. This makes
>> sense in that the stuff solidifies by outgasssing a [relatively low
>> refractive index] solvent. Since 0.002 steps in oil matter, it
>> appears that the Prlong Gold R.I. changes by at least 0.002 (maybe a
>> lot more) from edge to middle. This suggests that instead of spending
>> time to change the oil for different areas (distances), that it may
>> be more efficient to select an oil and then search the coverglass for
>> optimum R.I. match (best fring contrast and then final check on the
>> reconstructed image). ... I may decide to use Prolong Gold with "open
>> face" imaging dishes, or buy and test CytoSeal etc.
>> * The Applied Precision reconstruction software is called "SoftWoRx"
>> but a much more accurate name is "painfully tedious manual steps to
>> do everything anti-productivity WoRx".
>> * Plan V: I am scheduled for training on M.D. Anderson's Vutara
>> SR-200 on Monday (Tomasz and Anna doing the training again). Maybe
>> I'll end up optimizing sample preps for this instead of the OMX.
>>
>>
>

[Mark Cannell-2]

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

On 13/04/2013, at 7:51 AM, Mark Cannell <[hidden email]> wrote:

> Hi George
>
> Alison's comment about the immersion oil RI being needed to be adjusted by colour arises from the Abbe number of the media between the lens and plane of focus. The Abbe number of the immersion oil is rarely discussed, but is key to achieving achromat performance (the Abbe number is the change in RI with wavelength). There is a risk that you may worsen chromatic aberrations by using a different immersion oil because it will probably have the wrong Abbe number.  (Does the correcting immersion oil set also control Abbe number?) .  
>
> Cheers Mark
>
>
> On 13/04/2013, at 12:28 AM, George McNamara <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Alison,
>>
>> Thanks for clarifying for me about refractive index per specimen (I did mention that even across a specimen the mountant R.I. can change) - and for pointing out the room temperature and critical fluorescent channel are both critical issues as well.
>>
>> SoftWoRx - as far as I can see, Applied Precision / General Electric, has failed to "nail the landing" on the acquisition/recontruction/registration. Every step that I have to do manually, they could have automated years ago. probably the only way they will go to automation is when they start to lose sales because someone has a much more efficient workflow.
>>
>> I do agree with you that critical evaluation of the data is even more important for structured illumination (and the other nanoscopes) than it has been for confocal, deconvolution, and even plain old widefield.
>>
>> Thanks for the tip that I also need to learn to look at - and understand - the log files.
>>
>> Sincerely,
>>
>> George
>>
>>
>> On 4/12/2013 9:51 AM, Alison North wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear George,
>>>
>>> You are absolutely correct that the refractive index of the immersion oil is critical for 3D-SIM, which is why I feel I have to respond to your posting and expand it a bit, in case any new or potential OMX users out there get confused.  You say that the 1.512 oil was "the best oil" - I think it needs to be clarified that you mean this was the best oil for that particular sample, looking at the particular wavelength fluorochromes you were imaging, and working at the exact temperature of that room.   In picking the oil for any OMX experiment you need to first check the room temp (if only ours were perfectly stable that wouldn't be an issue, but sadly we do get 1 degree fluctuations from day to day depending on the weather etc., despite having stipulated that our engineers needed to give me the most stable room environment possible!).  Then you need to consider the sample prep - by encouraging people to use the high performance coverslips and to try to stick to ProLong Gold (talking about fixed samples only of course) we aim to minimize the amount of time we need to optimize the oil r.i. each time.  But you will still need to decide which is the most critical channel in your experiment - if you are most interested in the red signal then you will pick a different oil than if the blue or green signals are the most critical, as you won't get a perfectly optimal signal in every channel with just one oil.
>>>
>>> I also think you are being a tad unfair on SoftWorx - I can't say I find it at all painful to use.  Every software has its glitches and annoying features but of the many different softwares we have in our facility we actually find SoftWorx to be one of the most user-friendly - and it hardly ever crashes, which is a big plus in my book.  I don't know which tedious manual steps you are referring to, but the more I have worked with the system, the more I want to slow the users down and force them to look at each log file while the reconstruction is going along.  Checking numbers in the logs like the line spacing, the amplitude, and the ko angles gives a good initial indication of whether you have a decent data set or not.  If our users just ran everything as automatic batch files I fear that the number of artifacts published would shoot up.
>>>
>>> Anyway, that's just my two cents and I expect you will disagree vehemently! :-)
>>> All the best,
>>> Alison
>>>
>>>
>>>>
>>>> George
>>>> p.s. I had training on M.D. Anderson Cancer Center's OMX yesterday. A couple of observations (which may not be new to the listserv):
>>>> * refractive index of the immersion oil matters a lot. We (Tomasz and Anna Zal were the trainers) had best results with one of Michael Davidson's triple immunofluorescence slides mounted in CytoSeal. Olympus 100x/1.4 NA lens. The best oil was RI=1.512. The RI=1.510 and 1.514 oils were noticably worse in the reconstructions. The RI=1.518 was obviously a wrong choice from the acquisition screen (no or low contrast fringes).
>>>> * Michael's DAPI in CytoSeal photoconverted to green fluorescence after prolonged excitation with 405 nm laser (several of us have seen, and mentioned on listserv, this with Hoechst ... I suppose it is possible the slide was mislabeled and was actually Hoechst???).
>>>> * Prolong Gold (on Zeiss 18x18 mm coverglass) ... looks like the R.I. changes from the edge to the middle of the coverglass. This makes sense in that the stuff solidifies by outgasssing a [relatively low refractive index] solvent. Since 0.002 steps in oil matter, it appears that the Prlong Gold R.I. changes by at least 0.002 (maybe a lot more) from edge to middle. This suggests that instead of spending time to change the oil for different areas (distances), that it may be more efficient to select an oil and then search the coverglass for optimum R.I. match (best fring contrast and then final check on the reconstructed image). ... I may decide to use Prolong Gold with "open face" imaging dishes, or buy and test CytoSeal etc.
>>>> * The Applied Precision reconstruction software is called "SoftWoRx" but a much more accurate name is "painfully tedious manual steps to do everything anti-productivity WoRx".
>>>> * Plan V: I am scheduled for training on M.D. Anderson's Vutara SR-200 on Monday (Tomasz and Anna doing the training again). Maybe I'll end up optimizing sample preps for this instead of the OMX.
>>>>
>>>>
>>>
>
> 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]
>
>
>
>
>

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]

[Adrian Smith-6]

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

On 12/04/2013, at 10:59 PM, George McNamara <[hidden email]> wrote:

>  Michael's DAPI in CytoSeal photoconverted to green fluorescence after prolonged excitation with 405 nm laser (several of us have seen, and mentioned on listserv, this with Hoechst ... I suppose it is possible the slide was mislabeled and was actually Hoechst???).


Hi George,

I'm not 100% sure what you mean by "photoconverted to green fluorescence" but we have had issues with DAPI photoconversion after 405nm excitation on a couple of occasions in the last few years.

There have been a couple of publications in that last year that describe exactly what we have seen:-

> J Microsc. 2012 Apr;246(1):89-95. doi: 10.1111/j.1365-2818.2011.03591.x. Epub 2012 Jan 31.
> Photoconversion of DAPI following UV or violet excitation can cause DAPI to fluoresce with blue or cyan excitation.
> Piterburg M, Panet H, Weiss A.
> Abstract
> 4'-6-Diamidino-2-phenylindole is a fluorescent dye commonly used to visualize deoxyribonucleic acid or cell nuclei in fixed cell preparations, and is often used together with fluorescein or green fluorescent protein, which can be excited without exciting 4'-6-Diamidino-2-phenylindole. It is assumed that when using typical fluorescein or green fluorescent protein filter cubes, 4'-6-Diamidino-2-phenylindole will not be observed. In this paper, we show that following observation of 4'-6-Diamidino-2-phenylindole using UV or violet excitation, it may become sensitive to the blue/cyan excitation used in fluorescein/green fluorescent protein filter cubes. This has serious implications for the use of 4'-6-Diamidino-2-phenylindole together with widely used green fluorophores in double labelling experiments.
>
>




> Histochem Cell Biol. 2013 Jan;139(1):195-204. doi: 10.1007/s00418-012-1039-8. Epub 2012 Oct 14.
> The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem.
> Jež M, Bas T, Veber M, Košir A, Dominko T, Page R, Rožman P.
> Abstract
> Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4',6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.



Regards,

Adrian Smith
Centenary Institute, Sydney, Australia

[Vitaly Boyko]

*****
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*****

Using ProlongGold for fixed samples in SIM complicates matters, as RI of ProlongGold changes over (maturation) time, plus ProlongGold attenuates GFP and other dyes fluorescence significantly.
Also, 2x increase in resolution generates pretty pictures but adds little in understanding molecular aspects of protein-protein, protein-RNA, protein-ligand interactions. Also, SIM is very limited to studies of dynamic events (live samples) that is (time domain) crucial to modern Cell Molecular Biology. Unfortunately, PALM/STORM has similar limitations when talking LIVE...      

Good luck,

Vitaly


________________________________
 From: Mark Cannell <[hidden email]>
To: [hidden email]
Sent: Saturday, April 13, 2013 2:58 AM
Subject: Re: Responding to the OMX oil comments.....
 

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

On 13/04/2013, at 7:51 AM, Mark Cannell <[hidden email]> wrote:

> Hi George
>
> Alison's comment about the immersion oil RI being needed to be adjusted by colour arises from the Abbe number of the media between the lens and plane of focus. The Abbe number of the immersion oil is rarely discussed, but is key to achieving achromat performance (the Abbe number is the change in RI with wavelength). There is a risk that you may worsen chromatic aberrations by using a different immersion oil because it will probably have the wrong Abbe number.  (Does the correcting immersion oil set also control Abbe number?) . 
>
> Cheers Mark
>
>
> On 13/04/2013, at 12:28 AM, George McNamara <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Alison,
>>
>> Thanks for clarifying for me about refractive index per specimen (I did mention that even across a specimen the mountant R.I. can change) - and for pointing out the room temperature and critical fluorescent channel are both critical issues as well.
>>
>> SoftWoRx - as far as I can see, Applied Precision / General Electric, has failed to "nail the landing" on the acquisition/recontruction/registration. Every step that I have to do manually, they could have automated years ago. probably the only way they will go to automation is when they start to lose sales because someone has a much more efficient workflow.
>>
>> I do agree with you that critical evaluation of the data is even more important for structured illumination (and the other nanoscopes) than it has been for confocal, deconvolution, and even plain old widefield.
>>
>> Thanks for the tip that I also need to learn to look at - and understand - the log files.
>>
>> Sincerely,
>>
>> George
>>
>>
>> On 4/12/2013 9:51 AM, Alison North wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear George,
>>>
>>> You are absolutely correct that the refractive index of the immersion oil is critical for 3D-SIM, which is why I feel I have to respond to your posting and expand it a bit, in case any new or potential OMX users out there get confused.  You say that the 1.512 oil was "the best oil" - I think it needs to be clarified that you mean this was the best oil for that particular sample, looking at the particular wavelength fluorochromes you were imaging, and working at the exact temperature of that room.   In picking the oil for any OMX experiment you need to first check the room temp (if only ours were perfectly stable that wouldn't be an issue, but sadly we do get 1 degree fluctuations from day to day depending on the weather etc., despite having stipulated that our engineers needed to give me the most stable room environment possible!).  Then you need to consider the sample prep - by encouraging people to use the high performance coverslips and to try

 to stick to ProLong Gold (talking about fixed samples only of course) we aim to minimize the amount of time we need to optimize the oil r.i. each time.  But you will still need to decide which is the most critical channel in your experiment - if you are most interested in the red signal then you will pick a different oil than if the blue or green signals are the most critical, as you won't get a perfectly optimal signal in every channel with just one oil.

>>>
>>> I also think you are being a tad unfair on SoftWorx - I can't say I find it at all painful to use.  Every software has its glitches and annoying features but of the many different softwares we have in our facility we actually find SoftWorx to be one of the most user-friendly - and it hardly ever crashes, which is a big plus in my book.  I don't know which tedious manual steps you are referring to, but the more I have worked with the system, the more I want to slow the users down and force them to look at each log file while the reconstruction is going along.  Checking numbers in the logs like the line spacing, the amplitude, and the ko angles gives a good initial indication of whether you have a decent data set or not.  If our users just ran everything as automatic batch files I fear that the number of artifacts published would shoot up.
>>>
>>> Anyway, that's just my two cents and I expect you will disagree vehemently! :-)
>>> All the best,
>>> Alison
>>>
>>>
>>>>
>>>> George
>>>> p.s. I had training on M.D. Anderson Cancer Center's OMX yesterday. A couple of observations (which may not be new to the listserv):
>>>> * refractive index of the immersion oil matters a lot. We (Tomasz and Anna Zal were the trainers) had best results with one of Michael Davidson's triple immunofluorescence slides mounted in CytoSeal. Olympus 100x/1.4 NA lens. The best oil was RI=1.512. The RI=1.510 and 1.514 oils were noticably worse in the reconstructions. The RI=1.518 was obviously a wrong choice from the acquisition screen (no or low contrast fringes).
>>>> * Michael's DAPI in CytoSeal photoconverted to green fluorescence after prolonged excitation with 405 nm laser (several of us have seen, and mentioned on listserv, this with Hoechst ... I suppose it is possible the slide was mislabeled and was actually Hoechst???).
>>>> * Prolong Gold (on Zeiss 18x18 mm coverglass) ... looks like the R.I. changes from the edge to the middle of the coverglass. This makes sense in that the stuff solidifies by outgasssing a [relatively low refractive index] solvent. Since 0.002 steps in oil matter, it appears that the Prlong Gold R.I. changes by at least 0.002 (maybe a lot more) from edge to middle. This suggests that instead of spending time to change the oil for different areas (distances), that it may be more efficient to select an oil and then search the coverglass for optimum R.I. match (best fring contrast and then final check on the reconstructed image). ... I may decide to use Prolong Gold with "open face" imaging dishes, or buy and test CytoSeal etc.
>>>> * The Applied Precision reconstruction software is called "SoftWoRx" but a much more accurate name is "painfully tedious manual steps to do everything anti-productivity WoRx".
>>>> * Plan V: I am scheduled for training on M.D. Anderson's Vutara SR-200 on Monday (Tomasz and Anna doing the training again). Maybe I'll end up optimizing sample preps for this instead of the OMX.
>>>>
>>>>
>>>
>
> 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]
>
>
>
>
>

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]

[George McNamara]

*****
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*****

Hi Mark,

Thanks for the details on this. The Staudt 2007 MRT paper on
2,2'-thiodiethanol stated:

    "Another important feature of TDE is that its dispersion matches the
    dispersion of regular immersion oil (see appendix); hence no
    additional chromatic aberration is induced. As expected, embedding
    cells in a 97% TDE solution significantly reduces the phase contrast
    which demonstrates the improvement of the optical properties of
    fixed samples for high resolution imaging (Fig. 5)."

so I will likely test this. And I'll follow Stan Vitha's protocol
(Microscopy Today 2010, also posted on the listserv) for transitioning
from aqueous to TDE.

The OMX here is going to be modified by API/GE next month to be
optimized for 60x lenses, in particular the Olympus 60x/1.3NA silicon
oil immersion lens for S.I., refractive index 1.404, so hopefully that
oil has similar dispersion properties to the Cargille oil(s) that API/GE
sells for the OMX.

***

Our lab is getting interested in thick specimens (i.e. confocal imaging)
so I thought it worth mentioning on the listserv (for those who manage
to avoid press release blitzes) that Karl Deisseroth and colleagues (K.
Chung et al Nature 2013) just published on CLARITY - yet another optical
clearing scheme (re: Scale, Erturk&dodt's tetrahydrofuran ... not to
forget the classics of methyl salicylate=oil of wintergreen and of BABB
- see Robert Zucker's papers for more).

Enjoy,

George

On 4/13/2013 1:58 AM, Mark Cannell wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> On 13/04/2013, at 7:51 AM, Mark Cannell<[hidden email]>  wrote:
>
>    
>> Hi George
>>
>> Alison's comment about the immersion oil RI being needed to be adjusted by colour arises from the Abbe number of the media between the lens and plane of focus. The Abbe number of the immersion oil is rarely discussed, but is key to achieving achromat performance (the Abbe number is the change in RI with wavelength). There is a risk that you may worsen chromatic aberrations by using a different immersion oil because it will probably have the wrong Abbe number.  (Does the correcting immersion oil set also control Abbe number?) .
>>
>> Cheers Mark
>>
>>
>> On 13/04/2013, at 12:28 AM, George McNamara<[hidden email]>  wrote:
>>
>>      
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Alison,
>>>
>>> Thanks for clarifying for me about refractive index per specimen (I did mention that even across a specimen the mountant R.I. can change) - and for pointing out the room temperature and critical fluorescent channel are both critical issues as well.
>>>
>>> SoftWoRx - as far as I can see, Applied Precision / General Electric, has failed to "nail the landing" on the acquisition/recontruction/registration. Every step that I have to do manually, they could have automated years ago. probably the only way they will go to automation is when they start to lose sales because someone has a much more efficient workflow.
>>>
>>> I do agree with you that critical evaluation of the data is even more important for structured illumination (and the other nanoscopes) than it has been for confocal, deconvolution, and even plain old widefield.
>>>
>>> Thanks for the tip that I also need to learn to look at - and understand - the log files.
>>>
>>> Sincerely,
>>>
>>> George
>>>
>>>
>>> On 4/12/2013 9:51 AM, Alison North wrote:
>>>        
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Dear George,
>>>>
>>>> You are absolutely correct that the refractive index of the immersion oil is critical for 3D-SIM, which is why I feel I have to respond to your posting and expand it a bit, in case any new or potential OMX users out there get confused.  You say that the 1.512 oil was "the best oil" - I think it needs to be clarified that you mean this was the best oil for that particular sample, looking at the particular wavelength fluorochromes you were imaging, and working at the exact temperature of that room.   In picking the oil for any OMX experiment you need to first check the room temp (if only ours were perfectly stable that wouldn't be an issue, but sadly we do get 1 degree fluctuations from day to day depending on the weather etc., despite having stipulated that our engineers needed to give me the most stable room environment possible!).  Then you need to consider the sample prep - by encouraging people to use the high performance coverslips and to try to stick to ProLong Gold (talking about fixed samples only of course) we aim to minimize the amount of time we need to optimize the oil r.i. each time.  But you will still need to decide which is the most critical channel in your experiment - if you are most interested in the red signal then you will pick a different oil than if the blue or green signals are the most critical, as you won't get a perfectly optimal signal in every channel with just one oil.
>>>>
>>>> I also think you are being a tad unfair on SoftWorx - I can't say I find it at all painful to use.  Every software has its glitches and annoying features but of the many different softwares we have in our facility we actually find SoftWorx to be one of the most user-friendly - and it hardly ever crashes, which is a big plus in my book.  I don't know which tedious manual steps you are referring to, but the more I have worked with the system, the more I want to slow the users down and force them to look at each log file while the reconstruction is going along.  Checking numbers in the logs like the line spacing, the amplitude, and the ko angles gives a good initial indication of whether you have a decent data set or not.  If our users just ran everything as automatic batch files I fear that the number of artifacts published would shoot up.
>>>>
>>>> Anyway, that's just my two cents and I expect you will disagree vehemently! :-)
>>>> All the best,
>>>> Alison
>>>>
>>>>
>>>>          
>>>>> George
>>>>> p.s. I had training on M.D. Anderson Cancer Center's OMX yesterday. A couple of observations (which may not be new to the listserv):
>>>>> * refractive index of the immersion oil matters a lot. We (Tomasz and Anna Zal were the trainers) had best results with one of Michael Davidson's triple immunofluorescence slides mounted in CytoSeal. Olympus 100x/1.4 NA lens. The best oil was RI=1.512. The RI=1.510 and 1.514 oils were noticably worse in the reconstructions. The RI=1.518 was obviously a wrong choice from the acquisition screen (no or low contrast fringes).
>>>>> * Michael's DAPI in CytoSeal photoconverted to green fluorescence after prolonged excitation with 405 nm laser (several of us have seen, and mentioned on listserv, this with Hoechst ... I suppose it is possible the slide was mislabeled and was actually Hoechst???).
>>>>> * Prolong Gold (on Zeiss 18x18 mm coverglass) ... looks like the R.I. changes from the edge to the middle of the coverglass. This makes sense in that the stuff solidifies by outgasssing a [relatively low refractive index] solvent. Since 0.002 steps in oil matter, it appears that the Prlong Gold R.I. changes by at least 0.002 (maybe a lot more) from edge to middle. This suggests that instead of spending time to change the oil for different areas (distances), that it may be more efficient to select an oil and then search the coverglass for optimum R.I. match (best fring contrast and then final check on the reconstructed image). ... I may decide to use Prolong Gold with "open face" imaging dishes, or buy and test CytoSeal etc.
>>>>> * The Applied Precision reconstruction software is called "SoftWoRx" but a much more accurate name is "painfully tedious manual steps to do everything anti-productivity WoRx".
>>>>> * Plan V: I am scheduled for training on M.D. Anderson's Vutara SR-200 on Monday (Tomasz and Anna doing the training again). Maybe I'll end up optimizing sample preps for this instead of the OMX.
>>>>>
>>>>>
>>>>>            
>>>>          
>> 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]
>>
>>
>>
>>
>>
>>      
> 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]
>
>    

[George McNamara]

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

Hi Vitaly,

to quote one of my (many) mentors, Larry Phillips (now at
http://www.biovis.com/contact.htm , was referring to MetaMorph Imaging
Systems at the time ):

"we sell [data] shovels. Some customers shovel gold. Some shovel shit."

This is true of all of the equipment in researchers labs, not just
imaging systems (and not just MetaMorph customers).

Here is a web link to a blog from someone who is a fan of the OMX
('OMG') microscope,

http://www.govexec.com/excellence/promising-practices/2013/03/photo-omg-microscope-lives-its-name/62021/

Unfortunately the blog did not mention what day Jane Stout's "mitosis
image will light up a billboard in Times Square in New York City in
April. That is a wonderful celebration of science!"  (and I'm not going
to be visiting NYC anytime soon, so not going to look up the date -
should be on the GE web site).

As for " 2x increase in resolution ... ", I disagree with everythhing
you wrote:
1. improvement is 8x, we are talking three dimensions, 2^3 = 8.
2. understanding is done by the researcher, not the pixels.
3. live - read the SIM papers.
4. PALM/STORM/Vutara "limitations" - read the papers. In particular
check out Xiaowei Zhuang's neuronal synapse papers and Clare Waterman's
focal adhesions paper.

I also want to mention with respect to optical, computation
(deconvolution) and combined (S.I.) resolution improvements, a point
that Paul Goodwin of API/GE made to me: these are not just resolution
improvement(s), but increase in contrast. I suggest that better contrast
can lead a researcher(s) to better understanding.

George



On 4/13/2013 8:04 AM, Vitaly Boyko wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Using ProlongGold for fixed samples in SIM complicates matters, as RI of ProlongGold changes over (maturation) time, plus ProlongGold attenuates GFP and other dyes fluorescence significantly.
> Also, 2x increase in resolution generates pretty pictures but adds little in understanding molecular aspects of protein-protein, protein-RNA, protein-ligand interactions. Also, SIM is very limited to studies of dynamic events (live samples) that is (time domain) crucial to modern Cell Molecular Biology. Unfortunately, PALM/STORM has similar limitations when talking LIVE...      
>
> Good luck,
>
> Vitaly
>
>
> ________________________________
>   From: Mark Cannell<[hidden email]>
> To: [hidden email]
> Sent: Saturday, April 13, 2013 2:58 AM
> Subject: Re: Responding to the OMX oil comments.....
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> On 13/04/2013, at 7:51 AM, Mark Cannell<[hidden email]>  wrote:
>
>    
>> Hi George
>>
>> Alison's comment about the immersion oil RI being needed to be adjusted by colour arises from the Abbe number of the media between the lens and plane of focus. The Abbe number of the immersion oil is rarely discussed, but is key to achieving achromat performance (the Abbe number is the change in RI with wavelength). There is a risk that you may worsen chromatic aberrations by using a different immersion oil because it will probably have the wrong Abbe number.  (Does the correcting immersion oil set also control Abbe number?) .
>>
>> Cheers Mark
>>
>>
>> On 13/04/2013, at 12:28 AM, George McNamara<[hidden email]>  wrote:
>>
>>      
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Alison,
>>>
>>> Thanks for clarifying for me about refractive index per specimen (I did mention that even across a specimen the mountant R.I. can change) - and for pointing out the room temperature and critical fluorescent channel are both critical issues as well.
>>>
>>> SoftWoRx - as far as I can see, Applied Precision / General Electric, has failed to "nail the landing" on the acquisition/recontruction/registration. Every step that I have to do manually, they could have automated years ago. probably the only way they will go to automation is when they start to lose sales because someone has a much more efficient workflow.
>>>
>>> I do agree with you that critical evaluation of the data is even more important for structured illumination (and the other nanoscopes) than it has been for confocal, deconvolution, and even plain old widefield.
>>>
>>> Thanks for the tip that I also need to learn to look at - and understand - the log files.
>>>
>>> Sincerely,
>>>
>>> George
>>>
>>>
>>> On 4/12/2013 9:51 AM, Alison North wrote:
>>>        
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Dear George,
>>>>
>>>> You are absolutely correct that the refractive index of the immersion oil is critical for 3D-SIM, which is why I feel I have to respond to your posting and expand it a bit, in case any new or potential OMX users out there get confused.  You say that the 1.512 oil was "the best oil" - I think it needs to be clarified that you mean this was the best oil for that particular sample, looking at the particular wavelength fluorochromes you were imaging, and working at the exact temperature of that room.   In picking the oil for any OMX experiment you need to first check the room temp (if only ours were perfectly stable that wouldn't be an issue, but sadly we do get 1 degree fluctuations from day to day depending on the weather etc., despite having stipulated that our engineers needed to give me the most stable room environment possible!).  Then you need to consider the sample prep - by encouraging people to use the high performance coverslips and to try
>>>>          
>   to stick to ProLong Gold (talking about fixed samples only of course) we aim to minimize the amount of time we need to optimize the oil r.i. each time.  But you will still need to decide which is the most critical channel in your experiment - if you are most interested in the red signal then you will pick a different oil than if the blue or green signals are the most critical, as you won't get a perfectly optimal signal in every channel with just one oil.
>    
>>>> I also think you are being a tad unfair on SoftWorx - I can't say I find it at all painful to use.  Every software has its glitches and annoying features but of the many different softwares we have in our facility we actually find SoftWorx to be one of the most user-friendly - and it hardly ever crashes, which is a big plus in my book.  I don't know which tedious manual steps you are referring to, but the more I have worked with the system, the more I want to slow the users down and force them to look at each log file while the reconstruction is going along.  Checking numbers in the logs like the line spacing, the amplitude, and the ko angles gives a good initial indication of whether you have a decent data set or not.  If our users just ran everything as automatic batch files I fear that the number of artifacts published would shoot up.
>>>>
>>>> Anyway, that's just my two cents and I expect you will disagree vehemently! :-)
>>>> All the best,
>>>> Alison
>>>>
>>>>
>>>>          
>>>>> George
>>>>> p.s. I had training on M.D. Anderson Cancer Center's OMX yesterday. A couple of observations (which may not be new to the listserv):
>>>>> * refractive index of the immersion oil matters a lot. We (Tomasz and Anna Zal were the trainers) had best results with one of Michael Davidson's triple immunofluorescence slides mounted in CytoSeal. Olympus 100x/1.4 NA lens. The best oil was RI=1.512. The RI=1.510 and 1.514 oils were noticably worse in the reconstructions. The RI=1.518 was obviously a wrong choice from the acquisition screen (no or low contrast fringes).
>>>>> * Michael's DAPI in CytoSeal photoconverted to green fluorescence after prolonged excitation with 405 nm laser (several of us have seen, and mentioned on listserv, this with Hoechst ... I suppose it is possible the slide was mislabeled and was actually Hoechst???).
>>>>> * Prolong Gold (on Zeiss 18x18 mm coverglass) ... looks like the R.I. changes from the edge to the middle of the coverglass. This makes sense in that the stuff solidifies by outgasssing a [relatively low refractive index] solvent. Since 0.002 steps in oil matter, it appears that the Prlong Gold R.I. changes by at least 0.002 (maybe a lot more) from edge to middle. This suggests that instead of spending time to change the oil for different areas (distances), that it may be more efficient to select an oil and then search the coverglass for optimum R.I. match (best fring contrast and then final check on the reconstructed image). ... I may decide to use Prolong Gold with "open face" imaging dishes, or buy and test CytoSeal etc.
>>>>> * The Applied Precision reconstruction software is called "SoftWoRx" but a much more accurate name is "painfully tedious manual steps to do everything anti-productivity WoRx".
>>>>> * Plan V: I am scheduled for training on M.D. Anderson's Vutara SR-200 on Monday (Tomasz and Anna doing the training again). Maybe I'll end up optimizing sample preps for this instead of the OMX.
>>>>>
>>>>>
>>>>>            
>>>>          
>> 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]
>>
>>
>>
>>
>>
>>      
> 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]
>
>    

[George McNamara]

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

Hi Adrian,

Thanks for pointing out the Jez et al 2013 HCB paper (I had read the
Piterburg 2012 JM paper, thanks for the reminder of it as well).

I see Jez et al's abstract included,

"Hoechst 33342 exhibited the lowest photoconversion while that for DAPI
and Hoechst 33258 was much stronger. Different fixation methods did not
substantially affect the strength of photoconversion. We also suggest
avoiding the use of mounting medium with high glycerol concentrations
since glycerol showed the strongest impact on photoconversion."

I'll have to read the paper to see if H33342 is worth buying. Until I
read the paper, I won't know whether the authors claim that " glycerol
showed the strongest impact " is correct or (my guess) that "anti-fade"
reagent(s) added to commercial glycerol based mounting media are at fault.

for that matter, it is possible that an additive in the Cytoseal is
responsible for the observation I mentioned - MSDS for Cytoseal 60 is:

Toluene CAS# 108-88-3 -65%
Acrylic Resin CAS# 28262-63-7 -35%
Antioxidant CAS# 128-37-0
Butyl Benzyl Phthalate CAS# 85-68-7

The Antioxidant CAS# 128-37-0 is (according to various google search
results), 2-6-di-tert-butyl_p-cresol
also known as Butylhydroxytoluene;BHT
http://www.caslab.com/2-6-di-tert-butyl_p-cresol_CAS_128-37-0/
I believe BHT is a food additive.


Sincerely,

George


On 4/13/2013 5:25 AM, Adrian Smith wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> On 12/04/2013, at 10:59 PM, George McNamara<[hidden email]>  wrote:
>
>    
>>   Michael's DAPI in CytoSeal photoconverted to green fluorescence after prolonged excitation with 405 nm laser (several of us have seen, and mentioned on listserv, this with Hoechst ... I suppose it is possible the slide was mislabeled and was actually Hoechst???).
>>      
>
> Hi George,
>
> I'm not 100% sure what you mean by "photoconverted to green fluorescence" but we have had issues with DAPI photoconversion after 405nm excitation on a couple of occasions in the last few years.
>
> There have been a couple of publications in that last year that describe exactly what we have seen:-
>
>    
>> J Microsc. 2012 Apr;246(1):89-95. doi: 10.1111/j.1365-2818.2011.03591.x. Epub 2012 Jan 31.
>> Photoconversion of DAPI following UV or violet excitation can cause DAPI to fluoresce with blue or cyan excitation.
>> Piterburg M, Panet H, Weiss A.
>> Abstract
>> 4'-6-Diamidino-2-phenylindole is a fluorescent dye commonly used to visualize deoxyribonucleic acid or cell nuclei in fixed cell preparations, and is often used together with fluorescein or green fluorescent protein, which can be excited without exciting 4'-6-Diamidino-2-phenylindole. It is assumed that when using typical fluorescein or green fluorescent protein filter cubes, 4'-6-Diamidino-2-phenylindole will not be observed. In this paper, we show that following observation of 4'-6-Diamidino-2-phenylindole using UV or violet excitation, it may become sensitive to the blue/cyan excitation used in fluorescein/green fluorescent protein filter cubes. This has serious implications for the use of 4'-6-Diamidino-2-phenylindole together with widely used green fluorophores in double labelling experiments.
>>
>>
>>      
>
>
>
>    
>> Histochem Cell Biol. 2013 Jan;139(1):195-204. doi: 10.1007/s00418-012-1039-8. Epub 2012 Oct 14.
>> The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem.
>> Jež M, Bas T, Veber M, Košir A, Dominko T, Page R, Rožman P.
>> Abstract
>> Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4',6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.
>>      
>
>
> Regards,
>
> Adrian Smith
> Centenary Institute, Sydney, Australia
>
>    

[Vitaly Boyko]

Yes, I have seen all the papers. Most of the biomolecules r
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi George,

Yes, I have seen all the papers. Most of the biomolecules reported are physiologically relatively abundant within the cells, and I would like to see much broader coverage of (molecules) - thus limited, and not broad. The papers you have mentioned are from the top labs, which does not cover probably even 1% of all research labs who are actively engaged in Cell Molecular Biology. 

Have a nice weekend,

Vitaly



________________________________
 From: George McNamara <[hidden email]>
To: [hidden email]
Sent: Saturday, April 13, 2013 9:30 AM
Subject: Re: Responding to the OMX oil comments.....
 

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

Hi Vitaly,

to quote one of my (many) mentors, Larry Phillips (now at
http://www.biovis.com/contact.htm , was referring to MetaMorph Imaging
Systems at the time ):

"we sell [data] shovels. Some customers shovel gold. Some shovel shit."

This is true of all of the equipment in researchers labs, not just
imaging systems (and not just MetaMorph customers).

Here is a web link to a blog from someone who is a fan of the OMX
('OMG') microscope,

http://www.govexec.com/excellence/promising-practices/2013/03/photo-omg-microscope-lives-its-name/62021/

Unfortunately the blog did not mention what day Jane Stout's "mitosis
image will light up a billboard in Times Square in New York City in
April. That is a wonderful celebration of science!"  (and I'm not going
to be visiting NYC anytime soon, so not going to look up the date -
should be on the GE web site).

As for " 2x increase in resolution ... ", I disagree with everythhing
you wrote:
1. improvement is 8x, we are talking three dimensions, 2^3 = 8.
2. understanding is done by the researcher, not the pixels.
3. live - read the SIM papers.
4. PALM/STORM/Vutara "limitations" - read the papers. In particular
check out Xiaowei Zhuang's neuronal synapse papers and Clare Waterman's
focal adhesions paper.

I also want to mention with respect to optical, computation
(deconvolution) and combined (S.I.) resolution improvements, a point
that Paul Goodwin of API/GE made to me: these are not just resolution
improvement(s), but increase in contrast. I suggest that better contrast
can lead a researcher(s) to better understanding.

George



On 4/13/2013 8:04 AM, Vitaly Boyko wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Using ProlongGold for fixed samples in SIM complicates matters, as RI of ProlongGold changes over (maturation) time, plus ProlongGold attenuates GFP and other dyes fluorescence significantly.
> Also, 2x increase in resolution generates pretty pictures but adds little in understanding molecular aspects of protein-protein, protein-RNA, protein-ligand interactions. Also, SIM is very limited to studies of dynamic events (live samples) that is (time domain) crucial to modern Cell Molecular Biology. Unfortunately, PALM/STORM has similar limitations when talking LIVE...     
>
> Good luck,
>
> Vitaly
>
>
> ________________________________
>   From: Mark Cannell<[hidden email]>
> To: [hidden email]
> Sent: Saturday, April 13, 2013 2:58 AM
> Subject: Re: Responding to the OMX oil comments.....
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> On 13/04/2013, at 7:51 AM, Mark Cannell<[hidden email]>  wrote:
>
>   
>> Hi George
>>
>> Alison's comment about the immersion oil RI being needed to be adjusted by colour arises from the Abbe number of the media between the lens and plane of focus. The Abbe number of the immersion oil is rarely discussed, but is key to achieving achromat performance (the Abbe number is the change in RI with wavelength). There is a risk that you may worsen chromatic aberrations by using a different immersion oil because it will probably have the wrong Abbe number.  (Does the correcting immersion oil set also control Abbe number?) .
>>
>> Cheers Mark
>>
>>
>> On 13/04/2013, at 12:28 AM, George McNamara<[hidden email]>  wrote:
>>
>>     
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Alison,
>>>
>>> Thanks for clarifying for me about refractive index per specimen (I did mention that even across a specimen the mountant R.I. can change) - and for pointing out the room temperature and critical fluorescent channel are both critical issues as well.
>>>
>>> SoftWoRx - as far as I can see, Applied Precision / General Electric, has failed to "nail the landing" on the acquisition/recontruction/registration. Every step that I have to do manually, they could have automated years ago. probably the only way they will go to automation is when they start to lose sales because someone has a much more efficient workflow.
>>>
>>> I do agree with you that critical evaluation of the data is even more important for structured illumination (and the other nanoscopes) than it has been for confocal, deconvolution, and even plain old widefield.
>>>
>>> Thanks for the tip that I also need to learn to look at - and understand - the log files.
>>>
>>> Sincerely,
>>>
>>> George
>>>
>>>
>>> On 4/12/2013 9:51 AM, Alison North wrote:
>>>       
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Dear George,
>>>>
>>>> You are absolutely correct that the refractive index of the immersion oil is critical for 3D-SIM, which is why I feel I have to respond to your posting and expand it a bit, in case any new or potential OMX users out there get confused.  You say that the 1.512 oil was "the best oil" - I think it needs to be clarified that you mean this was the best oil for that particular sample, looking at the particular wavelength fluorochromes you were imaging, and working at the exact temperature of that room.   In picking the oil for any OMX experiment you need to first check the room temp (if only ours were perfectly stable that wouldn't be an issue, but sadly we do get 1 degree fluctuations from day to day depending on the weather etc., despite having stipulated that our engineers needed to give me the most stable room environment possible!).  Then you need to consider the sample prep - by encouraging people to use the high performance coverslips and to try
>>>>         
>   to stick to ProLong Gold (talking about fixed samples only of course) we aim to minimize the amount of time we need to optimize the oil r.i. each time.  But you will still need to decide which is the most critical channel in your experiment - if you are most interested in the red signal then you will pick a different oil than if the blue or green signals are the most critical, as you won't get a perfectly optimal signal in every channel with just one oil.
>   
>>>> I also think you are being a tad unfair on SoftWorx - I can't say I find it at all painful to use.  Every software has its glitches and annoying features but of the many different softwares we have in our facility we actually find SoftWorx to be one of the most user-friendly - and it hardly ever crashes, which is a big plus in my book.  I don't know which tedious manual steps you are referring to, but the more I have worked with the system, the more I want to slow the users down and force them to look at each log file while the reconstruction is going along.  Checking numbers in the logs like the line spacing, the amplitude, and the ko angles gives a good initial indication of whether you have a decent data set or not.  If our users just ran everything as automatic batch files I fear that the number of artifacts published would shoot up.
>>>>
>>>> Anyway, that's just my two cents and I expect you will disagree vehemently! :-)
>>>> All the best,
>>>> Alison
>>>>
>>>>
>>>>         
>>>>> George
>>>>> p.s. I had training on M.D. Anderson Cancer Center's OMX yesterday. A couple of observations (which may not be new to the listserv):
>>>>> * refractive index of the immersion oil matters a lot. We (Tomasz and Anna Zal were the trainers) had best results with one of Michael Davidson's triple immunofluorescence slides mounted in CytoSeal. Olympus 100x/1.4 NA lens. The best oil was RI=1.512. The RI=1.510 and 1.514 oils were noticably worse in the reconstructions. The RI=1.518 was obviously a wrong choice from the acquisition screen (no or low contrast fringes).
>>>>> * Michael's DAPI in CytoSeal photoconverted to green fluorescence after prolonged excitation with 405 nm laser (several of us have seen, and mentioned on listserv, this with Hoechst ... I suppose it is possible the slide was mislabeled and was actually Hoechst???).
>>>>> * Prolong Gold (on Zeiss 18x18 mm coverglass) ... looks like the R.I. changes from the edge to the middle of the coverglass. This makes sense in that the stuff solidifies by outgasssing a [relatively low refractive index] solvent. Since 0.002 steps in oil matter, it appears that the Prlong Gold R.I. changes by at least 0.002 (maybe a lot more) from edge to middle. This suggests that instead of spending time to change the oil for different areas (distances), that it may be more efficient to select an oil and then search the coverglass for optimum R.I. match (best fring contrast and then final check on the reconstructed image). ... I may decide to use Prolong Gold with "open face" imaging dishes, or buy and test CytoSeal etc.
>>>>> * The Applied Precision reconstruction software is called "SoftWoRx" but a much more accurate name is "painfully tedious manual steps to do everything anti-productivity WoRx".
>>>>> * Plan V: I am scheduled for training on M.D. Anderson's Vutara SR-200 on Monday (Tomasz and Anna doing the training again). Maybe I'll end up optimizing sample preps for this instead of the OMX.
>>>>>
>>>>>
>>>>>           
>>>>         
>> 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]
>>
>>
>>
>>
>>
>>     
> 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]
>
>