Posted by
Vitaly Boyko on
URL: http://confocal-microscopy-list.275.s1.nabble.com/what-fluorescent-proteins-are-working-best-for-precision-localization-microscopy-nanoscopy-in-your-a-tp7580157p7580188.html
Yes, I have seen all the papers. Most of the biomolecules r
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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.....
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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:
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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:
>>>>
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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.
>>>>>
>>>>>
>>>>>
>>>>
>> 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]
>
>