Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/what-fluorescent-proteins-are-working-best-for-precision-localization-microscopy-nanoscopy-in-your-a-tp7580157p7580185.html
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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:
>
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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]
>
>