[3dem] "leopard skin" ice

R.I.Koning at lumc.nl R.I.Koning at lumc.nl
Mon Nov 25 07:21:59 PST 2013


Hi all,

Nice to see that more labs seem to have similar problems as we sometimes encounter. My experience is also that it comes and goes  and does not have much to do with the vitrification itself or the grids. More likely to me is also mild exposure to "warm" air or surfaces during transfer. Having the goniometer opened and closed during inspection also sometimes affected the behaviour. My latest trick is pre-pumping the airlock on our T20 more than once before transfer of the holder... 

Best regards,

Roman

PS I have no preference for a name (though we use leopard skin) and here's my horrible ice picture for you to admire...

Dr. R.I.Koning

Leiden University Medical Center, Department of Cell Biology, Section Electron Microscopy
Postal Zone S1-P, P.O.Box 9600, 2300 RC, Leiden, The Netherlands

visiting address. Einthovenweg 20, Building 2, zone S1-P, room R-90-20
tel. (31) 71 526 9296 fax. (+31) 71 526 8270 mail. r.i.koning at lumc.nl web. electronmicroscopy.lumc.nl


-----Original Message-----
From: 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of Pasi Laurinmäki
Sent: maandag 25 november 2013 7:55
To: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] "leopard skin" ice

Hi all,

I believe the reason is minor exposure to warm & moist air, typically during the transfer of the cryo holder.
We see similar thing every now and then (on rare occasions), but in the past we were occasionally pestered by the "cobblestones" (term coined by someone in Boulder, I believe).
Our problem was linked to design of the original version of 914 cryotomo holder that left a tiny physical slit in the tip open even when the hatch was closed. After we got a small upgrade that blocked the slit the problem was solved.

A lively thread!
-Pasi


Quoting Dianne Taylor <dianne at bio.fsu.edu>:

> Here in Florida it has long been and will always be alligator, gator 
> for short, ice.
>
>  As the many good comments suggest, there may well be more than one 
> source of the problem. I would echo the observation that transfer of 
> the cryoholder is likely the most common one. We see it frequently on 
> the CM120 but not usually on the Titan. We see it with both manual and 
> Vitrobot freezing irrespective of ethane tank age or fullness. Some 
> samples seem to be more susceptable than others, our lipid monolayer 
> samples are very prone to the "dried mud" appearance.
>
> Dianne Taylor
> At 08:53 AM 11/22/2013, Bob Grassucci wrote:
>
>> I am getting a chuckle out of the image of a Leopard shaving ;)   
>> That being said that is why turtle ice may be more accurate.
>> Bob
>> On 11/21/2013 10:45 PM, Joachim Frank wrote:
>>> Hi all,
>>> just a note on the terminology:
>>> You mean leopard fur, maybe?
>>> Because the animal, shaved down to the skin, looks quite different 
>>> for sure.
>>>
>>> --Joachim.
>>>
>>> Quoting Stefan Bohn <Stefan.Bohn at ucsf.edu>:
>>>
>>>> Hi,
>>>>
>>>> sometimes this ice was appearing, when the ethane bottle was nearly empty.
>>>> Sometimes, ethane bottle was full and it still appeared (open 
>>>> plunger, F20,
>>>> identical(!) sample - 20mM Hepes, 20mM NaCl, 10mM MgCl2, ~15% 
>>>> glycerol) - in those instances I attributed it to not having filled 
>>>> the nitrogen high enough in the dewar, maybe not cooling the ethane 
>>>> long enough. Sometimes only part of the grid showed this ice, other 
>>>> parts were normal - particles were distributed evenly on those grids.
>>>>
>>>> But, in general, it was not reproducible. I couldn't correlate it 
>>>> to the phase of the moon, as was suggested to me several times...
>>>>
>>>>
>>>>
>>>> Most importantly, we referred to it as "turtle-ice" (from 
>>>> Schildkroete = plate toad).
>>>>
>>>> Hope that helps,
>>>>
>>>> Stefan.
>>>>
>>>>
>>>>
>>>>
>>>> On Thu, Nov 21, 2013 at 1:26 PM, Jacob Brink <jbrink at jeol.com> wrote:
>>>>
>>>>> Hi Paul and Frank,
>>>>>
>>>>> We've also seen this type of ice in Flu particle preps at NIBSC 
>>>>> during the cryo-EM course, but we'd see it in the middle of the 
>>>>> ice, away from particles (see below). The ethane tank had not been 
>>>>> moved. So, we never really could attribute it to anything unless 
>>>>> the ice would get really thin?
>>>>> Open air plunger (!) and imaged in a 2100-LaB6 TT.
>>>>>
>>>>> Jaap
>>>>>
>>>>>
>>>>>
>>>>> On Nov 21, 2013, at 2:36 PM, Paul Chipman <pchipman at ufl.edu> wrote:
>>>>>
>>>>> Hi Frank,
>>>>>
>>>>> At Purdue we called this "alligator ice" and would see it from 
>>>>> time to time, occasionally when an ethane tank was nearing empty.  
>>>>> As such we attributed to impurities that impeded freezing.  It 
>>>>> could also be just something in the buffer or sample that alters 
>>>>> the freezing. The recent image below displays this type of ice but only near the viral particles.
>>>>> Most of the ice was great except in areas of high particle concentration.
>>>>> When I told the student the name we gave this type of ice, I got a laugh.
>>>>>
>>>>> Go Gators! (Florida)
>>>>>
>>>>> Cheers,
>>>>> Paul
>>>>>
>>>>>
>>>>> On Wed, Nov 20, 2013 at 1:54 AM, <frankpolzer at physik.hu-berlin.de> wrote:
>>>>>
>>>>>> Dear all,
>>>>>>
>>>>>> During the last weeks, I frequently observe what I believe is 
>>>>>> so-called leopard skin ice in my vitrified samples (plunge frozen 
>>>>>> in ethane using Mark IV Vitrobot).
>>>>>>
>>>>>> What again is the reason for this type of ice and how can this be 
>>>>>> avoided?
>>>>>>
>>>>>> Thanks for your help in advance,
>>>>>>
>>>>>> Frank
>>>>>>
>>>>>>
>>>>>> Dr. Frank Polzer
>>>>>>
>>>>>> TEM Group
>>>>>> Insitute of Physics
>>>>>> Humboldt Universität zu Berlin
>>>>>> Newtonstraße 15
>>>>>> 12489 - Berlin
>>>>>> Tel.: +49 30 2093-4995 (office)
>>>>>> Tel.: +49 30 2093-7829 (TEM)
>>>>>> Fax: +49 30 2093-7886
>>>>>>
>>>>>>
>>>>>> _______________________________________________
>>>>>> 3dem mailing list
>>>>>> 3dem at ncmir.ucsd.edu
>>>>>> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem<http://cp.mcafe
>>>>>> e.com/d/2DRPoArhpuvKUU-ejdTdFET73AhOtXCQQrzxO8VcsedFEIEIffzDS3qqd
>>>>>> TAnQTQSjoygza1k52fRfVsS8_k_BPoVyUXTAT-LObbVEVKNRXBQn76ejouppjVqWt
>>>>>> AklrTjVkffGhBrwqrhdECXYyMCY-ehojd79KVIDeqR4IMjW5BphVKR4eZ1L1dnoov
>>>>>> aAVgtHza7GCSkflbAaJMJZ0l2OIEYTqy7uwSruud79EVsjdwLQzh0qmSkfldbFEwd
>>>>>> 4h_RmSa1EwElB0Qh6y2lLdTdCt0VWq79>
>>>>>
>>>>>
>>>>>
>>>>> --
>>>>> Paul Chipman
>>>>> Assistant Director of Research
>>>>> Biochemistry and Molecular Biology University of Florida
>>>>> 352-294-1790
>>>>>
>>>>> <alligator_ice.tif>_______________________________________________
>>>>> 3dem mailing list
>>>>> 3dem at ncmir.ucsd.edu
>>>>>
>>>>> http://cp.mcafee.com/d/5fHCNAedEILfTssv79CXCQQrzxO8VeZPqqdNMV4sCe7
>>>>> 6QQmkm7DNPX1Jd6XObWrWr9Ih8hB0G2x7WDYKr4vGvOVIsNstXOr_nV5BYQsToWZOW
>>>>> bzz79IfcIFYJteOaaJXFYG7DR8OJMddECQjt-hojuv78I9CzATsSjDdqymo9Z2OIEY
>>>>> Tqy7uwTwCHIcfBisEeRNB3Rjra7GBO5mUm-waxpmkurJh3LgrdLf6zAQsK9CMnWhEw
>>>>> dbra7GCBQQg6y8_WHr50QgkaOwq8zh1aTCXCZuhzHyMKD70s
>>>>> _______________________________________________
>>>>> 3dem mailing list
>>>>> 3dem at ncmir.ucsd.edu
>>>>> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>>>>>
>>>>
>>>>
>>>> --
>>>> Dr. Stefan Bohn
>>>> Department of Cellular and Molecular Pharmacology University of 
>>>> California
>>>> 1700 4th Street, Box 2530
>>>> San Francisco, CA. 94158
>>>>
>>>> Phone: +1 (415) 476 - 2980
>>>> Fax: +1 (415) 514 - 9736
>>>
>>>
>>
>>
>> _______________________________________________
>> 3dem mailing list
>> 3dem at ncmir.ucsd.edu
>> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>
> Dianne W. Taylor
> Associate in Research
> Institute of Molecular Biophysics
> Florida State University
> Tallahassee, FL 32306-4380
> Phone: (850) 644-4104
> E-mail:  dianne at bio.fsu.edu 
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem


<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>
Pasi Laurinmäki  Laboratory Engineer

Institute of Biotechnology
Electron Microscopy Unit
P.O.Box 56
FIN-00014 University of Helsinki
Finland

tel. +358 9 191 59502
fax. +358 9 191 59498
E-mail. pasi.laurinmaki at helsinki.fi
<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>

_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
-------------- next part --------------
A non-text attachment was scrubbed...
Name: horrible_ice.jpg
Type: image/jpeg
Size: 387948 bytes
Desc: horrible_ice.jpg
Url : https://mail.ncmir.ucsd.edu/mailman/private/3dem/attachments/20131125/6d843861/horrible_ice-0001.jpg


More information about the 3dem mailing list