I wanted to thank everyone for very insightful replies to my question!
To summarize the most important points:
1. Ice in the LN2 reservoir can be a problem.
Several people report that the source of the LN2 matters.
I was impressed by Ilme's advice to use coffee filters (we'll try this!)
2. Care should be taken to minimize ice formation
during freezing. Humidity in the room should be kept low
and the LN2 vessel should be covered all the time.
3. One can try to remove the ice from loops using a
LN2 'jet' (we tried this before with some success) or a fine paintbrush.
Trying to freeze as quickly as possible is certainly a good idea,
but in our limited experience this mostly does not resolve the
problem of ice formation on the surface...
Many thanks again,
Sergei
On 11-Aug-15 3:16 PM, Ilme Schlichting wrote:
> Hello Sergei,
>
> we often filter the liquid nitrogen before we use it. We use
> old-fashioned funnel-shaped paper filters in a plastic funnel, pretty
> much like making coffee. I think this also removes the tiny ice seeds.
>
> Good luck
> ilme
>
> On 8/11/2015 2:21 PM, Sergei Strelkov wrote:
>> Dear All,
>>
>> What I wanted to discuss specifically is the ice crystals that we
>> often find
>> on the outside of the loops rather than bulk ice formation which is due
>> to insufficient cryoprotection (although it is sometimes difficult to
>> distinguish
>> between the two cases). Sometimes these ice crystals on the surface can
>> be removed
>> by pouring LN2 onto the loop, but more often this has little effect.
>> 'Annealing' (thawing and freezing again) always resolves the problem
>> but this is of course not an option most of the time (as the protein
>> crystal gets damaged).
>>
>> We measure at synchrotrons on a regular basis and the ice formation
>> problem
>> seems to vary quite a bit from trip to trip. Thus far we could not quite
>> figure
>> out what causes it.
>>
>> We usually mount our crystals (with cryoprotectant...) by plunging them
>> into LN2 on a bench. Thereafter they are stored either on canes or
>> unipacks
>> in a dry shipper. As we travel by car or train, we always keep the dry
>> shipper filled
>> with LN2 anyway. At the beamline they are usually mounted by a robot.
>>
>> We mostly use the fiber loops (Hampton) but sometimes also the 'plastic'
>> loops
>> from Mitegen. I could not see much difference between them in terms of
>> ice formation,
>> but I wondered what the others' experiences are.
>>
>> The choice of cryoprotectant matters obviously but annoyingly we can
>> sometimes
>> happily use some cryoprotecting conditions initially, but suddenly have
>> problems
>> with exactly the same crystals and cryoprotectant on the next trip...
>>
>> The ice accumulation in any one of the LN2 vessels used at different
>> stages
>> (LN2 tank, the open reservoir we use for plunge-freezing, or the crystal
>> storage/shipping dewar)
>> /might/ be the cause. Specifically, there will /always/ be /some/ ice
>> accumulation in the open reservoir.
>> Thus far we were not too paranoic about refreshing LN2 every five
>> minutes -- but maybe we should?...
>>
>> Or could the (variable) presence of ice in the 100l LN2 tank that our
>> internal services fill for us
>> be a problem?...
>>
>> Thanks and best wishes,
>> Sergei
>>
>> --
>> Prof. Sergei V. Strelkov
>> Laboratory for Biocrystallography
>> Dept of Pharmaceutical and Pharmacological Sciences, KU Leuven
>> Herestraat 49 bus 822, 3000 Leuven, Belgium
>> Work phone: +32 16 330845 Mobile: +32 486 294132
>> Lab pages:http://pharm.kuleuven.be/Biocrystallography
>>
>
--
Prof. Sergei V. Strelkov
Laboratory for Biocrystallography
Dept of Pharmaceutical and Pharmacological Sciences, KU Leuven
Herestraat 49 bus 822, 3000 Leuven, Belgium
Work phone: +32 16 330845 Mobile: +32 486 294132
Lab pages: http://pharm.kuleuven.be/Biocrystallography
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