[3dem] how to do cryo-EM sample preparation with the sample heated to 60 degree

[Shaoxia]

Hi Yan Zhang,

Have you tried it at room temperature firstly? If it works at room 
temperature, the problem may just be the evaporation rate which is much 
high at 60 degree.

Shaoxia Chen
MRC-LMB

On 2012-5-15 17:16, David Belnap wrote:
> Yanzhang,
>
> Did you use 100% relative humidity, or get as close as you can to 100%?  That may be important for avoiding rapid evaporation at higher temperatures.
>
> David
>
>
> On May 14, 2012, at 6:49 PM, yanzhang wrote:
>
>> Dear all,
>>             I encountered a problem when I was doing the cryo-EM sample preparation: During the sample preparation process using FEI vitroboot system, I also needed to  heat the sample to 60 degree inside the cavity of vitroboot, because I wanted to see if the structure of my protein would be changed after heated to 60 degree. But when I observed the heated cryo-em sample using electron microscopy, I found a strange phenomenon: 1. there was no ice in most of the holes; 2. Only very very few holes located at the boundary of some squares of the grid have ice; 3. For the very few holes with ice, there were very little samples located at the inside boundary of the holes.
>>
>>             I tried many different conditions of blotting time and blotting force, but no chang for the sample.
>>
>>             I'm not sure why I can't get a good heated cryo-em sample. Then I tried to heated the sample in the water-bath first, then add the sample to the tweezers in vitroboot (vitroboot also heated to 60 degree), but nothing changed, I still can't get a good sample to collect data.
>>
>>            p.s. I used the quantiful film for cryo-em sample preparation. I once tried the carbon film for this heated cryo-em sample preparation, but the results even worse: the films were totally broken.
>>
>>            Even I repeat the experiment many times, but I got nothing. So please anyone who have the experiences of doing heated cryo-em sample preparation, please give me suggestions!
>>
>>           Thank you!
>> yanzhang
>>
>> yanzhang at moon.ibp.ac.cn
>>
>> Institute of Biophysics,
>> Chinese Academy of Sciences,
>>
>> 15 Datun Road,
>> Chaoyang District,
>> Beijing, China
>>
>> Tel(o): 64888137
>>
>>
>>
>>
>>
>>
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> ============================================
>   David M. Belnap
>   Department of Chemistry and Biochemistry
>   Brigham Young University
>   Provo, Utah 84602  USA
>
>   Phone:  801-422-9163
>   FAX:      801-422-0153
>   David.Belnap at byu.edu
> ============================================
>
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