[3DEM] A question about ice thickness of cryo-EM

Hu, Guobin guohu at utmb.edu
Thu Feb 17 09:34:44 PST 2005


Hi Paul,

 

Thanks for your information! You might have given me a basic idea what
the maximum of ice thickness could be.  Actually as indicated in my
earliest message, I'm not trying to figure out how thick the ice could
be in any specific case. What I'm trying to find out is an estimate of
the maximum of ice thickness of cryo-EM grids which is good enough for
imaging. I wonder if anybody has ever collected data with ice thicker
than 800 nm. 

Thanks for any further help!

 

Regards,

Guobin

 

 

 

-----Original Message-----
From: owner-3dem at ncmir.ucsd.edu [mailto:owner-3dem at ncmir.ucsd.edu] On
Behalf Of Paul Chipman
Sent: Thursday, February 17, 2005 10:50 AM
To: 3dem at ucsd.edu
Subject: RE: [3DEM] A question about ice thickness of cryo-EM

 

Hi Guobin,

 

The ice thickness is a product of the environmental  conditions
(humidity, temperature), the composition of the sample, the blotting
duration, pressure and paper and also the characteristics of the grid.
I don't think the grid hole size or periodicity alone dictates your ice
thickness.  With the Quantifoil 7/2 we can get vitreous ice that ranges
from ~25-800nm.  And, as Yifan mentioned, we can also get ice too thick
to image through.

 

Cheers,

Paul

 

	Hi Yifan,

	 

	Thanks for your response. You brought a really good suggestion.
I love to try it. However due to our very limited access to the scope
and due to the fact that we don't have a CCD camera, I prefer to get a
direct answer to the question from our 3DEM community. I guess some
people do have experience on this issue.

	By the way, as indicated in my previous message, the purpose for
me to estimate the maximum of ice thickness is for my image analysis,
which implied that the ice thickness is still appropriate for imaging.
On the other hand, my experience suggested that ice thickness varies on
different type of grids, for example, on 7/2 quantifoil holy grids, I
usually get thinner ice than that on 3.5/1 quantifoil holy grids. But I
have never tried to figure out how significant the difference of ice
thickness could be.

	I'm still waiting for more response and an answer. Thanks for
attention and help!

	 

	Regards,

	Guobin

	 

	 

	-----Original Message-----
	From: Yifan Cheng [mailto:Yifan_Cheng at hms.harvard.edu]
	Sent: Tuesday, February 15, 2005 6:39 PM
	To: Hu, Guobin ; 3dem at ucsd.edu
	Subject: Re: [3DEM] A question about ice thickness of cryo-EM

	 

	Hi

	 

	Well, the ice can be very very very thick no matter what grids
you use.

	Did you ever get cryo grids which is too thick to be seen
through?

	 

	What you can do to measure the ice thickness is to burn a small
hole in

	the middle of the ice. Then tilt you holder to a high tilt say
60 degree

	and measure the size of the hole. This can tell you how thick is
your

	ice. Then you can calibrate it with your CCD camera by measure
the

	counts over an empty hole and over ice. Next time just by
comparing the

	CCD counts over ice and empty hole will give you some fairly
good

	estimation about your ice thickness. Good luck

	 

	Yifan

	 

	 

	 

	Hu, Guobin wrote:

	 

	> Hi colleagues,

	>

	> 

	>

	> For our image analysis, we need to estimate the maximum ice
thickness in

	> our cryo-EM specimen on 3.5/1 quantifoil grids. We may be able
to

	> estimate that based on the thickness of carbon foil. As
indicated by the

	> quantifoil product catalogue, the standard thickness of carbon
foil is

	> 12 nm. While observations of our cryo-EM micrographs have
suggested that

	> the thickness of carbon foil is definitely much larger than 12
nm. Does

	> anybody have an idea of what the maximum thickness of ice
could be on

	> quantifoil grids (3.5/1)?

	>

	> Thanks for help!

	>

	> Guobin Hu

	>

	> 

	>

	> 

	>

	 

	--

	 

	************************************************************

	Yifan Cheng, Ph.D.

	Department of Cell Biology, Harvard Medical School

	240 Longwood Avenue, Boston, MA 02115, USA

	Phone: 1-617-432-4095 (office), 432-4097 (wet lab),

	              432-4708 (EM lab), 432-4107 (computer lab);

	Fax:   1-617-432-1144 (department)

	email: Yifan_Cheng at hms.harvard.edu

	************************************************************

	 

	 

	 

	-- 

	Paul Chipman
	Electron Microscopy Facility Manager
	Dept. of Biology, Purdue University
	Lilly Hall, Rm. B216
	Phone: 765-494-1487
	Fax:765-496-1189

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