[3dem] pressure failure with Leica EM Pact2?

Wed Oct 7 10:40:05 PDT 2015

Hi Gongpu,

I had issues with pressure failures previously and found that anything that's left on the 'shoulders' of the carriers (that contacts the black diamond), such as yeast paste or gel, can prevent a tight seal from forming - so the carrier and diamond pop apart during the freeze (hence the sudden drop in pressure on the pressure curve, and your curve looks similar to what I had). Not an issue in Bal-tec type freezers like the HPM100 since the whole chamber, not just the carrier interior, is pressurised.

Another possibility is an aging high pressure tube, which might not be contacting the cassette properly (if I remember rightly); in that case a session can start OK but might produce failures later on, as everything gets colder and the tube shortens.

Cheers,

Matt

Matt Russell, PhD
Electron Microscopy STP
The Francis Crick Institute
Lincoln’s Inn Fields Laboratory
44 Lincoln's Inn Fields
London
WC2A 3LY

T: 020 7269 3346
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On 7 Oct 2015, at 17:09, <3dem-request at ncmir.ucsd.edu<mailto:request at ncmir.ucsd.edu>>
 wrote:

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Today's Topics:

  1. Re: pressure failure with Leica EM Pact2? (Paul Verkade)
  2. Re: pressure failure with Leica EM Pact2?
     (benoit.zuber at ana.unibe.ch)

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Message: 1
Date: Wed, 7 Oct 2015 17:00:36 +0100
From: Paul Verkade <bixpv at bristol.ac.uk>
To: "Zhao, Gongpu" <goz1 at pitt.edu>
Cc: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] pressure failure with Leica EM Pact2?
Message-ID: <D7108C4B-8F6F-4FD4-A6B7-C7AEEA4BF112 at bristol.ac.uk>
Content-Type: text/plain; charset="us-ascii"

Hi Gongpu,

Indeed pressure should go up to around 2050 bar.

A few things to check (which you probably already did).
If you turn the blue knob on the side you can adjust the pressure, try adjusting that.
Is the incoming pressure from the compressor OK (about 10bar)?

Let me know if it works (or not), otherwise I can think of a few other things to try / check.

Good luck,
Paul
__________________________________
Paul Verkade PhD FRMS
Reader in Cell Imaging
Wolfson Bioimaging Facility
Schools of Biochemistry and Physiology & Pharmacology
Biomedical Sciences Building
University of Bristol
University Walk
BS8 1TD Bristol
United Kingdom

tel.+44-117-3312179
Fax. +44-117-3312168
mail: p.verkade at bristol.ac.uk <mailto:p.verkade at bristol.ac.uk>
websites:
http://www.bristol.ac.uk/biochemistry/research/pv.html <http://www.bristol.ac.uk/biochemistry/research/pv.html>
http://www.bris.ac.uk/phys-pharm/research/staffresearch/paulverkade.html <http://www.bris.ac.uk/phys-pharm/research/staffresearch/paulverkade.html>
http://www.bristol.ac.uk/biochemistry/wbif <http://www.bristol.ac.uk/biochemistry/wbif>

On 6 Oct 2015, at 20:42, Zhao, Gongpu <goz1 at pitt.edu> wrote:

Dear All,

We are experiencing a pressure failure problem during high pressure freezing using Leica EM Pact . For successful freezing, the curve is smooth and pressure should go up to 2000 bar. But right now, the curve is fuzzy, at some points it drops and the small display shows the pressure is failure. I have attached an image of the bad curve.  Any insights about what might cause the problem and how to fix it would be greatly appreciated? Thank you very much.

gongpu

GONGPU ZHAO, PH.D.
Research Associate
Department of Structural Biology
University of Pittsburgh School of Medicine
3501 5th Ave. RM2054, Pittsburgh, PA 15260

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Message: 2
Date: Wed, 7 Oct 2015 16:09:06 +0000
From: <benoit.zuber at ana.unibe.ch>
To: <bixpv at bristol.ac.uk>, <goz1 at pitt.edu>
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] pressure failure with Leica EM Pact2?
Message-ID: <D23B0EA8.17799%benoit.zuber at ana.unibe.ch>
Content-Type: text/plain; charset="iso-8859-1"

Hi Gongpu,

One more test suggestion: does this behaviour happen with more than one type of specimen carrier (e.g. Membrane carriers vs copper tubes)? I have experienced with two EMPACT2 machines that after a few years of usage the tubes burst if you shoot at 2000 bars and you get a complete pressure failure, but this is not the case here. The solution to this tube problem is simply to reduce a bit the pressure, to around 1900 bars. Since we do CEMOVIS, we can check by electron diffraction that the sample is indeed vitrified so we are not worried to work slightly below the optimal pressure value.

Best wishes
Ben

De : 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> on behalf of Paul Verkade <bixpv at bristol.ac.uk<mailto:bixpv at bristol.ac.uk>>
Date : mercredi, 7 octobre 2015 18:00
? : "Zhao, Gongpu" <goz1 at pitt.edu<mailto:goz1 at pitt.edu>>
Cc : "3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>" <3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>>
Objet : Re: [3dem] pressure failure with Leica EM Pact2?

Hi Gongpu,

Indeed pressure should go up to around 2050 bar.

A few things to check (which you probably already did).
If you turn the blue knob on the side you can adjust the pressure, try adjusting that.
Is the incoming pressure from the compressor OK (about 10bar)?

Let me know if it works (or not), otherwise I can think of a few other things to try / check.

Good luck,
Paul
__________________________________
Paul Verkade PhD FRMS
Reader in Cell Imaging
Wolfson Bioimaging Facility
Schools of Biochemistry and Physiology & Pharmacology
Biomedical Sciences Building
University of Bristol
University Walk
BS8 1TD Bristol
United Kingdom

tel.+44-117-3312179
Fax. +44-117-3312168
mail: p.verkade at bristol.ac.uk<mailto:p.verkade at bristol.ac.uk>
websites:
http://www.bristol.ac.uk/biochemistry/research/pv.html
http://www.bris.ac.uk/phys-pharm/research/staffresearch/paulverkade.html
http://www.bristol.ac.uk/biochemistry/wbif

On 6 Oct 2015, at 20:42, Zhao, Gongpu <goz1 at pitt.edu<mailto:goz1 at pitt.edu>> wrote:

Dear All,

We are experiencing a pressure failure problem during high pressure freezing using Leica EM Pact . For successful freezing, the curve is smooth and pressure should go up to 2000 bar. But right now, the curve is fuzzy, at some points it drops and the small display shows the pressure is failure. I have attached an image of the bad curve.  Any insights about what might cause the problem and how to fix it would be greatly appreciated? Thank you very much.

gongpu

GONGPU ZHAO, PH.D.
Research Associate
Department of Structural Biology
University of Pittsburgh School of Medicine
3501 5th Ave. RM2054, Pittsburgh, PA 15260

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