[3dem] Seeking Advice on BSL-2 CryoEM Laboratory Setup and Safety Protocols

Thu Jun 12 07:20:18 PDT 2025

Dear Jason,

there are to my knowledge (having worked for FEI and TF for quite a long time) only 2 scenarios:

-if the filling fails the nitrogen is not refilled. the microscope will slowly warm up over hours because there is no active heating.
-the sample warms up either in vacuum, which will result in freeze drying or the vacuum also fails which will result in a liquids state at some point.

However, the column is closed and has many protection valves. failing if the pumps will not result in instant loss of vacuum be a degraded state that is still below the triple point. I think you actively need to vent the column to break the vacuum of an autoloader system.

The only way that via an airflow one could get aerosols is if the sample goes through liquid state while the column is aired at exactly the same time? how would that work exactly? To my knowledge this scenario is impossible

making gas with the electron beam also needs to consider the area. the ultra high dose can only be reached by illuminating a very small area. but the still, which particles will come off? are you suggesting we can ionize full bacteria and virus particles and make then “fly” in a vacuum like a mass spec? or evaporation? infectious particles are not atoms and molecules one can evaporate like water. how would a herpes virus evaporate? and evaporate out of a solid block of ice, since we determined the liquid state can not be made.

For clarity, should the vacuum be broken, you cant get an electron beam because that would instantly destroy the tip and create a lightning bolt? without vacuum the safety prevents you to switch on the beam.

Then during imaging, if the sample would be exposed to a small area extreme high dose beam it won’t evaporate but be destroyed. The reason we image in low dose is to preserve the structure. too high dose and we actually see the damage in the total illuminated area destroying any pathogen and turning the structure into oxygen, hydrogen, carbon based gasses, etc, but anything you can push into the vacuum is not an infectious particles.

Mass spec has great difficulties developing methods of gentle ionization to make larger proteins “fly”, let alone a whole virus, bacteria or cell.

And finally should we be able to ionize intact pathogens into the vacuum system. what is the change that you get sick if only one or 10 in the bsl-2 space. in a bsl-2 lab you wear personal protection. service engineers due to tge vacuum already need to wear gloves. the airconditioning refresh rate on bsl-2 is much higher and what is the lower infectious dose.

And finally the pathogens are class two. any toilet visit would pose a higher risk, that service of the inside of the microscope in a bsl-2 lab wearing personal protection dealing with 1 sample of a volume of 1-2 nL.

I see a lot that because safety organisation don’t understand the technology that much stricter standards are imposed vs standard wet labs where much larger quantities are used and the risk are several orders of magnitude higher.

I hope this helps and i’m open for a physics explanation how inside an EM infections particles (so full full intact undamaged viruses and cells) can be made airport at sufficient dose and infect a service engineer splitting the column and wearing personal protection.

cheers

Matthijn

On Jun 12, 2025, at 15:38, Jason NG (CPOS) <jasonng.cpos at hku.hk> wrote:

Dear Matthijn and Kim,
Thank you both for your thoughtful responses and for sharing your valuable experiences.
Matthijn, I appreciate your clarification on the physics of cryo-TEM conditions. I apologize for not doing enough research before raising my initial question. As I understand now, under standard cryo-EM doses (such as 20–60 e⁻/Å² for single particle analysis or 100–120 e⁻/Å² for tomography), the risk is extremely low. However, I am curious about what might happen at much higher doses—on the order of thousands of e⁻/Å². Would such extreme irradiation potentially change the situation? From my current understanding, cryo-TEM operates far below both the triple point pressure and temperature of water, placing the sample firmly in the solid or vapor region of the phase diagram, where aerosol formation is not possible. Thus, the question is whether there might be any potential risks associated with the vapor that is formed under these conditions.
Additionally, I am interested in situations where the sample might inadvertently warm up in the TEM, for example, due to insufficient liquid nitrogen or a vacuum pump failure. Would there be any recommended follow-up procedures in such scenarios?
Kim, thank you for sharing your insights on the practical aspects of equipment disposal. It is certainly an important concern, and we will communicate with our local TFS engineer regarding this. Fortunately, our vacuum pumps have been quite stable over the past half year, and no replacement has been required so far.
Thank you both again for your guidance and support.
Best regards,
Jason
________________________________
From: Gibson, Kimberley <kimberley.gibson at yale.edu>
Sent: Thursday, June 12, 2025 21:04
To: Jason NG (CPOS) <jasonng.cpos at hku.hk>; Matthijn VOS <matthijn.vos at pasteur.fr>
Cc: 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Seeking Advice on BSL-2 CryoEM Laboratory Setup and Safety Protocols

Hi Jason,

Typically, at BSL-2, all equipment can be operated under normal conditions - i.e. in the open on a lab bench (Vitrobot or Leica  GPS) with special considerations for the evacuation of N2 gas or Ethane and Ethane/Propane mixtures.

Issues regarding BSL-2 have only arisen during disposal of equipment. TFS was not allowed to ship an old Ion Getter Pump overseas (from the US back to Eindhoven, NL) if the microscope operated in a BLS-2 lab. The IGP had to be disposed of locally and typically sprayed down with Ethanol prior to disposal. I have also sprayed an old Peltier device removed from a Vitrobot and sent it for electronic waste disposal.

Best,

Kim

________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Matthijn VOS via 3dem <3dem at ncmir.ucsd.edu>
Sent: Thursday, June 12, 2025 06:32
To: Jason NG <jasonng.cpos at hku.hk>
Cc: 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Seeking Advice on BSL-2 CryoEM Laboratory Setup and Safety Protocols

Dear Jason,

could you explain the physics hiw sn electron beam can create aerosols on a solid sample ar -196 degrees in ultra high vacuum (orders of magnitude under the tripple point of water)

I don’t see how this is possible.

cheers

Matthijn

On Jun 12, 2025, at 11:01, Jason NG (CPOS) via 3dem <3dem at ncmir.ucsd.edu> wrote:

Dear CryoEM Community,

We are currently evaluating the risks of becoming BSL-2 CryoEM laboratory and would greatly appreciate your guidance on several safety and setup considerations.

Specifically, we are interested in understanding:

  *   Given that the electron beam can potentially generate aerosols or cause warming within TEMs, what disinfection protocols or measures have you found effective for maintaining biosafety?
  *   Is implementing negative room pressure necessary in a CryoEM lab at BSL-2?
  *   For sample preparation instruments such as the TFS Vitrobot and Leica GP2, is it recommended to place them inside a biosafety cabinet? How about large instruments like the Leica ICE—are there specific safety measures we should consider?

Your insights and experiences would be extremely helpful as we evaluate the best practices for creating a safe and effective CryoEM environment. Thank you very much for your time and guidance.

Best Regards,

Jason NG

Senior Technical Assistant

LKS Cryo-EM Laboratory

<image001.jpg>

Centre for PanorOmic Sciences

LLG03, Laboratory Block

21 Sassoon Road, Pokfulam, Hong Kong

P: +852 3910-3528

E: jasonng.cpos at hku.hk<mailto:jasonng.cpos at hku.hk>

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