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

Thu Jun 12 12:37:16 PDT 2025

To supplement the nice answers from Matthijn, Kimberly, Jaap.

Our wet lab, grid prep room, and microscopes are all in BSL2. In my
opinion, the primary concern for BSL2 is grid recovery, as culturing
something from a thawed grid might actually be possible. We simply recover
BSL2 grids from LN2 into a tube of bleach, and eventually the tube is
discarded via the regular BSL2 biohazard waste stream. One can compare to
how pipette tips or cryopreservation tubes are handled in tissue culture.
We also wipe down inside the GP2 with 70% ethanol, as well as the
freezing/clipping tools, and use a slide warmer at a fairly high
temperature just inside the range for heat inactivation for good measure
(>45˚C). On the Vitrobot, an Aclar "anti-contamination" ring can be placed
behind the filter paper to prevent media soaking into the foam pads (also
makes papers behave better in the GP2, can be made by hand or purchased
from Subangstrom, they seem to be infinitely reusable). The filter papers
and so forth go in regular biohazard waste. Typically used grid boxes
already get soaked in ethanol or IPA to clean the labels (aside: colored
ink is easier to remove).

Best,
-da

On Thu, Jun 12, 2025 at 6:47 AM Jason NG (CPOS) via 3dem <
3dem at ncmir.ucsd.edu> 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
>
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