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<body lang="EN-HK" link="#467886" vlink="#96607D" style="word-wrap:break-word">
<div class="WordSection1">
<p class="MsoNormal"><span style="font-size:11.0pt">Dear all,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">As there is interest in this topic within the community, I would like to share a summary of the replies I have received and in 3dem. Below are some highlights:<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt">Important!! There is no international standard. All the requirements will depend on your country and university.<o:p></o:p></span></b></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">#1 Oxford Particle Imaging Centre, University of Oxford, UK (Thanks to Helen and especially OPIC for their contributions.)<o:p></o:p></span></p>
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<p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;color:black">Comments</span></b><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
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<tr style="height:26.3pt">
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<p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;color:black">Risk Assessment
</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l20 level1 lfo4"><span lang="EN-GB" style="font-size:11.0pt;color:black">Perform case-by-case risk assessments for each organism and share with the local health and safety team. Each institution tends to have a slightly
different risk assessment format.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l20 level1 lfo4"><span lang="EN-GB" style="font-size:11.0pt;color:black">For some cases, you will need to get HSE approval in the UK e.g. GMO Class 2 samples. This is best determined by your local health and safety team,
since your organisation might already have permission, for instance, to work with GMO Class 1 agents.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l20 level1 lfo4"><span lang="EN-GB" style="font-size:11.0pt;color:black">NOTE! Bacteria and prions are tricky and often not possible because they are difficult to inactivate without damaging the electron microscope. </span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l20 level1 lfo4"><span lang="EN-GB" style="font-size:11.0pt;color:black">NOTE! At CL3 it may be possible to avoid the requirement for a validated method for room fumigations for EM work.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l20 level1 lfo4"><span lang="EN-GB" style="font-size:11.0pt;color:black">It is also important to consider the use of sharps (tweezers) in your risk assessment.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
</td>
</tr>
<tr style="height:26.3pt">
<td width="145" valign="top" style="width:109.05pt;border:solid white 1.0pt;border-top:none;background:#E7EAED;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.3pt">
<p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;color:black">Validation </span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="834" valign="top" style="width:625.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#E7EAED;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.3pt">
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l11 level1 lfo5"><span lang="EN-GB" style="font-size:11.0pt;color:black">In house validation tests are ONLY necessary for CL3 work. For this, you would need to perform validation tests to demonstrate that the infectious
agent is inactivated after X time at room temperature. Additional validations are needed to demonstrate that the infectious agent is inactivated after X time with disinfectant (important to find the minimal time required to do this).</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l11 level1 lfo5"><span lang="EN-GB" style="font-size:11.0pt;color:black">For CL2 samples, it is sufficient to use evidence from disinfectant companies or peer reviewed articles
</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
<p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;color:black">(it is good to find the minimal time required for this). </span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
</tr>
<tr style="height:26.3pt">
<td width="145" valign="top" style="width:109.05pt;border:solid white 1.0pt;border-top:none;background:#CCD2D8;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.3pt">
<p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;color:black">Disinfection & Decontamination</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="834" valign="top" style="width:625.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.3pt">
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l7 level1 lfo6"><span lang="EN-GB" style="font-size:11.0pt;color:black">Use 70% ethanol (make sure that this is in excess as evaporates quickly) or 10% Chemgene with thorough rinsing after validated contact time (avoid
Virkon as this corrodes equipment).</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l7 level1 lfo6"><span lang="EN-GB" style="font-size:11.0pt;color:black">Validated contact time (e.g., 10 minutes) is essential.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l7 level1 lfo6"><span lang="EN-GB" style="font-size:11.0pt;color:black">Fumigation can be used for entire microbiological safety cabinets containing equipment e.g. loading station, nanocab if available (likely overkill
CL2 material) but avoid hydrogen peroxide for nanocab and cassette.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
</td>
</tr>
<tr style="height:26.3pt">
<td width="145" valign="top" style="width:109.05pt;border:solid white 1.0pt;border-top:none;background:#E7EAED;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.3pt">
<p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;color:black">Laboratory Design & Safety</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="834" valign="top" style="width:625.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#E7EAED;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.3pt">
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l14 level1 lfo7"><span lang="EN-GB" style="font-size:11.0pt;color:black">Number of air changes is important. Negative pressure is in place for CL3 laboratories.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l14 level1 lfo7"><span lang="EN-GB" style="font-size:11.0pt;color:black">Consult local health and safety regulations for specific requirements.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l14 level1 lfo7"><span lang="EN-GB" style="font-size:11.0pt;color:black">Keep laboratory as clear and clean as possible.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
</td>
</tr>
<tr style="height:26.3pt">
<td width="145" valign="top" style="width:109.05pt;border:solid white 1.0pt;border-top:none;background:#CCD2D8;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.3pt">
<p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;color:black">Others</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="834" valign="top" style="width:625.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.3pt">
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l18 level1 lfo8"><span lang="EN-GB" style="font-size:11.0pt;color:black">Prior to work, it is important to notify the suppliers of instruments that we are performing work and that we have risk assessed/performed the validations
such that the engineers are safe.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l18 level1 lfo8"><span lang="EN-GB" style="font-size:11.0pt;color:black">Keep a log of work performed with instruments at CL2.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l18 level1 lfo8"><span lang="EN-GB" style="font-size:11.0pt;color:black">Ensure that adequate PPE is available (for CL2, users should wear gloves, safety glasses and a laboratory coat).</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l18 level1 lfo8"><span lang="EN-GB" style="font-size:11.0pt;color:black">Document stored samples.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
</td>
</tr>
</tbody>
</table>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt"><o:p> </o:p></span></p>
<table class="MsoNormalTable" border="0" cellspacing="0" cellpadding="0" width="982" style="width:736.3pt;border-collapse:collapse">
<tbody>
<tr style="height:26.65pt">
<td width="104" style="width:78.25pt;border:solid white 1.0pt;border-bottom:solid white 3.0pt;background:#156082;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">OPIC Equipment</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="56" style="width:41.75pt;border-top:solid white 1.0pt;border-left:none;border-bottom:solid white 3.0pt;border-right:solid white 1.0pt;background:#156082;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Level</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="448" style="width:336.15pt;border-top:solid white 1.0pt;border-left:none;border-bottom:solid white 3.0pt;border-right:solid white 1.0pt;background:#156082;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Setup</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="374" style="width:280.15pt;border-top:solid white 1.0pt;border-left:none;border-bottom:solid white 3.0pt;border-right:solid white 1.0pt;background:#156082;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Decontamination</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
</tr>
<tr style="height:32.0pt">
<td width="104" style="width:78.25pt;border:solid white 1.0pt;border-top:none;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:32.0pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">GP2</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="56" style="width:41.75pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:32.0pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">CL2/CL3</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="448" style="width:336.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:32.0pt">
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l10 level1 lfo9"><span style="font-size:11.0pt;color:black">Within a Class 1,2 or 3 microbiological safety cabinet (this might require customisation) if pathogen of interest is transmitted by aerosol.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l10 level1 lfo9"><span style="font-size:11.0pt;color:black">For CL2 agents that are not aerosols, write a risk assessment to perform preparation outside of a cabinet.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
</td>
<td width="374" style="width:280.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:32.0pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">GP2 can be baked out after use. All surfaces and tools must be wiped with the validated disinfectant (e.g 10 % chemgene) and left for the validated contact time after use.</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
</tr>
<tr style="height:26.65pt">
<td width="104" style="width:78.25pt;border:solid white 1.0pt;border-top:none;background:#E7EAED;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Glacios</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="56" style="width:41.75pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#E7EAED;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">CL2</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="448" style="width:336.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#E7EAED;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l12 level1 lfo10"><span style="font-size:11.0pt;color:black">If transmitted by aerosol, samples are loaded into the nanocab within a microbiological safety cabinet. If not aerosol transmission, samples can be loaded in
a CL2 laboratory. As before, users must wear appropriate PPE, which includes gloves.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
</td>
<td width="374" style="width:280.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#E7EAED;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">After loading, nanocab must either be fumigated or heated (if validation tests show that can inactivate after x time at 37 degreesC for instance). All surfaces and tools must be wiped with the
validated disinfectant (e.g 10 % chemgene) and left for the validated contact time after use.</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
</tr>
<tr style="height:26.65pt">
<td width="104" style="width:78.25pt;border:solid white 1.0pt;border-top:none;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Krios and Arctis</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="56" style="width:41.75pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">CL2/CL3</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="448" style="width:336.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l2 level1 lfo11"><span style="font-size:11.0pt;color:black">Ensure that grids are counted in/out pre and post loading. For CL3 work it is necessary to transport the nanocab in a special transport box such that the target
is doubly contained without creating a bomb.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
</td>
<td width="374" style="width:280.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Validation tests for target indicate time that it takes to deactivate in a dry environment at room temperature. After experiments have completed, the system can be cryocycled and left at room temperature
for the indicated time. In emergency cases, and/or where inactivation cannot be achieved after a sensible time at room temperature, microscopes have heating elements within the housing such that there is an option to heat cycle the entire instrument to 60
¡ÆC.</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
</tr>
<tr style="height:26.65pt">
<td width="104" style="width:78.25pt;border:solid white 1.0pt;border-top:none;background:#E7EAED;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Ancillary (eg clipping/loading station)</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="56" style="width:41.75pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#E7EAED;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">CL2/CL3</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="448" style="width:336.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#E7EAED;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l6 level1 lfo12"><span style="font-size:11.0pt;color:black">As with everything, risk assess on a per target basis and depending on the target, it is possible to do outside of the cabinet e.g. no aerosols. Use of sharp tweezers
should be risk assessed.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
</td>
<td width="374" style="width:280.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#E7EAED;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Both can be placed in an oven.</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
</tr>
<tr style="height:26.65pt">
<td width="104" style="width:78.25pt;border:solid white 1.0pt;border-top:none;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Laboratory preparation</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="56" style="width:41.75pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">CL2/CL3</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="448" style="width:336.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l15 level1 lfo13"><span style="font-size:11.0pt;color:black">When CL3 experiments are desired, the suite access is changed to only CL3 trained users and PPE appropriate for CL3 is adopted. All laboratories in which CL3
work is to take place are cleared and deep cleaned at the end of work to comply with CL3 laboratory practices. A cold start where everything is treated as CL3 is scheduled before the actual experiments take place. The laboratory building is maintained to
comply with CL3 legislation e.g. operates under negative pressure.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
</td>
<td width="374" style="width:280.15pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:.75pt 5.4pt 0cm 5.4pt;height:26.65pt">
</td>
</tr>
</tbody>
</table>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">#2 A facility in the USA<o:p></o:p></span></p>
<table class="MsoNormalTable" border="0" cellspacing="0" cellpadding="0" width="991" style="width:743.2pt;border-collapse:collapse">
<tbody>
<tr style="height:26.05pt">
<td width="147" valign="top" style="width:110.4pt;border:solid white 1.0pt;border-bottom:solid white 3.0pt;background:#156082;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.05pt">
</td>
<td width="844" valign="top" style="width:632.8pt;border-top:solid white 1.0pt;border-left:none;border-bottom:solid white 3.0pt;border-right:solid white 1.0pt;background:#156082;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.05pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Comments</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
</tr>
<tr style="height:26.05pt">
<td width="147" valign="top" style="width:110.4pt;border:solid white 1.0pt;border-top:none;background:#CCD2D8;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.05pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Risk Assessment & Validation</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="844" valign="top" style="width:632.8pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#CCD2D8;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.05pt">
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l23 level1 lfo14"><span style="font-size:11.0pt;color:black">The risk studying human derived cell lines is generally low and setting up freezing equipment in a BSL2 space may be fine. Pathogens however are a different story.
</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l23 level1 lfo14"><span style="font-size:11.0pt;color:black">When working with pathogenic viruses, always perform work inside a biosafety cabinet (BSC) due to aerosol generation during freezing.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
</td>
</tr>
<tr style="height:26.05pt">
<td width="147" valign="top" style="width:110.4pt;border:solid white 1.0pt;border-top:none;background:#E7EAED;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.05pt">
<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Disinfection & Decontamination</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
</td>
<td width="844" valign="top" style="width:632.8pt;border-top:none;border-left:none;border-bottom:solid white 1.0pt;border-right:solid white 1.0pt;background:#E7EAED;padding:3.6pt 7.2pt 3.6pt 7.2pt;height:26.05pt">
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l19 level1 lfo15"><span style="font-size:11.0pt;color:black">Proper decontamination methods include autoclaving, bleach, aerosolized hydrogen peroxide, or UV exposure according to the US EPA.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l19 level1 lfo15"><span style="font-size:11.0pt;color:black">Ethanol is not approved by EPA for disinfecting pathogens and it was used only with follow-up bleach washes.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l19 level1 lfo15"><span style="font-size:11.0pt;color:black">Post-imaging, store grids in liquid nitrogen or disinfect (10% bleach solution) immediately to prevent contamination.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l19 level1 lfo15"><span style="font-size:11.0pt;color:black">The vacuum system in TEM would be pretty harsh on any pathogen. UV light on exhausted materials from the backing pump or TFS heating element to 60¡ÆC could assuage
such concerns. </span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Laboratory Design & Safety</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l8 level1 lfo16"><span style="font-size:11.0pt;color:black">BSL2 samples would be segregated in separate dewars and kept in secure areas</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Others</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l0 level1 lfo17"><span style="font-size:11.0pt;color:black">Safety measures should be able to protect everyone in all health status, including immunocompromised person.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l0 level1 lfo17"><span style="font-size:11.0pt;color:black">Electron beam imaging is likely to destroy pathogen due to radiation damage.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Equipment</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Level</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Setup</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">High Pressure Freezer</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">BSL2</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l3 level1 lfo18"><span style="font-size:11.0pt;color:black">Had extra security and the lab was under negative pressure</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l3 level1 lfo18"><span style="font-size:11.0pt;color:black">Heard other facilities may have some extra ventilation hoods being put over HPF devices
</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Krios</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">BSL2</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l5 level1 lfo19"><span style="font-size:11.0pt;color:black">Not under negative pressure as anything that entered the Krios room was cryofixed</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Krios</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">BSL3</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l21 level1 lfo20"><span style="font-size:11.0pt;color:black">Any sample that came out of the BSL3 either had to be UV exposed prior to cryo-fixation or chemically inactivated.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l21 level1 lfo20"><span style="font-size:11.0pt;color:black">All inactivation protocols had to show that the pathogen was rendered non-infectious through either plaque assay or some PCR based assay.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<span style="font-size:11.0pt;font-family:"Aptos",sans-serif;mso-ligatures:standardcontextual;mso-fareast-language:ZH-CN"><br clear="all" style="page-break-before:always">
</span>
<p class="MsoNormal"><span style="font-size:11.0pt">#3-8<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">#</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">From</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Comments</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">3</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">Switzerland</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l1 level1 lfo21"><span style="font-size:11.0pt;color:black">During sample preparation, all the tools and devices are BSL2 and reserved only for that.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l1 level1 lfo21"><span style="font-size:11.0pt;color:black">After fixation or frozen, it is considered just any other sample.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">4</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">USA</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l22 level1 lfo22"><span style="font-size:11.0pt;color:black">All equipment can be operated under normal conditions - i.e. in the open on a lab bench, with special considerations for the evacuation of N2 gas or Ethane.
</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l22 level1 lfo22"><span style="font-size:11.0pt;color:black">Issues regarding BSL-2 have only arisen during disposal of equipment.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">5</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">France</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l4 level1 lfo23"><span style="font-size:11.0pt;color:black">Insufficient liquid nitrogen or a vacuum pump failure in TEMs should not be an issue as the temperature and pressure are far bellow triple point.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l4 level1 lfo23"><span style="font-size:11.0pt;color:black">Electron beam that will heat up the sample will destroy the sample.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l4 level1 lfo23"><span style="font-size:11.0pt;color:black">With BSL-2 ventilation setting and personal protection equipment, the risk for service engineer in TEM rooms is low.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">6</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">USA</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l13 level1 lfo24"><span style="font-size:11.0pt;color:black">The risks regarding pathogens at the BSL-2 level is rather minimal. Having a cryo-TEM in a BSL-3 environment is a completely different story.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">7</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">N/A</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l9 level1 lfo25"><span style="font-size:11.0pt;color:black">BSL2 grids discard into a tube of bleach, and eventually the tube is discarded via the regular BSL2 biohazard waste stream.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l9 level1 lfo25"><span style="font-size:11.0pt;color:black">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¢ªC). </span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l9 level1 lfo25"><span style="font-size:11.0pt;color:black">An Aclar "anti-contamination" ring can be placed on Vitrobot foam pads to prevent media soaking into the pads.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">8</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:black">A lab in Switzerland</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoNormal" style="mso-list:l16 level1 lfo26"><span style="font-size:11.0pt;color:black">All freezing instruments, equipment and storage are in a dedicated BSL-2 lab. Freezing instruments are not in BSC but pipet under the cabinet, bring it to the
grid in the chamber. Use mask and gloves to reduce the risks.</span><span style="font-size:11.0pt"><o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l16 level1 lfo26"><span style="font-size:11.0pt;color:black">Work under the assumption that the risk of potential aerosol creation is negligeable as long as you are not milling and as long as your microscope remains cold.
Extra cautious when putting BSL-2 grids and avoid having them in the microscope over the weekend without close supervision.</span><span style="font-size:11.0pt"><o:p></o:p></span></li></ol>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">Thank you again to everyone who contributed. If you have further comments or experiences to share, please feel free to continue the discussion.</span><span lang="EN-US" style="font-size:11.0pt"><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">Regards,<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">Jason<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-ligatures:none">From:</span></b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-ligatures:none"> Jason NG (CPOS)
<br>
<b>Sent:</b> 16 June 2025 15:53<br>
<b>To:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> RE: [3dem] Seeking Advice on BSL-2 CryoEM Laboratory Setup and Safety Protocols<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><span style="font-size:11.0pt">Dear all,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">Thank you very much for your replies to my email and to those who messaged me privately regarding the BSL-2 lab setup. I sincerely appreciate the detailed information and experiences you have shared—they are
extremely helpful and valuable.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">I am currently summarizing all your comments and suggestions to present to my senior. Please feel free to continue the discussion and share any further opinions or advice you may have.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">Regards,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">Jason<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-ligatures:none">From:</span></b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-ligatures:none"> 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu">3dem-bounces@ncmir.ucsd.edu</a>>
<b>On Behalf Of </b>Jason NG (CPOS) via 3dem<br>
<b>Sent:</b> 12 June 2025 17:00<br>
<b>To:</b> <a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br>
<b>Subject:</b> [3dem] Seeking Advice on BSL-2 CryoEM Laboratory Setup and Safety Protocols<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><span style="font-size:11.0pt">Dear CryoEM Community,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">We are currently evaluating the risks of becoming BSL-2 CryoEM laboratory and would greatly appreciate your guidance on several safety and setup considerations.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">Specifically, we are interested in understanding:<o:p></o:p></span></p>
<ul style="margin-top:0cm" type="disc">
<li class="MsoNormal" style="mso-list:l24 level1 lfo3"><span style="font-size:11.0pt">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?<o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l24 level1 lfo3"><span style="font-size:11.0pt">Is implementing negative room pressure necessary in a CryoEM lab at BSL-2?<o:p></o:p></span></li><li class="MsoNormal" style="mso-list:l24 level1 lfo3"><span style="font-size:11.0pt">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?<o:p></o:p></span></li></ul>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">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.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal" style="background:white"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:#1F497D">Best Regards,<o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:#1F497D">Jason NG<o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:#548DD4">Senior Technical Assistant<o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:#548DD4">LKS Cryo-EM Laboratory</span><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#212121"><o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:black"> </span><span style="color:#212121"><o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span style="color:black;mso-ligatures:none"><img border="0" width="292" height="41" style="width:3.0416in;height:.427in" id="Picture_x0020_12" src="cid:image001.jpg@01DBE1C4.F71045C0"></span><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:black"> </span><span style="color:#212121"><o:p></o:p></span></p>
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<p class="MsoNormal" style="background:white"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:#1F4E79">Centre for PanorOmic Sciences</span><span style="color:#212121"><o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:#1F497D">LLG03, Laboratory Block<o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:#1F497D">21 Sassoon Road, Pokfulam, Hong Kong<o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:#1F497D">P: +852 3910-3528</span><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#212121"><o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:#1F497D">E: </span><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:#1F4E79"><a href="mailto:jasonng.cpos@hku.hk">jasonng.cpos@hku.hk</a><o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:#1F497D">W: </span><span style="color:#1F4E79"><a href="https://urldefense.com/v3/__https:/cpos.hku.hk/__;!!Mih3wA!B5co-igM_UHRmCF1lSC-8fHY1eebmRgfFqxjKo2KEWhA_VnVBHdVH1ZLRO9f_JY6lLds5D1C_SANjg3mKpVKQaHmXg$" target="_blank"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:blue">https://cpos.hku.hk</span></a></span><span style="color:#212121"><o:p></o:p></span></p>
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