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<p class="MsoNormal"><span style="font-size:11.0pt">Dear cryo colleagues,<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 wondering if anyone on this list has successfully frozen/imaged mammalian cells using continuous carbon grids, and if so whether they would be willing to provide tips regarding their freezing conditions?<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">Brief background, I’ve been trying to grow/image neurons (tried Hela as well) for tomography using continuous carbon grids of varying thickness (tested 3-10nm carbon layer, 150-200 mesh types, all gold), but
have not had success during freezing step. I’m using a Leica EMGP, and have attempted back and front blots, respectively (tested 5-20 sec blot times, as well as different aliquots of media from 0-2uL, in 90-99% humidity). I’ve even tried side blotting, but
still no success. All attempts have resulted in either broken grid holes or ice that is too thick (back blot generally causes more breakage, while front generally causes ice that’s too thick). I’ve verified the carbon remains (mostly) intact prior to freezing
(at least much more intact than what is seen after freezing), and am mindful of how I take the grid out of the liquid prior to freezing. I should also add I’ve successfully grown/imaged the neurons on holey carbon grids, which are my (and it seems the literature’s)
preferred grids for this system, but our order is delayed, and so that’s why I’m trying to make continuous carbon work.<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">If anyone has had success with these grids to image any kind of mammalian cell I’d be very appreciative of any advice/tips!<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,<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 style="font-size:11.0pt;font-family:"Times New Roman",serif;color:black">Erik Anderson<o:p></o:p></span></b></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Times New Roman",serif;color:black">MD-PhD Candidate | Baylor College of Medicine<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Times New Roman",serif;color:black">B.S. Microbiology and Music | University of Wisconsin-Madison | 2015<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Times New Roman",serif;color:black"><a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__www.bcm.edu_research_faculty-2Dlabs_steven-2Dludtke-2Dlab&d=DwMF-g&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=0pToNIycF_Lo1X5zhTGdAjwpMQnFME8apFLnrmQ7x6eIuOOouehtrBpEXZZVf5FJ&s=fil6vpim5X2wVq7qP-h05TIMTBY6OX5uLrldcv_CcKg&e="><span style="color:#0563C1">Ludtke Lab</span></a>
<o:p></o:p></span></p>
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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"><o:p> </o:p></span></p>
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