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<div><span style="font-size: 14px;" class="">Hi Juan,</span></div>
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<div><span style="font-size: 14px;" class="">Hope you are well.</span></div>
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<div><span style="font-size: 14px;" class="">I think it’s a general phenomenon that there are much more autofluorescence (fluo background as you said) detected in cryo temperature, especially mammalian cells. You may have seen or could check this paper reporting
the similar issue: <span style="text-indent: -36px;" class="">Carter, S. D., Mageswaran, S. K., Farino, Z. J., Mamede, J. I., Oikonomou, C. M., Hope, T. J., Freyberg, Z., & Jensen, G. J. (2018). Distinguishing signal from autofluorescence in cryogenic correlated
light and electron microscopy of mammalian cells. </span><i style="text-indent: -36px;" class="">J Struct Biol</i><span style="text-indent: -36px;" class="">,</span><i style="text-indent: -36px;" class=""> 201</i><span style="text-indent: -36px;" class="">(1),
15-25. <a href="https://urldefense.com/v3/__https://doi.org/10.1016/j.jsb.2017.10.009__;!!Mih3wA!HZbRUUWHg4Fs8iMo-y6cRGVbXFPHGIW5Qp6xuo_qTVJI5WC3HqxTEgGbJt_dzhutI0Yygx5fy1pUK6GOz6kda-M2WjAJd-Lsme0$" class="">https://doi.org/10.1016/j.jsb.2017.10.009</a></span><span style="text-indent: -36px;" class=""> </span></span></div>
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<div class=""><span style="font-size: 14px;" class="">I also see more autofluorescence in my sample in cryo temperature than RT, especially in green channel. My guess is that in RT the autofluorescence get quenched soon after imaging, but in cryo any fluorescence
gets barely quenched or very slow to get quenched (at least by non-laser light source as Leica cryoCLEM).</span></div>
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<div class=""><span style="font-size: 14px;" class="">Best wishes,</span></div>
<div class=""><span style="font-size: 14px;" class="">Joy</span></div>
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<div class="">On 25 Aug 2022, at 12:34, Juan Shen <<a href="mailto:juan.shen@strubi.ox.ac.uk" class="">juan.shen@strubi.ox.ac.uk</a>> wrote:</div>
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Hi all,</div>
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We are trying to image tagged VLPs in cells using Leica cryo CLEM. Our sample looks good under the regular confocal microscope with bright particles. However, the cell fluorescence background is relatively high <span style="background-color: rgb(255, 255, 255); display: inline !important;" class="">u</span><span style="margin: 0px; background-color: rgb(255, 255, 255); display: inline !important;" class="">nder
the Leica cryo CLEM microscopy.</span></div>
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We have tried using FluoroBrite DMEM media or different cell lines but the problem is still there. I was wondering if anyone had a similar problem when imaging cell samples using cryo CLEM? Thank you.</div>
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Best wishes,</div>
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Juan</div>
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<span style="margin: 0px; font-size: 10pt; font-family: Arial, Helvetica, sans-serif;" class="">Juan Shen</span></div>
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