[3dem] Background in cell samples under Leica cryo CLEM

[Jeongyoon Choi]

Hi Juan,

Hope you are well.

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:  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. J Struct Biol, 201(1), 15-25. https://urldefense.com/v3/__https://doi.org/10.1016/j.jsb.2017.10.009__;!!Mih3wA!HZbRUUWHg4Fs8iMo-y6cRGVbXFPHGIW5Qp6xuo_qTVJI5WC3HqxTEgGbJt_dzhutI0Yygx5fy1pUK6GOz6kda-M2WjAJd-Lsme0$  

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).


Best wishes,
Joy








On 25 Aug 2022, at 12:34, Juan Shen <juan.shen at strubi.ox.ac.uk<mailto:juan.shen at strubi.ox.ac.uk>> wrote:

Hi all,

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 under the Leica cryo CLEM microscopy.

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.

Best wishes,
Juan



-----------------------------------------------
Juan Shen



Lab manager of Prof Peijun Zhang's group



Wellcome Centre for Human Genetics
University of Oxford
Old Road Campus
Roosevelt Drive, Oxford OX3 7BN
Tel: 01865 287723

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