I usually use PFA 4%. However I must fix cells (Hs 578Bst) with a not hazard-toxic compound. I thought to use air flow comes of biological cape, but the morphology must be affected. Some suggestions are more than welcome.
Thanks to everybody.
Luca
Hello,
unfortunately fixation is toxic for cells by definition. Usually the fixative cross links some or all proteins or their parts, which ends in cell death. Our experience is that 4%PFA is slightly affecting morphology, but strongly affects apparent elasticity (~10 fold stiffer) and surface roughness for adherent cells. Maybe some combination of ethanol and acetic acid at -20 degree would be a bit milder for morphology and elasticity, but the exact ratio is hard to quantify.
Best,
A.G. Vegh
Hi Attila, thank you so much for your kind help. I understand your point and I usually use PFA4%, because is a good compromise between AFM performances and good cell properties. I'm interested in analysis of the cell cytoskeleton after cell membrane denaturation, just through the structural point of view. I'm not interested in assay some mechanical properties, even though they are very interesting and so appealing.
Best regards
Luca
Caro Luca, since you "must fix cells (Hs 578Bst) with a not hazard-toxic compound." I guess you'll have no real choice other than imaging "living cells in culture medium...." Also ethanol, methanol +/- acid (like acetic acid)....[keyword: "acid fixation"] are hazardous & toxic compounds [more effective to cells than to you personally, I guess (:-)) ] .....
" 3.2. Direct AFM imaging of cell-bound membrane vesicles
Our initial attempts to image live cells in liquid failed due to slight movements of the cells in response to the touch of the AFM tip during AFM scanning in the contact mode. So, we had to image the cells prefixed in 4% paraformaldehyde *) by AFM in liquid. The AFM topographical images of single cells were successfully obtained although.... "
[*) 4% paraformaldehyde for 30 min, washed twice with PBS and subjected to AFM imaging.....]"
NB: The Materials and Methods section does not indicate whether "4% paraformaldehyde solution" was "buffered" appropriately or not.
cited from: AFM visualization of cortical filaments/network under cell-bound membrane vesicles; Xiaojun Zhang, Qisheng Tang, Li Wub, Jie Huang, Yong Chen; Biochimica et Biophysica Acta (BBA) - Biomembranes
Volume 1848, Issue 10, Part A, October 2015, Pages 2225–2232; http://doi.org/10.1016/j.bbamem.2015.06.025, can be read via Open Access at: http://www.sciencedirect.com/science/article/pii/S0005273615002059
Effect of Cold Plasma on Glial Cell Morphology Studied by Atomic Force
Microscopy
Recek N, Cheng X, Keidar M, Cvelbar U, Vesel A, Mozetic M, et al. (2015) Effect of Cold Plasma on Glial Cell Morphology Studied by Atomic Force Microscopy. PLoS ONE 10(3): e0119111. doi:10.1371/journal.pone.0119111, at: http://hsrc.himmelfarb.gwu.edu/cgi/viewcontent.cgi?article=1086&context=smhs_neurosurg_facpubs
They use(d) 0.5% buffered glutaraldehyde for fixation....also in this article it isn't defined whether the GA-solution was appropriately buffered or not.
Studying early stages of fibronectin fibrillogenesis in living cells by atomic force microscopy.
Tetyana Gudzenko and Clemens M. Franz (2015?) Open access at: http://www.molbiolcell.org/content/early/2015/07/27/mbc.E14-05-1026.full.pdf. They used 1% glutaraldehyde/PBS prefixed cells [for 10 min], imaging in PBS
otherwise, as a "last" option:
i) "enabling live cell imaging with the highest resolution", cf: http://www.nanonics.co.il/solutions/bioafm#ImagingLiveCells
and (perhaps you already know this article and perhaps other specific literature on that matter):
[Citation:] "studies were performed on living, intact cells in cell culture medium..."
Lanzicher et al, 2015: AFM single-cell force spectroscopy links altered nuclear and cytoskeletal mechanics to defective cell adhesion in cardiac myocytes with a nuclear lamin mutation.
Nucleus. 2015; 6(5): 394–407.
Published online 2015 Aug 26. doi: 10.1080/19491034.2015.1084453, @ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915516/ pdf: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915516/pdf/kncl-06-05-1084453.pdf
I really would like to read that these references were of help anyway....Best wishes and good luck, WM
Hi Mohammad, tank for appreciating our past discussion. As far as cel fixed are concerned, I've mostly used PFA4%. It is the one of the best chemicals, used for cell fixation, recommended for AFM. I suggest you to scanned the cell at least 24 hour later cell fixation, unless the protocol required differently. AFM preserves cells from any kinds of changes made by time. I scanned cell samples fixated 8 years beforehand. Concerning the essication, made by biological cabinet airflow (30 minutes, no more), it depends what you want to look for. In my latest AFM experiments I fixed cells by airflow, and the samples were perfect. I would like to suggest to consider also the airflow fixation, not for long time or you must seriously shrink samples. I hope my reply will be helpful. :-)
Cheers
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