|State||Ex λ||Em λ||EC (M-1 cm-1)||QY||Brightness||pKa||Maturation (min)||Lifetime (ns)|
|% Normal Cells||OSER/NE ratio||Cell Type||Reference|
|54.0 ± 6.8 (10000 cells)||-||HeLa||Paez-Segala et al. (2015)|
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mEos4a was derived from mEos2 with the following mutations: M1_S2insV/K9R/F34Y/S39T/A69V/I102Y/C195A
Note: Figure Sup. 6A of the original paper (Paez-Segala et al. 2015) erroneously shows mEos4a as lacking Met-14. The sequence below includes M14 and should be considered the correct sequence (Paez-Segala, personal communication)
We mutated surface residues on mEos2 to remove nucleophilic groups, which are involved in cross-linking with aldehydes and OsO4. This resulted in the selection of two mutants, mEos4a and mEos4b, each with substantially improved resistance to OsO4 fixation and fluorescence properties unchanged from those of the starting scaffold mEos2. These proteins facilitated the development of two protocols: (i) a ‘consecutive-section’ approach in which adjacent ultrathin sections cut from resin are separately split between PALM imaging and EM fixation and imaging; and (ii) a ‘same-section’ approach, in which a single resin-cut section is subjected to both PALM and TEM and/or SEM. In both cases, plastic resin embedding markedly decreases tissue distortion from dehydration and secondary fixation, and additionally improves performance of the specimen under the electron beam. We also found that this protocol is appropriate for use with HPF-FS (e.g., Fig. 1d) or without it (e.g., Fig. 1e). We found ultrastructure preservation to be comparable between the two.
Osmium-resistant probes for fluorescence and electron microscopy—the fluorescent EosFP derivative mEos4 has recently been described to withstand osmium treatment during EM processing. The mEos4 molecule is therefore still fluorescent in a well-stained resin-embedded sample. In addition, it is compatible with super resolution photoactivated localization microscopy.
(2018). Journal of Physics D: Applied Physics, 51(44) , 443001. doi: 10.1088/1361-6463/aad055. Article