@article {68463, title = {Chemically stable fluorescent proteins for advanced microscopy.}, journal = {Nature Methods}, volume = {19}, year = {2022}, month = {2022 Nov 07}, pages = {1612-21}, abstract = {

We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), {\textquoteright}hyperfolder YFP{\textquoteright} (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-{\r A} resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 {\r A}) and an mGreenLantern mutant (1.2 {\r A}), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.

}, issn = {1548-7105}, doi = {10.1038/s41592-022-01660-7}, author = {Campbell, Benjamin C and Paez-Segala, Maria G and Looger, Loren L and Petsko, Gregory A and Liu, Ce Feng} } @article {48416, title = {Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples.}, journal = {Nature Protocols}, volume = {12}, year = {2017}, month = {2017 May}, pages = {916-946}, abstract = {

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replication creates high-contrast, 3D images of the cytoplasmic surface of the plasma membrane but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (\~{}10-50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2-7 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.

}, issn = {1750-2799}, doi = {10.1038/nprot.2017.017}, author = {Kopek, Benjamin G and Paez-Segala, Maria G and Shtengel, Gleb and Sochacki, Kem A and Sun, Mei G and Wang, Yalin and Xu, C Shan and Van Engelenburg, Schuyler B and Taraska, Justin W and Looger, Loren L and Hess, Harald F} } @article {45844, title = {Fixation-resistant photoactivatable fluorescent proteins for CLEM.}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Mar}, pages = {215-8}, abstract = {

Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1\% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

}, issn = {1548-7105}, doi = {10.1038/nmeth.3225}, author = {Paez-Segala, Maria G and Sun, Mei G and Shtengel, Gleb and Viswanathan, Sarada and Baird, Michelle A and Macklin, John J and Patel, Ronak and Allen, John R and Howe, Elizabeth S and Piszczek, Grzegorz and Hess, Harald F and Davidson, Michael W and Wang, Yalin and Looger, Loren L} }