title: High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions.
abstract: Superresolution microscopy has fundamentally altered our ability to resolve subcellular proteins, but improving on these techniques to study dense structures composed of single-molecule-sized elements has been a challenge. One possible approach to enhance superresolution precision is to use cryogenic fluorescent imaging, reported to reduce fluorescent protein bleaching rates, thereby increasing the precision of superresolution imaging. Here, we describe an approach to cryogenic photoactivated localization microscopy (cPALM) that permits the use of a room-temperature high-numerical-aperture objective lens to image frozen samples in their native state. We find that cPALM increases photon yields and show that this approach can be used to enhance the effective resolution of two photoactivatable/switchable fluorophore-labeled structures in the same frozen sample. This higher resolution, two-color extension of the cPALM technique will expand the accessibility of this approach to a range of laboratories interested in more precise reconstructions of complex subcellular targets.
authors.affiliation: Department of Biology, Brandeis University, Waltham, MA 02454.
authors.forename: Marc
authors.initials: M
authors.lastname: Nahmani
authors.affiliation: Department of Biology, Brandeis University, Waltham, MA 02454.
authors.forename: Conor
authors.initials: C
authors.lastname: Lanahan
authors.affiliation: Department of Biology, Brandeis University, Waltham, MA 02454.
authors.forename: David
authors.initials: D
authors.lastname: DeRosier
authors.affiliation: Department of Biology, Brandeis University, Waltham, MA 02454 turrigiano@brandeis.edu.
authors.forename: Gina G
authors.initials: GG
authors.lastname: Turrigiano
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins
keywords: cryogenic microscopy; localization microscopy; photoactivated localization microscopy; single molecule; superresolution