title: Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes.
abstract: We demonstrate three-dimensional (3D) super-resolution microscopy in whole fixed cells using photoactivated localization microscopy (PALM). The use of the bright, genetically expressed fluorescent marker photoactivatable monomeric (m)Cherry (PA-mCherry1) in combination with near diffraction-limited confinement of photoactivation using two-photon illumination and 3D localization methods allowed us to investigate a variety of cellular structures at <50 nm lateral and <100 nm axial resolution. Compared to existing methods, we have substantially reduced excitation and bleaching of unlocalized markers, which allows us to use 3D PALM imaging with high localization density in thick structures. Our 3D localization algorithms, which are based on cross-correlation, do not rely on idealized noise models or specific optical configurations. This allows instrument design to be flexible. By generating appropriate fusion constructs and expressing them in Cos7 cells, we could image invaginations of the nuclear membrane, vimentin fibrils, the mitochondrial network and the endoplasmic reticulum at depths of greater than 8 μm.
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, USA. andrew.g.york+naturemethods@gmail.com
authors.forename: Andrew G
authors.initials: AG
authors.lastname: York
authors.forename: Alireza
authors.initials: A
authors.lastname: Ghitani
authors.forename: Alipasha
authors.initials: A
authors.lastname: Vaziri
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.forename: Hari
authors.initials: H
authors.lastname: Shroff
mesh_terms: D000465:Algorithms; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D021621:Imaging, Three-Dimensional; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D008857:Microscopy, Interference; D017785:Photons; D021381:Protein Transport / Q000502:physiology*