title: Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM.
abstract: Many biomolecules in cells can be visualized with high sensitivity and specificity by fluorescence microscopy. However, the resolution of conventional light microscopy is limited by diffraction to ~200-250 nm laterally and >500 nm axially. Here, we describe superresolution methods based on single-molecule localization analysis of photoswitchable fluorophores (PALM: photoactivated localization microscopy) as well as our recent three-dimensional (3D) method (iPALM: interferometric PALM) that allows imaging with a resolution better than 20 nm in all three dimensions. Considerations for their implementations, applications to multicolor imaging, and a recent development that extend the imaging depth of iPALM to ~750 nm are discussed. As the spatial resolution of superresolution fluorescence microscopy converges with that of electron microscopy (EM), direct imaging of the same specimen using both approaches becomes feasible. This could be particularly useful for cross validation of experiments, and thus, we also describe recent methods that were developed for correlative superresolution fluorescence and EM.
authors: Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.
authors: Gleb
authors: G
authors: Shtengel
authors: Mechanobiology Institute, National University of Singapore, Singapore.
authors: Yilin
authors: Y
authors: Wang
authors: Mechanobiology Institute, National University of Singapore, Singapore.
authors: Zhen
authors: Z
authors: Zhang
authors: Mechanobiology Institute, National University of Singapore, Singapore.
authors: Wah Ing
authors: WI
authors: Goh
authors: Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.
authors: Harald F
authors: HF
authors: Hess
authors: Mechanobiology Institute, National University of Singapore, Singapore; Department of Biomedical Engineering, National University of Singapore, Singapore.
authors: Pakorn
authors: P
authors: Kanchanawong
mesh_terms: D000818:Animals; D002460:Cell Line; D057918:Fiducial Markers; D005456:Fluorescent Dyes / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence / Q000379:methods; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D006046:Gold
keywords: CLEM; PALM; Photoactivatable fluorescent proteins; Photoswitchable fluorophores; Single-molecule localization; Superresolution microscopy; iPALM; iPALM-FIB-SEM