Three-dimensional photoactivated localization microscopy with genetically expressed probes.
Photoactivated localization microscopy (PALM) and related single-molecule imaging techniques enable biological image acquisition at ~20 nm lateral and ~50-100 nm axial resolution. Although such techniques were originally demonstrated on single imaging planes close to the coverslip surface, recent technical developments now enable the 3D imaging of whole fixed cells. We describe methods for converting a 2D PALM into a system capable of acquiring such 3D images, with a particular emphasis on instrumentation that is compatible with choosing relatively dim, genetically expressed photoactivatable fluorescent proteins (PA-FPs) as PALM probes. After reviewing the basics of 2D PALM, we detail astigmatic and multiphoton imaging approaches well suited to working with PA-FPs. We also discuss the use of open-source localization software appropriate for 3D PALM.
Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, 13 South Drive, Bethesda, MD, 20892, USA.
Kelsey
K
Temprine
Andrew G
AG
York
Hari
H
Shroff
D003584:Cytological Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D012984:Software
D005456:Fluorescent Dyes