Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples

• Published in: Nature methods Vol. 5; no. 6; pp. 527 - 529
• Main Authors: Juette, Manuel F, Gould, Travis J, Lessard, Mark D, Mlodzianoski, Michael J, Nagpure, Bhupendra S, Bennett, Brian T, Hess, Samuel T, Bewersdorf, Joerg
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.06.2008; Nature Publishing Group
• Subjects: Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biophysics - economics; Biophysics - instrumentation; Biophysics - methods; brief-communication; Cell physiology; Cellular biology; Fluorescein - pharmacology; Fluorescence; Fluorescence microscopy; Fluorescent Dyes - pharmacology; Image Enhancement; Image Interpretation, Computer-Assisted - methods; Imaging, Three-Dimensional - instrumentation; Imaging, Three-Dimensional - methods; Innovations; Lasers; Life Sciences; Light; Methods; Microscope and microscopy; Microscopes; Microscopy - methods; Microscopy, Fluorescence - instrumentation; Microscopy, Fluorescence - methods; Photoactivation; Proteomics; Research methodology; Scientific apparatus & instruments; Software; Three dimensional imaging; United States
• ISSN: 1548-7091; 1548-7105; 1548-7105
• DOI: 10.1038/nmeth.1211

The ability to image thick volumes with invariant high axial and lateral resolution is a challenge for existing super-resolution fluorescence microscopy techniques. The combination of a double-plane detection scheme with fluorescence photoactivation microscopy (FPALM) allows three-dimensional sub-diffraction resolution imaging of samples as thick as whole cells. Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 × 30 × 75nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.