Abstract
Diffraction limits resolution in optical microscopy, but many biological phenomena occur on molecular length scales. Recently, methods to circumvent the diffraction limit have been presented. Fluorescence photoactivation localization microscopy (FPALM) uses multiple iterations of photoactivation, imaging, and photobleaching of subsets of photoactivatable fluorescent probe (PAFP) molecules to generate images with resolution of 10-40 nanometers. The three-dimensional position, brightness, polarization, and other single molecule information are obtained from the raw image(s) of each PAFP. The process is repeated for many subsets to build up data on many molecules. The positions of all localized molecules are used to construct an image of the sample with nanoscale resolution. Results will be shown for a variety of biological systems, including live and fixed cells expressing PAFP fusions of membrane, cytoplasmic, cytoskeletal, and nuclear proteins. Bi-plane FPALM can image in three dimensions, and polarization FPALM can image molecular positions and anisotropies simultaneously. Using these powerful capabilities, many potentially interesting biological problems can be addressed.
© 2009 Optical Society of America
PDF ArticleMore Like This
Travis J. Gould, Manasa V. Gudheti, Mudalige S. Gunewardene, Julie A. Gosse, and Samuel T. Hess
LSWA2 Laser Science (LS) 2009
Joerg Bewersdorf, Michael J. Mlodzianoski, Stefanie E. K. Kirschbaum, and Manuel F. Juette
LSThB2 Laser Science (LS) 2009
Tao Xu
NSu1C.1 Nanophotonics, Nanoelectronics and Nanosensor (N3) 2013