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Fluorescent speckle microscopy of microtubules: how low can you go?
Author(s) -
WatermanStorer Clare M.,
Salmon E. D.
Publication year - 1999
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.13.9002.s225
Subject(s) - fluorescence , speckle pattern , microscopy , fluorescence microscope , fluorophore , biophysics , photoactivated localization microscopy , microtubule , light sheet fluorescence microscopy , microscope , chemistry , optics , biology , super resolution microscopy , microbiology and biotechnology , physics
Fluorescent speckle microscopy (FSM) is a new technique for visualizing the movement, assembly, and turnover of macromolecular assemblies like the cytoskeleton in living cells. In this method, contrast is created by coassembly of a small fraction of fluorescent subunits in a pool of unlabeled subunits. Random variation in association creates a nonuniform “fluorescent speckle” pattern. Fluorescent speckle movements in time‐lapse recordings stand out to the eye and can be measured. Because fluorescent speckles represent fiduciary marks on the polymer lattice, FSM provides the opportunity for the first time to see the 2‐ and 3‐dimensional trajectories of lattice movements within large arrays of polymers as well as identifying sites of assembly and disassembly of individual polymers. The technique works with either microinjection of fluorescently labeled subunits or expression of subunits ligated to green fluorescent protein (GFP). We have found for microtubules assembled in vitro that speckles containing one fluorophore can be detected and recorded using a conventional wide‐field epi‐fluorescence light microscope and digital imaging with a low noise cooled CCD camera. In living cells, optimal speckle contrast occurs at fractions of labeled tubulin of ∼0.1–0.5% where the fluorescence of each speckle corresponds to one to seven fluorophores per resolvable unit (∼0.27 µm) in the microscope. This small fraction of labeled subunits significantly reduces out‐of‐focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells. Waterman‐Storer, C. M., Salmon, E. D. Fluorescent speckle microscopy of microtubules: how low can you go? FASEB J. 13, S225–S230 (1999)