Subdiffraction-Limit Two-Photon Fluorescence Microscopy for GFP-Tagged Cell Imaging

We report applications of two-photon excitation fluorescence (2PEF) microscopy with subdiffraction-limit resolution for green-fluorescent-protein-tagged cell imaging. The microscope integrates 2PEF microscopy and stimulated emission depletion microscopy in one microscope that has the benefits of bot...

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Bibliographic Details
Published in:Biophysical journal 2009-12, Vol.97 (12), p.3224-3228
Main Authors: Li, Qifeng, Wu, Sherry S.H., Chou, Keng C.
Format: Article
Language:English
Subjects:
Animals
Atoms & subatomic particles
Cells
CHO Cells
Cricetinae
Cricetulus
Diffraction
Fluorescence
Green Fluorescent Proteins - metabolism
Microscopes
Microscopy
Microscopy, Fluorescence - methods
Molecular Imaging - methods
Photons
Proteins
Spectroscopy, Imaging, and Other Techniques
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Summary:We report applications of two-photon excitation fluorescence (2PEF) microscopy with subdiffraction-limit resolution for green-fluorescent-protein-tagged cell imaging. The microscope integrates 2PEF microscopy and stimulated emission depletion microscopy in one microscope that has the benefits of both techniques: intrinsic three-dimensional resolution, confined photobleaching, and subdiffraction-limit resolution. The subdiffraction-limit resolution was demonstrated by resolving green-fluorescent-protein-tagged caveolar vesicles located within a distance shorter than the diffraction limit of a regular 2PEF microscope, which is ∼250 nm even with the best optics. The full width at half-maximum of the effective point-spread function for the 2PEF microscope was estimated to be ∼54 nm.
ISSN:0006-3495
1542-0086
DOI:10.1016/j.bpj.2009.09.038