Ultrahigh-resolution multicolor colocalization of single fluorescent probes.
An optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described in this paper. It relies on the use of two unique families of fluorophores, namely energy-transfer fluorescent beads (TransFluoSpheres) and semiconductor nanocrystal quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A multicolor sample-scanning confocal microscope was constructed that allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps with a piezo-scanner. The resulting spots are accurately localized by fitting them to the known shape of the excitation point-spread function of the microscope. We present results of two-dimensional colocalization of TransFluoSpheres (40 nm in diameter) and of nanocrystals (3-10 nm in diameter) and demonstrate distance-measurement accuracy of better than 10 nm using conventional far-field optics. This ruler bridges the gap between fluorescence resonance energy transfer, near- and far-field imaging, spanning a range of a few nanometers to tens of micrometers.
Lacoste
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TD
Material Sciences and Physical Biosciences Divisions, Lawrence Berkeley National Laboratory, CA 94720, USA.
Michalet
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X
Pinaud
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Chemla
D S
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Alivisatos
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AP
Weiss
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S
D003116:Color; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes