Abstract

In microscopy, single fluorescence point sources can be localized with a precision several times greater than the resolution limit of the microscope. We show that the intermittent fluorescence or �??blinking�?? of quantum dots can analyzed by an Independent Component Analysis so as to identify the light emitted by each individual nanoparticle, localize it precisely, and thereby resolve groups of closely spaced (< λ / 30) quantum dots. Both simulated and experimental data demonstrate that this technique is superior to localization based on Maximum Likelihood Estimation of the sum image under the assumption of point emitters. This technique has general application to any emitter with non-Gaussian temporal intensity distribution, including triplet state blinking. When applied to the labeling of structures, a high resolution �??image�?? consisting of individually localized points may be reconstructed leading to the term �??Pointillism�??.

© 2005 Optical Society of America

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Anal. Chem. (1)

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. �??High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,�?? Anal. Chem. 74, 3511�??3517 (2002).
[CrossRef]

Appl. Phys. Lett. (2)

S.W. Hell, S. Lindek, C. Cremer, and E.H.K. Stelzer. �??Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,�?? Appl. Phys. Lett. 64, 1335�??1337 (1994).
[CrossRef]

T.A. Klar, M. Dyba, and S.W. Hell. �??Stimulated emission depletion microscopy with an offset depleting beam,�?? Appl. Phys. Lett. 78, 393�??395 (2001).
[CrossRef]

Biophys. J. (1)

R.J. Ober, S. Ram, and S.E. Ward. �??Localization accuracy in single-molecule microscopy,�?? Biophys. J. 86, 1185�??1200 (2004).

IEEE Trans. Neural Networks (1)

A. Hyvärinen. �??Fast and robust fixed-point algorithms for independent component analysis,�?? IEEE Trans. Neural Networks 10, 626�??634 (1999).
[CrossRef]

J. Am. Chem. Soc. (1)

S. Hohng and T. Ha. �??Near-complete suppression of quantum dot blinking in ambient conditions,�?? J. Am. Chem. Soc. 126, 1324�??1325 (2004).
[CrossRef]

J. Microsc. (2)

A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. �??Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,�?? J. Microsc. 199, 96�??105 (2000).
[CrossRef]

M.G.L. Gustafsson, D.A. Agard, and J.W. Sedat. �??I5M-3D widefield light microscopy with better than 100 nm axial resolution,�?? J. Microsc. 195, 10�??16 (1999).
[CrossRef]

J. Opt. Soc. Am. A (1)

Microsc. Res. Tech. (1)

H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. �??Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,�?? Microsc. Res. Tech. 65, 169�??179 (2004).
[CrossRef]

Nat. Biotechnol. (1)

D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. �??Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,�?? Nat. Biotechnol. 22, 198�??203 (2004).
[CrossRef]

Nature (1)

M. Nirmal, B.O. Dabbausi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris, and L.E. Brus. �??Fluoroscence intermittency in single cadmium selenide nanocrystals,�?? Nature 383, 802�??805 (1996).
[CrossRef]

Neural Networks (1)

M. Funaro, E. Oja, and H. Valpula. �??Independent component analysis for artefact separation in astrophysical images,�?? Neural Networks 16, 469�??478 (2003).
[CrossRef]

Opt. Lett. (1)

Proc. IEEE (1)

J.-F. Cardoso. �??Blind signal separation: statistical principles,�?? Proc. IEEE 86, 2009�??2025 (1998).

Proc. Natl. Acad. Sci. U.S.A. (1)

M.P. Gordon, T. Ha, and P.R. Selvin. �??Single-molecule high-resolution imaging with photobleaching,�?? Proc. Natl. Acad. Sci. U.S.A. 101, 6462�??6465 (2004).
[CrossRef]

Proc. R. Soc. London A (1)

B. Richards and E. Wolf. �??Electromagnetic diffraction in optical systems. II. structure of the image field in an aplanatic system,�?? Proc. R. Soc. London A 253, 358�??379 (1959).

Proc. SPIE (1)

T.Q. Pham, M. Bezuijen, L.J. van Vliet, K. Schutte, and C.L. Luengo Hendriks. �??Performance of optimal registration estimators,�?? Proc. SPIE 5817, 133�??144 (2005).

Science (3)

R. Ando, H. Mizuno, and A. Miyawaki �??Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting,�?? Science 306, 1370-1373 (2004).
[CrossRef]

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M.Wu, S. S. Gambhir, and S. Weiss. �??Quantum dots for live cells, in vivo imaging, and diagnostics,�?? Science 307, 538�??544 (2005).
[CrossRef]

A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin. �??Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization,�?? Science 300, 2061�??2066 (2003).
[CrossRef]

Other (1)

J. Hurri, H. Gävert, J. Särelä, and A. Hyvärinen. FastICA: �??An implementation of the fast fixed-point algorithm for independent component analysis in MATLAB, version 2.3, 2004,�?? Helsinki University of Technology, <a href="http://www.cis.hut.fi/projects/ica/fastica/.">http://www.cis.hut.fi/projects/ica/fastica/.</a>

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