Four-focus single-particle position determination in a confocal microscope

We discuss the capabilities for sub-diffraction, single-nanoparticle position determination in a confocal one- or twophoton microscope with four-focus pulse-interleaved excitation and time-gated single-photon counting. As the technique is scalable to multiple detectors for multi-color observations, it can be used to find the separations of differently colored molecules over a distance range that is complementary to that achievable by FRET. Also, there is a possibility for improved spatial localization by using the nonlinearity of saturation of the excitation or by using the technique together with imaging of the point spread function. Applications of two experimental set-ups for four-focus fluorescence excitation for studies of quantum dots and single-particle manipulation and trapping are also discussed.

[1]  S. Ram,et al.  Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions , 2004, IEEE Transactions on NanoBioscience.

[2]  Mark Bates,et al.  Super-resolution fluorescence microscopy. , 2009, Annual review of biochemistry.

[3]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[4]  H. P. Kao,et al.  Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. , 1994, Biophysical journal.

[5]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[6]  James R McBride,et al.  White-light emission from magic-sized cadmium selenide nanocrystals. , 2005, Journal of the American Chemical Society.

[7]  J. Enderlein.  Tracking of fluorescent molecules diffusing within membranes , 2000 .

[8]  Enrico Gratton,et al.  Exploring dynamics in living cells by tracking single particles , 2007, Cell Biochemistry and Biophysics.

[9]  Brian K. Canfield,et al.  Electrokinetic delivery of single fluorescent biomolecules in fluidic nanochannels , 2008, NanoScience + Engineering.

[10]  Enrico Gratton,et al.  3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells. , 2005, Biophysical journal.

[11]  Michael Unser,et al.  Super-resolution orientation estimation and localization of fluorescent dipoles using 3-D steerable filters. , 2009, Optics express.

[12]  S. Ram,et al.  Localization accuracy in single-molecule microscopy. , 2004, Biophysical journal.

[13]  Jerry Chao,et al.  Quantitative study of single molecule location estimation techniques. , 2009, Optics express.

[14]  J. Lippincott-Schwartz,et al.  Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure , 2009, Proceedings of the National Academy of Sciences.

[15]  Guoqing Shen,et al.  Accounting for triplet and saturation effects in FCS measurements. , 2006, Current pharmaceutical biotechnology.

[16]  James H. Werner,et al.  Three-dimensional tracking of individual quantum dots , 2007 .

[17]  Lloyd M. Davis,et al.  Maximum-likelihood position sensing and actively controlled electrokinetic transport for single-molecule trapping , 2008, SPIE BiOS.

[18]  W. Webb,et al.  Precise nanometer localization analysis for individual fluorescent probes. , 2002, Biophysical journal.

[19]  Rafael Piestun,et al.  Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system , 2009 .

[20]  S. Ram,et al.  Improved single particle localization accuracy with dual objective multifocal plane microscopy. , 2009, Optics express.

[21]  Paul R Selvin,et al.  Fluorescence imaging with one nanometer accuracy: application to molecular motors. , 2005, Accounts of chemical research.

[22]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.