Three-dimensional tracking of a single fluorescent nanoparticle using four-focus excitation in a confocal microscope.

We report high sensitivity detection and tracking of a single fluorescent nanoparticle in solution by use of four alternately pulsed laser diodes for fluorescence excitation in a confocal microscope. Slight offsets between the centers of the overlapping laser foci together with time-resolved photon counting enable sub-micron precision position measurements. Real-time correction for diffusional motion with a xyz-piezo stage then enables tracking of a nanoparticle with diffusivity up to ~12 μm(2) s(-1). Fluorescence correlation spectroscopy and calibration measurements indicate a net fluorescence photon detection efficiency of ~6-9%, comparable to that of an optimized single-molecule microscope.

[1]  Jason K. King,et al.  Microfluidic device for the electrokinetic manipulation of single molecules , 2009 .

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

[3]  Aurélie Dupont,et al.  Nanoscale three-dimensional single particle tracking. , 2011, Nanoscale.

[4]  Lyle Richard Middendorf,et al.  Imaging of single-chromophore molecules in aqueous solution near a fused-silica interface , 2001, SPIE BiOS.

[5]  Matthew D. Lew,et al.  Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane. , 2010, Applied physics letters.

[6]  Haw Yang,et al.  Progress in single-molecule tracking spectroscopy , 2008 .

[7]  Lloyd M. Davis,et al.  Extension of multidimensional microscopy to ultrasensitive applications with maximum-likelihood analysis , 2007, SPIE BiOS.

[8]  Lloyd M. Davis,et al.  Engineering of illumination and collection field profiles for single-molecule orientational imaging , 2007, SPIE OPTO.

[9]  Haw Yang,et al.  Guiding a confocal microscope by single fluorescent nanoparticles. , 2007, Optics letters.

[10]  Hideo Mabuchi,et al.  Feedback localization of freely diffusing fluorescent particles near the optical shot-noise limit. , 2007, Optics letters.

[11]  M. Strano,et al.  Three-dimensional tracking of carbon nanotubes within living cells. , 2012, ACS nano.

[12]  L. Davis,et al.  Monte carlo simulation of a single-molecule detection experiment. , 1998, Applied optics.

[13]  Brian K. Canfield,et al.  Four-focus single-particle position determination in a confocal microscope , 2010, BiOS.

[14]  You Li.  Single Molecule Detection of Near-Infrared Phthalocyanine Dyes , 2018 .

[15]  J. Jett,et al.  High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules. , 1989, Journal of biomolecular structure & dynamics.

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

[17]  Lloyd M. Davis,et al.  Actively quenched single‐photon avalanche diode for high repetition rate time‐gated photon counting , 1996 .

[18]  L. Davis,et al.  An in Situ Method for Detection of Lipid Peroxidation Effects , 1987 .

[19]  J. Bewersdorf,et al.  Three-dimensional tracking of single fluorescent particles with submillisecond temporal resolution. , 2010, Nano letters.

[20]  W E Moerner,et al.  New directions in single-molecule imaging and analysis , 2007, Proceedings of the National Academy of Sciences.

[21]  J. Bechhoefer.  Feedback for physicists: A tutorial essay on control , 2005 .

[22]  D. P. Fromm,et al.  Methods of single-molecule fluorescence spectroscopy and microscopy , 2003 .

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

[24]  N. Cheng.  Formula for the Viscosity of a Glycerol−Water Mixture , 2008 .

[25]  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.

[26]  L. Davis,et al.  Picosecond resolved evolution of laser breakdown in gases , 1993 .

[27]  Lloyd M. Davis,et al.  Computer simulation of gene detection without PCR by single molecule detection , 1999, European Conference on Biomedical Optics.

[28]  Sripad Ram,et al.  3D single molecule tracking with multifocal plane microscopy reveals rapid intercellular transferrin transport at epithelial cell barriers. , 2012, Biophysical journal.

[29]  J. Nichols,et al.  Tuning electronic structure via epitaxial strain in Sr2IrO4 thin films , 2013, 1302.0918.

[30]  Steven A. Soper,et al.  Multiplexed analysis using time-resolved near-IR fluorescence for the detection of genomic material , 2002, SPIE BiOS.

[31]  Brian K. Canfield,et al.  Three-dimensional anti-Brownian electrokinetic trapping of a single nanoparticle in solution , 2013 .

[32]  Hideo Mabuchi,et al.  Quantum dot photon statistics measured by three-dimensional particle tracking. , 2007, Nano letters.

[33]  Lloyd M. Davis,et al.  THE PHOTOPHYSICAL CONSTANTS OF SEVERAL FLUORESCENT DYES PERTAINING TO ULTRASENSITIVE FLUORESCENCE SPECTROSCOPY , 1993 .

[34]  Capabilities of high-sensitivity spectral fluorescence-lifetime imaging for resolving spectroscopically overlapping species , 2009 .

[35]  D. Toomre,et al.  Adaptive optics enables three-dimensional single particle tracking at the sub-millisecond scale , 2013 .

[36]  Markus B. Raschke,et al.  Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science Conference and Photonic Applications Systems Technologies , 2006 .

[37]  Massimo Ghioni,et al.  Avalanche detector with ultraclean response for time-resolved photon counting , 1998 .

[38]  Electro-optics.  Conference on lasers and electro-optics (CLEO) , 2003 .

[39]  Koichi Baba,et al.  Single-Molecule Tracking in Living Cells Using Single Quantum Dot Applications , 2012, Theranostics.

[40]  E. Gratton,et al.  Real-time nanomicroscopy via three-dimensional single-particle tracking. , 2009, Chemphyschem : a European journal of chemical physics and physical chemistry.

[41]  L. Davis,et al.  Rapid and efficient detection of single chromophore molecules in aqueous solution. , 1995, Applied optics.

[42]  L. Davis,et al.  Rate equation simulation of a synchronously pumped dye laser , 1984 .

[43]  Guoqing Shen,et al.  Fabrication and Characterization of Nanofluidics Device Using Fused Silica for Single Protein Molecule Detection , 2008 .

[44]  Steven A. Soper,et al.  Detection and identification of single molecules in solution , 1992 .

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

[46]  Lloyd M. Davis,et al.  Single photon avalanche diode for single molecule detection , 1993, Optical Society of America Annual Meeting.

[47]  George A. Lampropoulos,et al.  Applications of photonic technology , 1995 .

[48]  Lloyd M. Davis.  Efficient Counting of Single Molecules with Sub-100 Microsecond Transit Times , 1997 .

[49]  M. E. Phipps,et al.  Time-resolved three-dimensional molecular tracking in live cells. , 2010, Nano letters.

[50]  L. Davis,et al.  Single-molecule detection with axial flow into a micrometer-sized capillary. , 2007, Applied optics.

[51]  L. Davis,et al.  Use of streak camera for time-resolved photon counting fluorimetry , 1992 .

[52]  Sergey I. Bozhevolnyi,et al.  Conference on Lasers and Electro-Optics, 2008 and 2008 Conference on Quantum Electronics and Laser Science. CLEO/QELS 2008 , 2008 .

[53]  B. Baguley,et al.  Electron donor properties of the antitumour drug amsacrine as studied by fluorescence quenching of DNA-bound ethidium. , 1987, Chemico-biological interactions.

[54]  Y. Katayama,et al.  Three-dimensional single-particle tracking in live cells: news from the third dimension , 2013 .

[55]  Enrico Gratton,et al.  Real-time multi-parameter spectroscopy and localization in three-dimensional single-particle tracking , 2009, Journal of The Royal Society Interface.

[56]  E Gratton,et al.  Scanning FCS, a novel method for three-dimensional particle tracking. , 2003, Biochemical Society transactions.

[57]  James H Werner,et al.  Confocal, three-dimensional tracking of individual quantum dots in high-background environments. , 2008, Analytical chemistry.