Abstract

Multiparticle tracking with scanning confocal and multiphoton fluorescence imaging is increasingly important for elucidating biological function, as in the transport of intracellular cargo-carrying vesicles. We demonstrate a simple rapid-sampling stochastic scanning multiphoton multifocal microscopy (SS-MMM) fluorescence imaging technique that enables multiparticle tracking without specialized hardware at rates 1,000 times greater than conventional single point raster scanning. Stochastic scanning of a diffractive optic generated 10x10 hexagonal array of foci with a white noise driven galvanometer yields a scan pattern that is random yet space-filling. SS-MMM creates a more uniformly sampled image with fewer spatio-temporal artifacts than obtained by conventional or multibeam raster scanning. SS-MMM is verified by simulation and experimentally demonstrated by tracking microsphere diffusion in solution.

©2006 Optical Society of America

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  1. V. Levi, Q. Ruan, and E. Gratton, “3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J. 88, 2919–2928 (2005).
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref]
  12. R. Jin, J. E. Jureller, H. Y. Kim, and N. F. Scherer, “Correlating second harmonic optical responses of single Ag nanoparticles with morphology,” J. Amer. Chem. Soc. 127, 12482–12483 (2005).
    [Crossref]
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    [Crossref]
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  16. J. E. Jureller, H. Y. Kim, L. Ma, A. Kuznetsov, L. H. Philipson, and N. F. Scherer, are preparing a manuscript entitled “Vesicular transport measured with stochastic scanning multiphoton multifocal microscopy.”

2005 (5)

V. Levi, Q. Ruan, and E. Gratton, “3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J. 88, 2919–2928 (2005).
[Crossref] [PubMed]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[Crossref] [PubMed]

K. C. Toussaint, S. Park, J. E. Jureller, and N. F. Scherer, “Generation of optical vector beams using a diffractive optical element interferometer,” Opt. Lett. 30, 2846–2848 (2005).
[Crossref] [PubMed]

V. Levi, Q. Ruan, and E. Gratton, “3-d particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J. 88, 29192928 (2005).
[Crossref]

R. Jin, J. E. Jureller, H. Y. Kim, and N. F. Scherer, “Correlating second harmonic optical responses of single Ag nanoparticles with morphology,” J. Amer. Chem. Soc. 127, 12482–12483 (2005).
[Crossref]

2003 (1)

2001 (1)

2000 (2)

1999 (1)

1998 (1)

1996 (1)

J. C. Crocker and D. G. Grier. “Methods of digital video microscopy for colloidal studies,” J. Coll. Inter. Sci. 179, 298310 (1996).
[Crossref]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

Andresen, V.

Antolini, R.

Bewersdorf, J.

Brown, C. M.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[Crossref] [PubMed]

Buehler, C.

Choudhury, A.

Crocker, J. C.

J. C. Crocker and D. G. Grier. “Methods of digital video microscopy for colloidal studies,” J. Coll. Inter. Sci. 179, 298310 (1996).
[Crossref]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

Diaspro, A.

A. Diaspro, Confocal and two-photon microscopy (Wiley, New York, 2002).

Digman, M. A.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[Crossref] [PubMed]

Egner, A.

Fittinghoff, D. N.

Froner, E.

Gratton, E.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[Crossref] [PubMed]

V. Levi, Q. Ruan, and E. Gratton, “3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J. 88, 2919–2928 (2005).
[Crossref] [PubMed]

V. Levi, Q. Ruan, and E. Gratton, “3-d particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J. 88, 29192928 (2005).
[Crossref]

Grier, D. G.

J. C. Crocker and D. G. Grier. “Methods of digital video microscopy for colloidal studies,” J. Coll. Inter. Sci. 179, 298310 (1996).
[Crossref]

Hell, S. W.

Horwitz, A. R.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[Crossref] [PubMed]

Jin, R.

R. Jin, J. E. Jureller, H. Y. Kim, and N. F. Scherer, “Correlating second harmonic optical responses of single Ag nanoparticles with morphology,” J. Amer. Chem. Soc. 127, 12482–12483 (2005).
[Crossref]

Jureller, J. E.

R. Jin, J. E. Jureller, H. Y. Kim, and N. F. Scherer, “Correlating second harmonic optical responses of single Ag nanoparticles with morphology,” J. Amer. Chem. Soc. 127, 12482–12483 (2005).
[Crossref]

K. C. Toussaint, S. Park, J. E. Jureller, and N. F. Scherer, “Generation of optical vector beams using a diffractive optical element interferometer,” Opt. Lett. 30, 2846–2848 (2005).
[Crossref] [PubMed]

J. E. Jureller, “Nonlinear and correlation microscopies for dyanmics and function in heterogeneous systems,” Ph.D. Dissertation, The University of Chicago (2006).

J. E. Jureller, H. Y. Kim, L. Ma, A. Kuznetsov, L. H. Philipson, and N. F. Scherer, are preparing a manuscript entitled “Vesicular transport measured with stochastic scanning multiphoton multifocal microscopy.”

Kim, H. Y.

R. Jin, J. E. Jureller, H. Y. Kim, and N. F. Scherer, “Correlating second harmonic optical responses of single Ag nanoparticles with morphology,” J. Amer. Chem. Soc. 127, 12482–12483 (2005).
[Crossref]

J. E. Jureller, H. Y. Kim, L. Ma, A. Kuznetsov, L. H. Philipson, and N. F. Scherer, are preparing a manuscript entitled “Vesicular transport measured with stochastic scanning multiphoton multifocal microscopy.”

Kim, K.

Kuznetsov, A.

J. E. Jureller, H. Y. Kim, L. Ma, A. Kuznetsov, L. H. Philipson, and N. F. Scherer, are preparing a manuscript entitled “Vesicular transport measured with stochastic scanning multiphoton multifocal microscopy.”

Levi, V.

V. Levi, Q. Ruan, and E. Gratton, “3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J. 88, 2919–2928 (2005).
[Crossref] [PubMed]

V. Levi, Q. Ruan, and E. Gratton, “3-d particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J. 88, 29192928 (2005).
[Crossref]

Ma, L.

J. E. Jureller, H. Y. Kim, L. Ma, A. Kuznetsov, L. H. Philipson, and N. F. Scherer, are preparing a manuscript entitled “Vesicular transport measured with stochastic scanning multiphoton multifocal microscopy.”

Park, S.

Pavone, F. S.

Philipson, L. H.

J. E. Jureller, H. Y. Kim, L. Ma, A. Kuznetsov, L. H. Philipson, and N. F. Scherer, are preparing a manuscript entitled “Vesicular transport measured with stochastic scanning multiphoton multifocal microscopy.”

Pick, R.

Ruan, Q.

V. Levi, Q. Ruan, and E. Gratton, “3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J. 88, 2919–2928 (2005).
[Crossref] [PubMed]

V. Levi, Q. Ruan, and E. Gratton, “3-d particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J. 88, 29192928 (2005).
[Crossref]

Sacconi, L.

Scherer, N. F.

K. C. Toussaint, S. Park, J. E. Jureller, and N. F. Scherer, “Generation of optical vector beams using a diffractive optical element interferometer,” Opt. Lett. 30, 2846–2848 (2005).
[Crossref] [PubMed]

R. Jin, J. E. Jureller, H. Y. Kim, and N. F. Scherer, “Correlating second harmonic optical responses of single Ag nanoparticles with morphology,” J. Amer. Chem. Soc. 127, 12482–12483 (2005).
[Crossref]

J. E. Jureller, H. Y. Kim, L. Ma, A. Kuznetsov, L. H. Philipson, and N. F. Scherer, are preparing a manuscript entitled “Vesicular transport measured with stochastic scanning multiphoton multifocal microscopy.”

Sengupta, P.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[Crossref] [PubMed]

So, P.

Squier, J. A.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

Taghizadeh, M. R.

Toussaint, K. C.

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

Wiseman, P. W.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[Crossref] [PubMed]

Appl. Opt. (1)

Biophys. J. (3)

V. Levi, Q. Ruan, and E. Gratton, “3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J. 88, 2919–2928 (2005).
[Crossref] [PubMed]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[Crossref] [PubMed]

V. Levi, Q. Ruan, and E. Gratton, “3-d particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J. 88, 29192928 (2005).
[Crossref]

J. Amer. Chem. Soc. (1)

R. Jin, J. E. Jureller, H. Y. Kim, and N. F. Scherer, “Correlating second harmonic optical responses of single Ag nanoparticles with morphology,” J. Amer. Chem. Soc. 127, 12482–12483 (2005).
[Crossref]

J. Coll. Inter. Sci. (1)

J. C. Crocker and D. G. Grier. “Methods of digital video microscopy for colloidal studies,” J. Coll. Inter. Sci. 179, 298310 (1996).
[Crossref]

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

Opt. Lett. (5)

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

Other (3)

A. Diaspro, Confocal and two-photon microscopy (Wiley, New York, 2002).

J. E. Jureller, “Nonlinear and correlation microscopies for dyanmics and function in heterogeneous systems,” Ph.D. Dissertation, The University of Chicago (2006).

J. E. Jureller, H. Y. Kim, L. Ma, A. Kuznetsov, L. H. Philipson, and N. F. Scherer, are preparing a manuscript entitled “Vesicular transport measured with stochastic scanning multiphoton multifocal microscopy.”

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Figures (5)

Fig. 1.
Fig. 1.

(a) SS-MMM setup. (b) DOE generated 10x10 hexagonal multifocal array. The smaller image is the actual array after L1. (c) False color two-photon fluorescence image of the multifocal array in a solution of Rhodamine 6G at the sample plane.

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Fig. 2.
Fig. 2.

Log-log plot of the measured galvanometer response power spectral density driven by white noise (blue) and 100 Hz raster (red) waveforms.

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Fig. 3.
Fig. 3.

(a) Single frame two-photon fluorescence multifocal raster scan image of a packed monolayer of 500 nm diameter microspheres at 1 fps. The unit cell boundaries are clearly oversampled, yielding artifacts in a brickwork pattern. (b) Single frame SS-MMM fluorescence image of the same area. The brickwork artifacts in (a) are not observed.

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Fig. 4.
Fig. 4.

Histograms (bin width = 1 pixel) of simulated fast axis residency times in units of array spacing at 1 fps for (a) raster and (b) stochastic scanning. (c and d) Histograms of scanning coverage (pixel intensities normalized by the mean image intensity) for simulated full images at 1 fps. (c) Raster scanning shows a broad distribution and large oversampling. (d) Stochastic scanning shows a much tighter distribution and a small undersampled tail.

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Fig. 5.
Fig. 5.

(a) Representative experimental microsphere trajectories measured with SS-MMM at 9 fps and (b) simulated microsphere trajectories measured with SS-MMM at 100 fps. (c) MSD from the experimentally measured trajectories in (a) and linear fit (solid line). (d) MSD from the simulations in (b) and linear fit (solid line).

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Tables (1)

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Table 1. Imaging simulation coverage results

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