Abstract

The life sciences require new highly sensitive imaging tools, which allow the quantitative measurement of molecular parameters within a physiological three-dimensional (3D) environment. Therefore, we combined single plane illumination microscopy (SPIM) with camera based fluorescence correlation spectroscopy (FCS). SPIM-FCS provides contiguous particle number and diffusion coefficient images with a high spatial resolution in homo- and heterogeneous 3D specimens and live zebrafish embryos. Our SPIM-FCS recorded up to 4096 spectra within 56 seconds at a laser power of 60 μW without damaging the embryo. This new FCS modality provides more measurements per time and more, less photo-toxic measurements per sample than confocal based methods. In essence, SPIM-FCS offers new opportunities to observe biomolecular interactions quantitatively and functions in a highly multiplexed manner within a physiologically relevant 3D environment.

© 2010 OSA

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2009

S. Chiantia, J. Ries, and P. Schwille, “Fluorescence correlation spectroscopy in membrane structure elucidation,” Biochim. Biophys. Acta 1788(1), 225–233 (2009).
[CrossRef]

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[CrossRef] [PubMed]

J. Sankaran, M. Manna, L. Guo, R. Kraut, and T. Wohland, “Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy,” Biophys. J. 97(9), 2630–2639 (2009).
[CrossRef] [PubMed]

J. Huisken and D. Y. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development 136(12), 1963–1975 (2009).
[CrossRef] [PubMed]

2008

P. J. Keller and E. H. Stelzer, “Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy,” Curr. Opin. Neurobiol. 18(6), 624–632 (2008).
[CrossRef]

G. Persson, P. Thyberg, and J. Widengren, “Modulated fluorescence correlation spectroscopy with complete time range information,” Biophys. J. 94(3), 977–985 (2008).
[CrossRef]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

J. G. Ritter, R. Veith, J. P. Siebrasse, and U. Kubitscheck, “High-contrast single-particle tracking by selective focal plane illumination microscopy,” Opt. Express 16(10), 7142–7152 (2008).
[CrossRef] [PubMed]

J. Ries, E. P. Petrov, and P. Schwille, “Total internal reflection fluorescence correlation spectroscopy: effects of lateral diffusion and surface-generated fluorescence,” Biophys. J. 95(1), 390–399 (2008).
[CrossRef] [PubMed]

L. Guo, J. Y. Har, J. Sankaran, Y. M. Hong, B. Kannan, and T. Wohland, “Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study,” ChemPhysChem 9(5), 721–728 (2008).
[CrossRef] [PubMed]

J. T. Groves, R. Parthasarathy, and M. B. Forstner, “Fluorescence imaging of membrane dynamics,” Annu. Rev. Biomed. Eng. 10(1), 311–338 (2008).
[CrossRef] [PubMed]

J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. 85, 471–484 (2008).
[CrossRef]

P. Liu, S. Ahmed, and T. Wohland, “The F-techniques: advances in receptor protein studies,” Trends Endocrinol. Metab. 19(5), 181–190 (2008).
[CrossRef] [PubMed]

A. V. Orden and J. Jung, “Review fluorescence correlation spectroscopy for probing the kinetics and mechanisms of DNA hairpin formation,” Biopolymers 89(1), 1–16 (2008).
[CrossRef]

M. Weiss, “Probing the interior of living cells with fluorescence correlation spectroscopy,” Ann. N. Y. Acad. Sci. 1130(1), 21–27 (2008).
[CrossRef]

2007

F. Pampaloni, E. G. Reynaud, and E. H. Stelzer, “The third dimension bridges the gap between cell culture and live tissue,” Nat. Rev. Mol. Cell Biol. 8(10), 839–845 (2007).
[CrossRef] [PubMed]

P. J. Keller, F. Pampaloni, and E. H. Stelzer, “Three-dimensional preparation and imaging reveal intrinsic microtubule properties,” Nat. Methods 4(10), 843–846 (2007).
[CrossRef] [PubMed]

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. 78(2), 023705 (2007).
[CrossRef] [PubMed]

M. H. Malone, N. Sciaky, L. Stalheim, K. M. Hahn, E. Linney, and G. L. Johnson, “Laser-scanning velocimetry: a confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae,” BMC Biotechnol. 7(1), 40 (2007).
[CrossRef] [PubMed]

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

2006

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

D. L. Kolin, D. Ronis, and P. W. Wiseman, “k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics,” Biophys. J. 91(8), 3061–3075 (2006).
[CrossRef] [PubMed]

M. Burkhardt and P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express 14(12), 5013–5020 (2006).
[CrossRef] [PubMed]

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

2005

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(2), 1317–1327 (2005).
[CrossRef] [PubMed]

2004

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P. A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

1999

M. Brinkmeier, K. Dorre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[CrossRef] [PubMed]

1998

K. Starchev, J. Zhang, and J. Buffle, “Applications of Fluorescence Correlation Spectroscopy—Particle Size Effect,” J. Colloid Interface Sci. 203(1), 189–196 (1998).
[CrossRef]

1972

D. Magde, E. Elson, and W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Ahmed, S.

P. Liu, S. Ahmed, and T. Wohland, “The F-techniques: advances in receptor protein studies,” Trends Endocrinol. Metab. 19(5), 181–190 (2008).
[CrossRef] [PubMed]

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Anhut, T.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P. A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Arevalo, R.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

Besse, P. A.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P. A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Blom, H.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P. A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Brinkmeier, M.

M. Brinkmeier, K. Dorre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[CrossRef] [PubMed]

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(2), 1317–1327 (2005).
[CrossRef] [PubMed]

Buffle, J.

K. Starchev, J. Zhang, and J. Buffle, “Applications of Fluorescence Correlation Spectroscopy—Particle Size Effect,” J. Colloid Interface Sci. 203(1), 189–196 (1998).
[CrossRef]

Burkhardt, M.

Chiantia, S.

S. Chiantia, J. Ries, and P. Schwille, “Fluorescence correlation spectroscopy in membrane structure elucidation,” Biochim. Biophys. Acta 1788(1), 225–233 (2009).
[CrossRef]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Dertinger, T.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

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(2), 1317–1327 (2005).
[CrossRef] [PubMed]

Ding, J. L.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Dorre, K.

M. Brinkmeier, K. Dorre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[CrossRef] [PubMed]

Eigen, M.

M. Brinkmeier, K. Dorre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[CrossRef] [PubMed]

Elson, E.

D. Magde, E. Elson, and W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Enderlein, J.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

Erdel, F.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

Forstner, M. B.

J. T. Groves, R. Parthasarathy, and M. B. Forstner, “Fluorescence imaging of membrane dynamics,” Annu. Rev. Biomed. Eng. 10(1), 311–338 (2008).
[CrossRef] [PubMed]

Gösch, M.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P. A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

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(2), 1317–1327 (2005).
[CrossRef] [PubMed]

Graves, C.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

Greger, K.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. 78(2), 023705 (2007).
[CrossRef] [PubMed]

Gregor, I.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

Groves, J. T.

J. T. Groves, R. Parthasarathy, and M. B. Forstner, “Fluorescence imaging of membrane dynamics,” Annu. Rev. Biomed. Eng. 10(1), 311–338 (2008).
[CrossRef] [PubMed]

Guo, L.

J. Sankaran, M. Manna, L. Guo, R. Kraut, and T. Wohland, “Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy,” Biophys. J. 97(9), 2630–2639 (2009).
[CrossRef] [PubMed]

L. Guo, J. Y. Har, J. Sankaran, Y. M. Hong, B. Kannan, and T. Wohland, “Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study,” ChemPhysChem 9(5), 721–728 (2008).
[CrossRef] [PubMed]

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Hahn, K. M.

M. H. Malone, N. Sciaky, L. Stalheim, K. M. Hahn, E. Linney, and G. L. Johnson, “Laser-scanning velocimetry: a confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae,” BMC Biotechnol. 7(1), 40 (2007).
[CrossRef] [PubMed]

Har, J. Y.

L. Guo, J. Y. Har, J. Sankaran, Y. M. Hong, B. Kannan, and T. Wohland, “Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study,” ChemPhysChem 9(5), 721–728 (2008).
[CrossRef] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Hartmann, R.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

Heuvelman, G.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

Hong, Y. M.

L. Guo, J. Y. Har, J. Sankaran, Y. M. Hong, B. Kannan, and T. Wohland, “Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study,” ChemPhysChem 9(5), 721–728 (2008).
[CrossRef] [PubMed]

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(2), 1317–1327 (2005).
[CrossRef] [PubMed]

Huisken, J.

J. Huisken and D. Y. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development 136(12), 1963–1975 (2009).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Johnson, G. L.

M. H. Malone, N. Sciaky, L. Stalheim, K. M. Hahn, E. Linney, and G. L. Johnson, “Laser-scanning velocimetry: a confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae,” BMC Biotechnol. 7(1), 40 (2007).
[CrossRef] [PubMed]

Jung, J.

A. V. Orden and J. Jung, “Review fluorescence correlation spectroscopy for probing the kinetics and mechanisms of DNA hairpin formation,” Biopolymers 89(1), 1–16 (2008).
[CrossRef]

Kannan, B.

L. Guo, J. Y. Har, J. Sankaran, Y. M. Hong, B. Kannan, and T. Wohland, “Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study,” ChemPhysChem 9(5), 721–728 (2008).
[CrossRef] [PubMed]

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Keller, P. J.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

P. J. Keller and E. H. Stelzer, “Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy,” Curr. Opin. Neurobiol. 18(6), 624–632 (2008).
[CrossRef]

P. J. Keller, F. Pampaloni, and E. H. Stelzer, “Three-dimensional preparation and imaging reveal intrinsic microtubule properties,” Nat. Methods 4(10), 843–846 (2007).
[CrossRef] [PubMed]

Kolin, D. L.

D. L. Kolin, D. Ronis, and P. W. Wiseman, “k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics,” Biophys. J. 91(8), 3061–3075 (2006).
[CrossRef] [PubMed]

Kraut, R.

J. Sankaran, M. Manna, L. Guo, R. Kraut, and T. Wohland, “Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy,” Biophys. J. 97(9), 2630–2639 (2009).
[CrossRef] [PubMed]

Kubitscheck, U.

Langowski, J.

J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. 85, 471–484 (2008).
[CrossRef]

Lasser, T.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P. A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Linney, E.

M. H. Malone, N. Sciaky, L. Stalheim, K. M. Hahn, E. Linney, and G. L. Johnson, “Laser-scanning velocimetry: a confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae,” BMC Biotechnol. 7(1), 40 (2007).
[CrossRef] [PubMed]

Liu, P.

P. Liu, S. Ahmed, and T. Wohland, “The F-techniques: advances in receptor protein studies,” Trends Endocrinol. Metab. 19(5), 181–190 (2008).
[CrossRef] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Magde, D.

D. Magde, E. Elson, and W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Malone, M. H.

M. H. Malone, N. Sciaky, L. Stalheim, K. M. Hahn, E. Linney, and G. L. Johnson, “Laser-scanning velocimetry: a confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae,” BMC Biotechnol. 7(1), 40 (2007).
[CrossRef] [PubMed]

Manna, M.

J. Sankaran, M. Manna, L. Guo, R. Kraut, and T. Wohland, “Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy,” Biophys. J. 97(9), 2630–2639 (2009).
[CrossRef] [PubMed]

Marcello, M.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Maruyama, I.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

McAllister, R.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

Mitchison, T. J.

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[CrossRef] [PubMed]

Needleman, D. J.

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[CrossRef] [PubMed]

Orden, A. V.

A. V. Orden and J. Jung, “Review fluorescence correlation spectroscopy for probing the kinetics and mechanisms of DNA hairpin formation,” Biopolymers 89(1), 1–16 (2008).
[CrossRef]

Pacheco, V.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

Pampaloni, F.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

P. J. Keller, F. Pampaloni, and E. H. Stelzer, “Three-dimensional preparation and imaging reveal intrinsic microtubule properties,” Nat. Methods 4(10), 843–846 (2007).
[CrossRef] [PubMed]

F. Pampaloni, E. G. Reynaud, and E. H. Stelzer, “The third dimension bridges the gap between cell culture and live tissue,” Nat. Rev. Mol. Cell Biol. 8(10), 839–845 (2007).
[CrossRef] [PubMed]

Parthasarathy, R.

J. T. Groves, R. Parthasarathy, and M. B. Forstner, “Fluorescence imaging of membrane dynamics,” Annu. Rev. Biomed. Eng. 10(1), 311–338 (2008).
[CrossRef] [PubMed]

Persson, G.

G. Persson, P. Thyberg, and J. Widengren, “Modulated fluorescence correlation spectroscopy with complete time range information,” Biophys. J. 94(3), 977–985 (2008).
[CrossRef]

Petrov, E. P.

J. Ries, E. P. Petrov, and P. Schwille, “Total internal reflection fluorescence correlation spectroscopy: effects of lateral diffusion and surface-generated fluorescence,” Biophys. J. 95(1), 390–399 (2008).
[CrossRef] [PubMed]

Popovic, R. S.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P. A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Reynaud, E. G.

F. Pampaloni, E. G. Reynaud, and E. H. Stelzer, “The third dimension bridges the gap between cell culture and live tissue,” Nat. Rev. Mol. Cell Biol. 8(10), 839–845 (2007).
[CrossRef] [PubMed]

Ries, J.

S. Chiantia, J. Ries, and P. Schwille, “Fluorescence correlation spectroscopy in membrane structure elucidation,” Biochim. Biophys. Acta 1788(1), 225–233 (2009).
[CrossRef]

J. Ries, E. P. Petrov, and P. Schwille, “Total internal reflection fluorescence correlation spectroscopy: effects of lateral diffusion and surface-generated fluorescence,” Biophys. J. 95(1), 390–399 (2008).
[CrossRef] [PubMed]

Rigler, R.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P. A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Rippe, K.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

Ritter, J. G.

Rochas, A.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P. A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Ronis, D.

D. L. Kolin, D. Ronis, and P. W. Wiseman, “k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics,” Biophys. J. 91(8), 3061–3075 (2006).
[CrossRef] [PubMed]

Sankaran, J.

J. Sankaran, M. Manna, L. Guo, R. Kraut, and T. Wohland, “Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy,” Biophys. J. 97(9), 2630–2639 (2009).
[CrossRef] [PubMed]

L. Guo, J. Y. Har, J. Sankaran, Y. M. Hong, B. Kannan, and T. Wohland, “Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study,” ChemPhysChem 9(5), 721–728 (2008).
[CrossRef] [PubMed]

Schmidt, A. D.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Schwille, P.

S. Chiantia, J. Ries, and P. Schwille, “Fluorescence correlation spectroscopy in membrane structure elucidation,” Biochim. Biophys. Acta 1788(1), 225–233 (2009).
[CrossRef]

J. Ries, E. P. Petrov, and P. Schwille, “Total internal reflection fluorescence correlation spectroscopy: effects of lateral diffusion and surface-generated fluorescence,” Biophys. J. 95(1), 390–399 (2008).
[CrossRef] [PubMed]

M. Burkhardt and P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express 14(12), 5013–5020 (2006).
[CrossRef] [PubMed]

Sciaky, N.

M. H. Malone, N. Sciaky, L. Stalheim, K. M. Hahn, E. Linney, and G. L. Johnson, “Laser-scanning velocimetry: a confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae,” BMC Biotechnol. 7(1), 40 (2007).
[CrossRef] [PubMed]

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(2), 1317–1327 (2005).
[CrossRef] [PubMed]

Serov, A.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P. A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Siebrasse, J. P.

Sisan, D. R.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

Stainier, D. Y.

J. Huisken and D. Y. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development 136(12), 1963–1975 (2009).
[CrossRef] [PubMed]

Stalheim, L.

M. H. Malone, N. Sciaky, L. Stalheim, K. M. Hahn, E. Linney, and G. L. Johnson, “Laser-scanning velocimetry: a confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae,” BMC Biotechnol. 7(1), 40 (2007).
[CrossRef] [PubMed]

Starchev, K.

K. Starchev, J. Zhang, and J. Buffle, “Applications of Fluorescence Correlation Spectroscopy—Particle Size Effect,” J. Colloid Interface Sci. 203(1), 189–196 (1998).
[CrossRef]

Stelzer, E. H.

P. J. Keller and E. H. Stelzer, “Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy,” Curr. Opin. Neurobiol. 18(6), 624–632 (2008).
[CrossRef]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

F. Pampaloni, E. G. Reynaud, and E. H. Stelzer, “The third dimension bridges the gap between cell culture and live tissue,” Nat. Rev. Mol. Cell Biol. 8(10), 839–845 (2007).
[CrossRef] [PubMed]

P. J. Keller, F. Pampaloni, and E. H. Stelzer, “Three-dimensional preparation and imaging reveal intrinsic microtubule properties,” Nat. Methods 4(10), 843–846 (2007).
[CrossRef] [PubMed]

Stelzer, E. H. K.

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. 78(2), 023705 (2007).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Stephan, J.

M. Brinkmeier, K. Dorre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[CrossRef] [PubMed]

Sudhaharan, T.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Swoger, J.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. 78(2), 023705 (2007).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Thyberg, P.

G. Persson, P. Thyberg, and J. Widengren, “Modulated fluorescence correlation spectroscopy with complete time range information,” Biophys. J. 94(3), 977–985 (2008).
[CrossRef]

Urbach, J. S.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

Veith, R.

Verveer, P. J.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

von der Hocht, I.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

Wachsmuth, M.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

Webb, W.

D. Magde, E. Elson, and W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Weiss, M.

M. Weiss, “Probing the interior of living cells with fluorescence correlation spectroscopy,” Ann. N. Y. Acad. Sci. 1130(1), 21–27 (2008).
[CrossRef]

Widengren, J.

G. Persson, P. Thyberg, and J. Widengren, “Modulated fluorescence correlation spectroscopy with complete time range information,” Biophys. J. 94(3), 977–985 (2008).
[CrossRef]

Wiseman, P. W.

D. L. Kolin, D. Ronis, and P. W. Wiseman, “k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics,” Biophys. J. 91(8), 3061–3075 (2006).
[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(2), 1317–1327 (2005).
[CrossRef] [PubMed]

Wittbrodt, J.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Wohland, T.

J. Sankaran, M. Manna, L. Guo, R. Kraut, and T. Wohland, “Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy,” Biophys. J. 97(9), 2630–2639 (2009).
[CrossRef] [PubMed]

L. Guo, J. Y. Har, J. Sankaran, Y. M. Hong, B. Kannan, and T. Wohland, “Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study,” ChemPhysChem 9(5), 721–728 (2008).
[CrossRef] [PubMed]

P. Liu, S. Ahmed, and T. Wohland, “The F-techniques: advances in receptor protein studies,” Trends Endocrinol. Metab. 19(5), 181–190 (2008).
[CrossRef] [PubMed]

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Xu, Y.

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[CrossRef] [PubMed]

Zhang, J.

K. Starchev, J. Zhang, and J. Buffle, “Applications of Fluorescence Correlation Spectroscopy—Particle Size Effect,” J. Colloid Interface Sci. 203(1), 189–196 (1998).
[CrossRef]

Anal. Chem.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

M. Brinkmeier, K. Dorre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[CrossRef] [PubMed]

Ann. N. Y. Acad. Sci.

M. Weiss, “Probing the interior of living cells with fluorescence correlation spectroscopy,” Ann. N. Y. Acad. Sci. 1130(1), 21–27 (2008).
[CrossRef]

Annu. Rev. Biomed. Eng.

J. T. Groves, R. Parthasarathy, and M. B. Forstner, “Fluorescence imaging of membrane dynamics,” Annu. Rev. Biomed. Eng. 10(1), 311–338 (2008).
[CrossRef] [PubMed]

Biochim. Biophys. Acta

S. Chiantia, J. Ries, and P. Schwille, “Fluorescence correlation spectroscopy in membrane structure elucidation,” Biochim. Biophys. Acta 1788(1), 225–233 (2009).
[CrossRef]

Biophys. J.

D. L. Kolin, D. Ronis, and P. W. Wiseman, “k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics,” Biophys. J. 91(8), 3061–3075 (2006).
[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(2), 1317–1327 (2005).
[CrossRef] [PubMed]

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[CrossRef] [PubMed]

G. Persson, P. Thyberg, and J. Widengren, “Modulated fluorescence correlation spectroscopy with complete time range information,” Biophys. J. 94(3), 977–985 (2008).
[CrossRef]

J. Ries, E. P. Petrov, and P. Schwille, “Total internal reflection fluorescence correlation spectroscopy: effects of lateral diffusion and surface-generated fluorescence,” Biophys. J. 95(1), 390–399 (2008).
[CrossRef] [PubMed]

J. Sankaran, M. Manna, L. Guo, R. Kraut, and T. Wohland, “Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy,” Biophys. J. 97(9), 2630–2639 (2009).
[CrossRef] [PubMed]

Biopolymers

A. V. Orden and J. Jung, “Review fluorescence correlation spectroscopy for probing the kinetics and mechanisms of DNA hairpin formation,” Biopolymers 89(1), 1–16 (2008).
[CrossRef]

BMC Biotechnol.

M. H. Malone, N. Sciaky, L. Stalheim, K. M. Hahn, E. Linney, and G. L. Johnson, “Laser-scanning velocimetry: a confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae,” BMC Biotechnol. 7(1), 40 (2007).
[CrossRef] [PubMed]

ChemPhysChem

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

L. Guo, J. Y. Har, J. Sankaran, Y. M. Hong, B. Kannan, and T. Wohland, “Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study,” ChemPhysChem 9(5), 721–728 (2008).
[CrossRef] [PubMed]

Curr. Opin. Neurobiol.

P. J. Keller and E. H. Stelzer, “Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy,” Curr. Opin. Neurobiol. 18(6), 624–632 (2008).
[CrossRef]

Development

J. Huisken and D. Y. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development 136(12), 1963–1975 (2009).
[CrossRef] [PubMed]

Eur. Biophys. J.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

J. Biomed. Opt.

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Figures (4)

Fig. 1
Fig. 1

Schematic drawing of the SPIM sample chamber. The sample chamber can contain both, illumination and detection, objectives as shown here. In our case only the water dipping detection objective was contained in the sample chamber while the light sheet from the illumination objective was coupled over a thin glass window into the sample chamber. The particular setup can be adapted to the NA required for the application. The sample chamber itself is filled with aqueous solution which is chosen according to the nature of the sample. The yellow 3D specimen, held in agarose by a capillary coming from the top (capillary is not shown for clarity), is positioned in the excitation light sheet (cyan). The emitted light (green) from the illuminated plane is collected by the detection objective and imaged over a tube lens on an EMCCD camera (camera not shown).

Fig. 2
Fig. 2

(A) Comparison of the light sheet profiles of the 10x/0.2 and 20x/0.4 illumination objective lenses. An image of the light sheet was recorded with the EMCCD camera by placing a mirror at an angle of 45° into the focal plane of the detection objective (100x/1.0 W). The full widths at half maximum (FWHM) of the light sheets are 2.4 µm and 1.4 μm, respectively. The 1/e2 radii are 1.9 µm and 1.4 μm, respectively. (B) Comparison of the normalized autocorrelation functions obtained with three different combinations of illumination and detection objectives. The measurement with the 10x/0.2 illumination objective was taken at an exposure time of 1 ms. The two measurements with the 20x/0.4 objective were taken with an exposure time of 5 ms. All three measurements result in similar diffusion coefficients between 0.9 μm2/s and 1.5 μm2/s. Using the same detection objective (100x/1.0 W) but different illumination objectives (10x/0.2 or 20x/0.4), the autocorrelation functions show little difference since diffusion along the z-direction contribute less significantly to the autocorrelation function than along the xy-directions. However, for different detection objectives (100x/1.0 W or 40x/0.75 W), but the same illumination objective (20x/0.4), the differences are significant since the detection objective influences the xy resolutions of the system. (C) The 0.2 μm bead sample is homogeneous. All 1024 ACFs are very similar. (D) The ACFs of the 1.0 μm bead sample allow us to distinguish single microspheres as well as aggregates. (E) Examples of the different ACFs for the 1.0 μm bead sample (solid gray lines) including their fits (black lines). (F) Comparison of ACFs for single 0.2 µm and 1.0 μm microspheres quantitate the different ACFs. The average diffusion coefficients are 1.1 ± 0.5 μm2/s and 0.28 ± 0.18 μm2/s for the two bead populations.

Fig. 3
Fig. 3

Diffusion measurement of 0.2 μm microspheres at a water/agarose border. During the course of the experiment, the microspheres were injected into the aqueous medium. (A) Intensity image averaged over 10,000 frames. (B) Particle number N extracted from the single fits to each ACF of the 1343 pixels. (C) The diffusion coefficient D extracted from the fits to each ACF. (D) Diffusion coefficients of all 17 lines (each with 79 pixels). The diffusion coefficient D within the aqueous phase sample agree well while D decreases towards the water/agarose border (from left to right). Beyond the border (pixel position ~50), no consistent diffusion coefficients can be found due to a lack of correlations. (E-G) Depicted are all experimental ACFs for areas from position 0-20 (solution, E), 21-54 transition region from solution to agarose (F), and 55-79 (agarose, G). The average D for position 1-5 (solution) is D = 1.2 ± 0.1 μm2/s, while the average D for position 30-35 (transition region) is D = 0.23 ± 0.02 μm2/s due to the decrease in diffusion towards the water/agarose border. Beyond position 55 no correlations are discernible.

Fig. 4
Fig. 4

SPIM-FCS measurements within a live zebrafish 48 hpf. Microspheres with adiameter of 0.2 μm were injected into the blood circulation 68 seconds prior to the start of the experiment. (A) Fast blood flow within a blood vessel. The ACFs are narrower and steeper compared to solution measurements since flow transports the molecules with a speed of about 60-170 μm/s through the observation volumes. (B) This figure shows cross-correlation functions (CCFs) between the central pixel of the 20 × 20 pixel ROI and the surrounding pixels at a distance of 3 pixels (same experiments as A). A prominent peak in the CCF confirms the transport of particles from the central pixel to the surrounding pixels. The development of the peak is direction dependent, with the flow going from the green marked pixels to the central pixel in the direction of the red marked pixels, giving the blood flow profile. (C) The ACFs were been recorded close to the heart and show dominant peaks due to the heart beat, which is about 3 per second in this example as observed from the ACFs. They show little transport since flow is slow in the large vessels. (D) Comparison between measurements of 0.2 μm microspheres in solution and at different parts in the zebrafish.

Tables (2)

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Table 1 Diffusion coefficients in μm2/s for 0.2 μm multicolor microspheres in aqueous solution measured by the 128 × 128 pixel chip (24 μm pixel size) with different combinations of illumination and detection objectives.

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Table 2 Dependence of diffusion coefficient and particle in the observation volume on binning

Equations (9)

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G(τ)=F(t)F(t+τ)F(t)2=g(τ)+G
gxy(τ)=14Ca4(2aerf(a2Dτ+σxy2)+4Dτ+σxy2π(ea24(Dτ+σxy2)1))2
P(z|z')I(z)I(z')dzdz'=12π(Dτ+σz2)
P(z|z')=12πDτe(zz')4Dτ
I(z)=12πσze(zm)22σz2
G(τ)=14a2πN(2aerf(a2Dτ+σxy2)+4Dτ+σxy2π(ea24(Dτ+σxy2)1))2(1+Dτσz2)12+G
G(τ)=G+14a2N(1+Dτσz2)12×Gx×Gy
Gx=2Dτ+σxy2π(e(a+rxvxτ)24(Dτ+σxy2)+e(arx+vxτ)24(Dτ+σxy2)2e(rxvxτ)24(Dτ+σxy2))+(a+rxvxτ)erf((a+rxvxτ)2Dτ+σxy2)+(arx+vxτ)erf((arx+vxτ)2Dτ+σxy2)2(rxvxτ)erf((rxvxτ)2Dτ+σxy2)
Gy=2Dτ+σxy2π(e(a+ryvyτ)24(Dτ+σxy2)+e(ary+vyτ)24(Dτ+σxy2)2e(ryvyτ)24(Dτ+σxy2))+(a+ryvyτ)erf((a+ryvyτ)2Dτ+σxy2)+(ary+vyτ)erf((ary+vyτ)2Dτ+σxy2)2(ryvyτ)erf((ryvyτ)2Dτ+σxy2)

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