Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments.

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 microW 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.

[1]  C. Landes,et al.  Dye diffusion at surfaces: charge matters. , 2010, Langmuir : the ACS journal of surfaces and colloids.

[2]  R. Kraut,et al.  Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy. , 2009, Biophysical journal.

[3]  K. Rippe,et al.  Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy , 2009, European Biophysics Journal.

[4]  T. Mitchison,et al.  Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy. , 2009, Biophysical journal.

[5]  Jan Huisken,et al.  Selective plane illumination microscopy techniques in developmental biology , 2009, Development.

[6]  Philipp J. Keller,et al.  Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy , 2008, Current Opinion in Neurobiology.

[7]  Philipp J. Keller,et al.  Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy , 2008, Science.

[8]  J. Groves,et al.  Fluorescence imaging of membrane dynamics. , 2008, Annual review of biomedical engineering.

[9]  T. Wohland,et al.  The F-techniques: advances in receptor protein studies , 2008, Trends in Endocrinology & Metabolism.

[10]  P. Schwille,et al.  Total internal reflection fluorescence correlation spectroscopy: effects of lateral diffusion and surface-generated fluorescence. , 2008, Biophysical journal.

[11]  Ulrich Kubitscheck,et al.  High-contrast single-particle tracking by selective focal plane illumination microscopy. , 2008, Optics express.

[12]  M. Weiss.  Probing the Interior of Living Cells with Fluorescence Correlation Spectroscopy , 2008, Annals of the New York Academy of Sciences.

[13]  Thorsten Wohland,et al.  Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study. , 2008, Chemphyschem : a European journal of chemical physics and physical chemistry.

[14]  Gustav Persson,et al.  Modulated fluorescence correlation spectroscopy with complete time range information. , 2008, Biophysical journal.

[15]  F. Pampaloni,et al.  The third dimension bridges the gap between cell culture and live tissue , 2007, Nature Reviews Molecular Cell Biology.

[16]  Philipp J. Keller,et al.  Three-dimensional preparation and imaging reveal intrinsic microtubule properties , 2007, Nature Methods.

[17]  Noah Sciaky,et al.  Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae , 2007, BMC biotechnology.

[18]  T. Wohland,et al.  Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera. , 2007, Analytical chemistry.

[19]  P. Verveer,et al.  High-resolution three-dimensional imaging of large specimens with light sheet–based microscopy , 2007, Nature Methods.

[20]  J. Swoger,et al.  Basic building units and properties of a fluorescence single plane illumination microscope. , 2007, The Review of scientific instruments.

[21]  Thomas Dertinger,et al.  Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. , 2007, Chemphyschem : a European journal of chemical physics and physical chemistry.

[22]  D. Sisan,et al.  Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope. , 2006, Biophysical journal.

[23]  P. Wiseman,et al.  k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics. , 2006, Biophysical journal.

[24]  Petra Schwille,et al.  Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy. , 2006, Optics express.

[25]  Thorsten Wohland,et al.  Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy. , 2006, Analytical chemistry.

[26]  Enrico Gratton,et al.  Measuring fast dynamics in solutions and cells with a laser scanning microscope. , 2005, Biophysical journal.

[27]  Theo Lasser,et al.  Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array. , 2004, Journal of biomedical optics.

[28]  F. Del Bene,et al.  Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy , 2004, Science.

[29]  M. Eigen,et al.  Two-beam cross-correlation:  a method to characterize transport phenomena in micrometer-sized structures. , 1999, Analytical chemistry.

[30]  J. Buffle,et al.  Applications of Fluorescence Correlation Spectroscopy— Particle Size Effect , 1998 .

[31]  W. Webb,et al.  Thermodynamic Fluctuations in a Reacting System-Measurement by Fluorescence Correlation Spectroscopy , 1972 .

[32]  P. Schwille,et al.  Fluorescence correlation spectroscopy in membrane structure elucidation. , 2009, Biochimica et biophysica acta.

[33]  J. Langowski,et al.  Protein-protein interactions determined by fluorescence correlation spectroscopy. , 2008, Methods in cell biology.

[34]  A. Orden,et al.  Review fluorescence correlation spectroscopy for probing the kinetics and mechanisms of DNA hairpin formation. , 2008, Biopolymers.

[35]  J. Lakowicz.  Topics in fluorescence spectroscopy , 2002 .

[36]  M. Westerfield.  The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio) , 1995 .