Scanning total internal reflection fluorescence imaging

Cell adhesion and focal complex formation require signalling complexes linking cell adhesion molecules to the cytoskeleton. To understand morphogenetic changes associated with tumour cell spreading, migration and tumour cell metastasis, the molecular mechanisms responsible for the regulation, formation and dissolution at the cell-extracellular matrix (ECM) interface need to be identified. In order to achieve this, an improved axial resolution is desirable. We report on the development of a multi-photon (MP) total internal reflection (TIR) fluorescence lifetime imaging (FLIM) system that allows the selective excitation of fluorophores, with such an improved axial resolution. Results from initial experiments are presented. High excitation efficiency is achieved by the use of a Nikon 1.45 NA TIRF objective using annular illumination.

[1]  R. Pepperkok,et al.  Observing proteins in their natural habitat: the living cell. , 2000, Trends in biochemical sciences.

[2]  S. Hell,et al.  Annular aperture two-photon excitation microscopy , 1995 .

[3]  H. Horstmann,et al.  Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells , 1995, Neuron.

[4]  I. Yamazaki,et al.  Time-resolved total internal reflection fluorescence spectroscopy for surface photophysics studies , 1986 .

[5]  P J Verveer,et al.  Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane. , 2000, Science.

[6]  P J Verveer,et al.  Global analysis of fluorescence lifetime imaging microscopy data. , 2000, Biophysical journal.

[7]  Thomas Zimmer,et al.  FRET measurements by TCSPC laser scanning microscopy , 2001, European Conference on Biomedical Optics.

[8]  James Monypenny,et al.  Ezrin is a downstream effector of trafficking PKC–integrin complexes involved in the control of cell motility , 2001, The EMBO journal.

[9]  S. Ameer-Beg,et al.  Imaging protein-protein interactions in cell motility using fluorescence resonance energy transfer (FRET). , 2004, Biochemical Society transactions.

[10]  Alan J. Brown.  Time resolved evanescent wave induced fluorescence spectroscopy , 1990 .

[11]  G. Alvarez de Toledo,et al.  The exocytotic event in chromaffin cells revealed by patch amperometry , 1997, Nature.

[12]  M. Oheim,et al.  REVIEW Imaging Transmitter Release. II. A Practical Guide to Evanescent-wave Imaging , 2001, Lasers in Medical Science.

[13]  P J Verveer,et al.  Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data , 2003, Journal of microscopy.

[14]  Marion Peter,et al.  Imaging molecular interactions by multiphoton FLIM , 2004, Biology of the cell.

[15]  F. Wouters,et al.  Imaging biochemistry inside cells. , 2001, Trends in cell biology.

[16]  J M Beechem,et al.  Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements. , 1989, Biophysical journal.

[17]  D. Axelrod,et al.  Evanescent field excitation of fluorescence by epi-illumination microscopy. , 1989, Applied optics.

[18]  D Barnes,et al.  Imaging protein kinase Calpha activation in cells. , 1999, Science.

[19]  Peter J. Parker,et al.  Imaging Protein Kinase Cα Activation in Cells , 1999 .

[20]  Min Gu,et al.  Scanning total internal reflection fluorescence microscopy , 2003, International Commission for Optics.

[21]  S W Hell,et al.  Four-dimensional multiphoton microscopy with time-correlated single-photon counting. , 2000, Applied optics.

[22]  D. Loerke,et al.  The last few milliseconds in the life of a secretory granule , 1998, European Biophysics Journal.

[23]  J. Lakowicz,et al.  Two-photon excitation by the evanescent wave from total internal reflection. , 1997, Analytical biochemistry.

[24]  Neil,et al.  Wide‐field optically sectioning fluorescence microscopy with laser illumination , 2000, Journal of microscopy.

[25]  K. Fujita.  [Two-photon laser scanning fluorescence microscopy]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[26]  P. Bastiaens,et al.  PKCα regulates β1 integrin‐dependent cell motility through association and control of integrin traffic , 1999 .

[27]  D. Axelrod.  Cell-substrate contacts illuminated by total internal reflection fluorescence , 1981, The Journal of cell biology.

[28]  William E. Hughes,et al.  Site-Directed Perturbation of Protein Kinase C- Integrin Interaction Blocks Carcinoma Cell Chemotaxis , 2002, Molecular and Cellular Biology.

[29]  Stefan Seeger,et al.  Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy. , 2004, Optics letters.

[30]  A. Rohrbach,et al.  Observing secretory granules with a multiangle evanescent wave microscope. , 2000, Biophysical journal.

[31]  D. O'connor,et al.  Time-Correlated Single Photon Counting , 1984 .

[32]  P. Bastiaens,et al.  Three dimensional image restoration in fluorescence lifetime imaging microscopy , 1999, Journal of microscopy.

[33]  G. Schiavo,et al.  Lipid rafts act as specialized domains for tetanus toxin binding and internalization into neurons. , 2001, Molecular biology of the cell.

[34]  Maddy Parsons,et al.  A novel PKC-regulated mechanism controls CD44–ezrin association and directional cell motility , 2002, Nature Cell Biology.

[35]  John White,et al.  Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability , 1999, Nature Biotechnology.

[36]  T M Jovin,et al.  Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation , 1995, The Journal of cell biology.

[37]  Min Gu,et al.  Two-photon fluorescence scanning near-field microscopy based on a focused evanescent field under total internal reflection. , 2003, Optics letters.

[38]  Maddy Parsons,et al.  Interaction of fascin and protein kinase Cα: a novel intersection in cell adhesion and motility , 2003, The EMBO journal.

[39]  H. Horstmann,et al.  Transport, docking and exocytosis of single secretory granules in live chromaffin cells , 1997, Nature.

[40]  Andrew G. Glen,et al.  APPL , 2001 .

[41]  A Liljeborg,et al.  A confocal laser microscope scanner for digital recording of optical serial sections , 1989, Journal of Microscopy.

[42]  P J Verveer,et al.  Improved spatial discrimination of protein reaction states in cells by global analysis and deconvolution of fluorescence lifetime imaging microscopy data , 2001, Journal of microscopy.