Towards high-throughput FLIM for protein-protein interaction screening of live cells and tissue microarrays

Studying cellular protein-protein interactions in situ requires a technique such as fluorescence resonance energy transfer (FRET) which is sensitive on the nanometer scale. Observing FRET is significantly simplified if the fluorescence lifetime of the donor can be monitored. Results from live cells and tissue micro arrays are presented from an automated microscope incorporating time-domain TCSPC fluorescence lifetime imaging (FLIM). Novel hardware and software with a modular approach and scripting abilities allow us to work towards speed-optimized acquisition and ease of use to bring FLIM into the high-throughput regime.

[1]  Ignacio A. Demarco,et al.  Quantitative imaging of protein interactions in the cell nucleus. , 2005, BioTechniques.

[2]  A. Ridley Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. , 2006, Trends in cell biology.

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

[4]  J. Kononen,et al.  Tissue microarrays for high-throughput molecular profiling of tumor specimens , 1998, Nature Medicine.

[5]  Horst Wallrabe,et al.  Imaging protein molecules using FRET and FLIM microscopy. , 2005, Current opinion in biotechnology.

[6]  K. Aoki,et al.  Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes. , 2005, Methods.

[7]  Fred S Wouters,et al.  Unsupervised Fluorescence Lifetime Imaging Microscopy for High Content and High Throughput Screening *S , 2007, Molecular & Cellular Proteomics.

[8]  L. Lim,et al.  The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts , 1995, Molecular and cellular biology.

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

[10]  B Vojnovic,et al.  Automated counting of mammalian cell colonies , 2001, Physics in medicine and biology.

[11]  Borivoj Vojnovic,et al.  Dynamic imaging of protein-protein interactions by MP-FLIM , 2005, SPIE BiOS.

[12]  Zhenbiao Yang,et al.  RHO Gtpases and the Actin Cytoskeleton , 2000 .

[13]  Borivoj Vojnovic,et al.  Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM , 2005, SPIE BiOS.

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

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

[16]  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.

[17]  P Dean,et al.  Proposed standard for image cytometry data files. , 1990, Cytometry.