Intracellular imaging of host‐pathogen interactions using combined CARS and two‐photon fluorescence microscopies

Intracellular imaging is a key tool in the investigation of host-pathogen interactions. Advances in this area are particularly sought to understand the effect of viral infection processes on the host cell and its metabolic functions including those cases where host cell lipid metabolism is modulated as a result of infection. We demonstrate the use of combined coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence microscopies to image fibroblast cells infected by cytomegalovirus. CARS is used to image the host cell membrane, lipid droplets and morphology of the nucleus. Cell nuclei are found to expand during infection, approximately doubling in area. Some cells also show accumulations of lipid droplets at the nuclear periphery. Using a genetically modified virus strain expressing the green fluorescent protein also enables two-photon imaging of the same cells to reveal the location, nature and extent of viral protein expression.

[1]  R. Irvine.  Nuclear lipid signalling , 2003, Nature Reviews Molecular Cell Biology.

[2]  P. Ghazal,et al.  The Major Immediate-Early Gene ie3 of Mouse Cytomegalovirus Is Essential for Viral Growth , 2000, Journal of Virology.

[3]  R Gauderon,et al.  Three-dimensional second-harmonic generation imaging with femtosecond laser pulses. , 1998, Optics letters.

[4]  Kishan Dholakia,et al.  Optical detection and grading of lung neoplasia by Raman microspectroscopy , 2009, International journal of cancer.

[5]  A. Yeh,et al.  Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices. , 2009, Experimental cell research.

[6]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[7]  Conor L Evans,et al.  Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Jürgen Popp,et al.  A comparative Raman and CARS imaging study of colon tissue , 2009, Journal of biophotonics.

[9]  K. Mühlemann,et al.  Transcriptome analysis of fibroblast cells immediate-early after human cytomegalovirus infection. , 2000, Journal of molecular biology.

[10]  K. Yeow,et al.  Cellular imaging in drug discovery , 2006, Nature Reviews Drug Discovery.

[11]  X. Xie,et al.  Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy Published, JLR Papers in Press, August 16, 2003. DOI 10.1194/jlr.D300022-JLR200 , 2003, Journal of Lipid Research.

[12]  B. Klupp,et al.  Herpesvirus assembly: a tale of two membranes. , 2006, Current opinion in microbiology.

[13]  Jun Ye,et al.  High-sensitivity coherent anti-Stokes Raman scattering microscopy with two tightly synchronized picosecond lasers. , 2002, Optics letters.

[14]  H. Hamaguchi,et al.  Vibrationally resonant imaging of a single living cell by supercontinuum-based multiplex coherent anti-Stokes Raman scattering microspectroscopy. , 2005, Optics express.

[15]  W. R. Wiley,et al.  Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering , 1999 .

[16]  J P Korterik,et al.  Background free CARS imaging by phase sensitive heterodyne CARS. , 2008, Optics express.

[17]  Andreas Volkmer,et al.  Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy , 2002 .

[18]  J. McLauchlan.  Lipid droplets and hepatitis C virus infection. , 2009, Biochimica et biophysica acta.

[19]  Coherent Raman Imaging of Human Living Cells Using a Supercontinuum Light Source , 2007 .

[20]  M. Bonn,et al.  Imaging of chemical and physical state of individual cellular lipid droplets using multiplex CARS microscopy , 2009 .

[21]  Andreas Volkmer,et al.  An Epi-Detected Coherent Anti-Stokes Raman Scattering (E-CARS) Microscope with High Spectral Resolution and High Sensitivity , 2001 .

[22]  G. McConnell,et al.  Advances in laser sources for confocal and multiphoton microscopy , 2005, Microscopy research and technique.

[23]  M. Reddehase.  Cytomegaloviruses : molecular biology and immunology , 2006 .

[24]  X. Xie,et al.  Peroxisome Proliferator-Activated Receptor α Antagonism Inhibits Hepatitis C Virus Replication , 2006 .

[25]  M. Trese,et al.  Congenital blindness and osteoporosis-pseudoglioma syndrome. , 2003, Journal of AAPOS : the official publication of the American Association for Pediatric Ophthalmology and Strabismus.

[26]  E. Seeberg,et al.  Human cytomegalovirus infection modulates DNA base excision repair in fibroblast cells. , 2006, Virology.

[27]  Satoshi Kawata,et al.  Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors , 2006 .

[28]  R. Weissleder,et al.  Imaging in the era of molecular oncology , 2008, Nature.

[29]  Julian Moger,et al.  Imaging metal oxide nanoparticles in biological structures with CARS microscopy. , 2008, Optics express.

[30]  Y. Takagi,et al.  Multiple- and single-shot autocorrelator based on two-photon conductivity in semiconductors. , 1992, Optics letters.

[31]  Laura Moody,et al.  Hepatitis C Virus Core Protein Induces Lipid Droplet Redistribution in a Microtubule‐ and Dynein‐Dependent Manner , 2008, Traffic.

[32]  I. Johnson,et al.  Practical Considerations in the Selection and Application of Fluorescent Probes , 2006 .

[33]  Michitaka Suzuki,et al.  Lipid droplets: a classic organelle with new outfits , 2008, Histochemistry and Cell Biology.

[34]  Feruz Ganikhanov,et al.  Broadly tunable dual-wavelength light source for coherent anti-Stokes Raman scattering microscopy. , 2006, Optics letters.

[35]  Jun Ye,et al.  Sub-10-femtosecond active synchronization of two passively mode-locked Ti:sapphire oscillators , 2001 .

[36]  T. T. Le,et al.  Label-free Imaging of Arterial Cells and Extracellular Matrix Using a Multimodal CARS Microscope. , 2008, Optics communications.

[37]  X. Xie,et al.  Intracellular Imaging of HCV RNA and Cellular Lipids by Using Simultaneous Two‐Photon Fluorescence and Coherent Anti‐Stokes Raman Scattering Microscopies , 2006, Chembiochem : a European journal of chemical biology.

[38]  X. Xie,et al.  Coherent Anti-Stokes Raman Scattering Microscopy: Instrumentation, Theory, and Applications , 2004 .

[39]  B. Helms.  Host-Pathogen interactions: Lipids grease the way , 2006 .

[40]  Sanjay Tyagi,et al.  Imaging intracellular RNA distribution and dynamics in living cells , 2009, Nature Methods.