Journal of the Optical Society of Korea

Optical Society of Korea (한국광학회)
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Multimodal Nonlinear Optical Microscopy for Simultaneous 3-D Label-Free and Immunofluorescence Imaging of Biological Samples

  • Park, Joo Hyun (Department of Nano and Bio Surface Science, Korea University of Science and Technology) ;
  • Lee, Eun-Soo (Center for Nanosafety Metrology, Korea Research Institute of Standards and Science) ;
  • Lee, Jae Yong (Department of Nano and Bio Surface Science, Korea University of Science and Technology) ;
  • Lee, Eun Seong (Center for Nanometrology, Korea Research Institute of Standards and Science) ;
  • Lee, Tae Geol (Department of Nano and Bio Surface Science, Korea University of Science and Technology) ;
  • Kim, Se-Hwa (Department of Nano and Bio Surface Science, Korea University of Science and Technology) ;
  • Lee, Sang-Won (Department of Nano and Bio Surface Science, Korea University of Science and Technology)
  • Received : 2014.07.02
  • Accepted : 2014.09.23
  • Published : 2014.10.25
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Abstract

In this study, we demonstrated multimodal nonlinear optical (NLO) microscopy integrated simultaneously with two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), and coherent anti-Stokes Raman scattering (CARS) in order to obtain targeted cellular and label-free images in an immunofluorescence assay of the atherosclerotic aorta from apolipoprotein E-deficient mice. The multimodal NLO microscope used two laser systems: picosecond (ps) and femtosecond (fs) pulsed lasers. A pair of ps-pulsed lights served for CARS (817 nm and 1064 nm) and SHG (817 nm) images; light from the fs-pulsed laser with the center wavelength of 720 nm was incident into the sample to obtain autofluorescence and targeted molecular TPEF images for high efficiency of fluorescence intensity without cross-talk. For multicolor-targeted TPEF imaging, we stained smooth-muscle cells and macrophages with fluorescent dyes (Alexa Fluor 350 and Alexa Fluor 594) for an immunofluorescence assay. Each depth-sectioned image consisted of $512{\times}512$ pixels with a field of view of $250{\times}250{\mu}m^2$, a lateral resolution of $0.4{\mu}m$, and an axial resolution of $1.3{\mu}m$. We obtained composite multicolor images with conventional label-free NLO images and targeted TPEF images in atherosclerotic-plaque samples. Multicolor 3-D imaging of atherosclerotic-plaque structural and functional composition will be helpful for understanding the pathogenesis of cardiovascular disease.

Keywords

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