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 <TEX>$512{\times}512$</TEX> pixels with a field of view of <TEX>$250{\times}250{\mu}m^2$</TEX>, a lateral resolution of <TEX>$0.4{\mu}m$</TEX>, and an axial resolution of <TEX>$1.3{\mu}m$</TEX>. 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.

© 2014 Optical Society of Korea

Differentiating atherosclerotic plaque burden in arterial tissues using femtosecond CARS-based multimodal nonlinear optical imaging

Leila B. Mostaço-Guidolin, Michael G. Sowa, Andrew Ridsdale, Adrian F. Pegoraro, Michael S. D. Smith, Mark D. Hewko, Elicia K. Kohlenberg, Bernie Schattka, Masashi Shiomi, Albert Stolow, and Alex C.-T. Ko
Biomed. Opt. Express 1(1) 59-73 (2010)

Label-free imaging of atherosclerotic plaques using third-harmonic generation microscopy

David M. Small, Jason S. Jones, Irwin I. Tendler, Paul E. Miller, Andre Ghetti, and Nozomi Nishimura
Biomed. Opt. Express 9(1) 214-229 (2018)

Label-free multimodal microscopy using a single light source and detector for biological imaging

Juehyung Kang, Ungyo Kang, Hyeong Soo Nam, Wooseop Kim, Hyun Jung Kim, Ryeong Hyeon Kim, Jin Won Kim, and Hongki Yoo
Opt. Lett. 46(4) 892-895 (2021)

Label-free multimodal nonlinear optical microscopy reveals fundamental insights of skeletal muscle development

Qiqi Sun, Yanfeng Li, Sicong He, Chenghao Situ, Zhenguo Wu, and Jianan Y. Qu
Biomed. Opt. Express 5(1) 158-166 (2014)

Multimodal microscopy for the simultaneous visualization of five different imaging modalities using a single light source

Jiheun Ryu, Ungyo Kang, Joon Woo Song, Junyoung Kim, Jin Won Kim, Hongki Yoo, and Bomi Gweon
Biomed. Opt. Express 12(9) 5452-5469 (2021)