Multimodal Nonlinear Optical Microscopy for Simultaneous 3-D Label-Free and Immunofluorescence Imaging of Biological Samples

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.