title: Integrated femtosecond stimulated Raman scattering and two-photon fluorescence imaging of subcellular lipid and vesicular structures.
abstract: The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.
authors.affiliation: Hong Kong University of Science and Technology, Department of Electronic and Computer Engineering, Biophotonics Research Laboratory, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Xuesong
authors.initials: X
authors.lastname: Li
authors.affiliation: Hong Kong University of Science and Technology, Division of Life Science, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Wen Jiun
authors.initials: WJ
authors.lastname: Lam
authors.affiliation: Hong Kong University of Science and Technology, Division of Life Science, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Cao
authors.affiliation: Hong Kong University of Science and Technology, Division of Life Science, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Hao
authors.affiliation: Hong Kong University of Science and Technology, Department of Electronic and Computer Engineering, Biophotonics Research Laboratory, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Qiqi
authors.initials: Q
authors.lastname: Sun
authors.affiliation: Hong Kong University of Science and Technology, Department of Electronic and Computer Engineering, Biophotonics Research Laboratory, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Sicong
authors.initials: S
authors.lastname: He
authors.affiliation: Hong Kong University of Science and Technology, Division of Life Science, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Ho Yi
authors.initials: HY
authors.lastname: Mak
authors.affiliation: Hong Kong University of Science and Technology, Department of Electronic and Computer Engineering, Biophotonics Research Laboratory, Clear Water Bay, Kowloon, Hong Kong SAR, ChinacHong Kong University of Science and Technology, School of Science and Insti.
authors.forename: Jianan Y
authors.initials: JY
authors.lastname: Qu
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D066292:Lipid Droplets / Q000378:metabolism* / Q000648:ultrastructure*; D050356:Lipid Metabolism / Q000502:physiology; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation*; D057054:Molecular Imaging / Q000295:instrumentation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013059:Spectrum Analysis, Raman / Q000295:instrumentation*; D018511:Systems Integration; D014018:Tissue Distribution; D022161:Transport Vesicles / Q000000981:diagnostic imaging* / Q000378:metabolism*; D014463:Ultrasonography