Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy

Abstract

Lipid bodies have an important role in energy storage and lipid regulation. Here we show that lipid bodies are a major source of contrast in third-harmonic generation (THG) microscopy of cells and tissues. In hepatocytes, micrometer-sized lipid bodies produce a THG signal 1-2 orders of magnitude larger than other structures, which allows one to image them with high specificity. THG microscopy with approximately 1,200 nm excitation can be used to follow the distribution of lipid bodies in a variety of unstained samples including insect embryos, plant seeds and intact mammalian tissue (liver, lung). We found that epi-THG imaging is possible in weakly absorbing tissues because bulk scattering redirects a substantial fraction of the forward-generated harmonic light toward the objective. Finally, we show that the combination of THG microscopy with two-photon and second-harmonic imaging provides a new tool for exploring the interactions between lipid bodies, extracellular matrix and fluorescent compounds (vitamin A, NADH and others) in tissues.

Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images

“…Third harmonic generation (THG) microscopy [1][2][3][4] shows great potential for real-time pathology of brain tumor tissue [5,6]. THG microscopy is also establishing itself as an important tool for studying intact tissue.…”

“…THG microscopy is also establishing itself as an important tool for studying intact tissue. It has been successfully applied to image unstained samples such as insect embryos, plant seeds and intact mammalian tissue [3], epithelial tissues [7][8][9], zebrafish embryos [10], zebrafish nervous system [4], and mouse brain [5]. THG is a nonlinear optical process that depends on the third-order susceptibility c (3) of the tissue and the phase-matching conditions.…”

“…It has been successfully applied to image unstained samples such as insect embryos, plant seeds and intact mammalian tissue [3], epithelial tissues [7][8][9], zebrafish embryos [10], zebrafish nervous system [4], and mouse brain [5]. THG is a nonlinear optical process that depends on the third-order susceptibility c (3) of the tissue and the phase-matching conditions. Three incident photons are converted into one photon with triple energy and one third of the wavelength [1][2][3][4][5][6].…”

“…THG is a nonlinear optical process that depends on the third-order susceptibility c (3) of the tissue and the phase-matching conditions. Three incident photons are converted into one photon with triple energy and one third of the wavelength [1][2][3][4][5][6].…”

“…Like other multi-photon techniques [10][11][12][13][14][15][16][17][18], THG not only enables the recording of label-free images of unfixed 3D volumes of tissue [1][2][3][4][5][6][7][8][9][10] free from spatial distortion artifacts inherent to histopathology, but also potentially allows feedback on the nature of the tissue, i. e. whether it is healthy or tumorous, to the surgeon during surgery, as the relative speed of the imaging modalities approaches 'real' time, and no preparation steps of the tissue are required [5,6,[19][20][21][22][23]. THG was shown recently to yield label-free images of ex-vivo human tumor tissue of histopathological quality, in real-time [6].…”