Chemical contrast for imaging living systems: molecular vibrations drive CARS microscopy

Abstract

Cellular biomolecules contain unique molecular vibrations that can be visualized by coherent anti-Stokes Raman scattering (CARS) microscopy without the need for labels. Here we review the application of CARS microscopy for label-free imaging of cells and tissues using the natural vibrational contrast that arises from biomolecules like lipids as well as for imaging of exogenously added probes or drugs. High-resolution CARS microscopy combined with multimodal imaging has allowed for dynamic monitoring of cellular processes such as lipid metabolism and storage, the movement of organelles, adipogenesis and host-pathogen interactions and can also be used to track molecules within cells and tissues. The CARS imaging modality provides a unique tool for biological chemists to elucidate the state of a cellular environment without perturbing it and to perceive the functional effects of added molecules.

Figure 1. How CARS microscopy works.

(a) Schematic diagram of the transitions involved for Raman spectroscopy (top) and CARS spectroscopy (bottom). Both the pump beam and the Stokes beam are required to obtain a CARS signal. (b) Schematic illustration showing linear Raman scattering of the probe beam (left) and nonlinear coherent anti-Stokes Raman scattering (right). (c) Raman spectra corresponding to various cellular components.

Figure 2. Applications of CARS microscopy.

Applications of CARS microscopy are shown for the label-free imaging of molecules in various systems. Clockwise from top left: (i) Lipid droplet detection: CARS image shows naïve Huh7 cells' lipid content. (ii) Monitoring host and viral factor localization and their influence on the lipid phenotype using immunofluorescence with CARS: CARS/TPF overlay image shows overexpressed liver protein CES1 that is involved in processing neutral lipids in lipid droplets and implicated in HCV replication and infection. (iii) Tracking chemical probes: CARS active functionalities provide direct detection of molecules, such as paclitaxel, label-free. (iv) Detection of lipid phenotype changes due to small molecules, or due to cellular processes, as shown by the schematic of mouse fibroblast differentiation into adipocytes. (v) Tissue imaging: image shows rabbit aorta (green, smooth muscle elastin; red, lipid droplets; blue, collagen); image reprinted with permission from ref. . (vi) Tracking dynamics of lipid droplets: image shows Alexa-488 labeling of tubulin with adjacent lipid droplets in cargo transport; in conjunction with CARS enables measurement of changes in lipid droplet dynamics. The scale bars for the cell images are 10 μm and 50 μm for the tissue image in (v).

Figure 3. Digestion of a glycerol trioleate droplet by porcine pancreatic lipase.

(a) Bright-field microscopy images. (b) False-color images obtained by CARS microspectroscopy showing glyceryl trioleate (red) and its lipolytic products (green). Scale bar = 5 μm. Reproduced with permission from ref. . Copyright 2010 American Chemical Society.

Figure 4. Identifying carboxylesterase 1 as a host factor involved in the hepatitis C virus life cycle.

(a,b) CES1 was discovered to be an important enzyme for HCV infection through the use of the fluorophosphonate activity–based protein profiling probe (a), following the general scheme in b. CES1 is a trimeric serine hydrolase enzyme that modifies lipid droplet content. (c) The enlargement of lipid droplets (red) is shown in Huh7.5 human hepatoma cell lines by the human protein CES1 (green). CES1 enzyme activity was shown to give rise to very large lipid droplets that both enhance HCV replication and favor HCV viral particle assembly.

Figure 5. Monitoring lipid storage in Caenorhabditis elegans.

(a) The schematic shows the advantages of CARS microscopy over fluorescence methods in imaging lipid droplets and lipid metabolism in C. elegans. Differences in lipid droplet distributions that can be observed by CARS microscopy and that are not possible to visualize by fluorescence in genetic mutants of C. elegans are represented schematically^,. (b–e) CARS microscopy images show differences in lipid droplet density for the wild-type C. elegans (b), the pha-3 mutant (c), the daf-2 (d) and the daf-4 (e) mutants. The scale bars are 10 μm. The images were reproduced with permission from ref. .

Figure 6. Tissue imaging using CARS microscopy.

(a,b) White matter from a rat brain. (a) CARS microscopy images of fixed myelin sheaths from the coronal plane of the lumbar spinal enlargement and (b) from a mechanically cut transverse section, both acquired with the excitation beams circularly polarized to remove polarization artifacts. Images courtesy of E. Bélanger, S. Laffray and D. Côté. (c–f) Label-free, lipid-selective CARS imaging of atherosclerotic lipids are shown for atherosclerotic lesions with four types of atherosclerotic lipids shown: (c) intracellular lipid droplets in foam cells, (d) needle-shaped lipid crystals, (e) extracellular lipid deposits and (f) plate-shape lipid crystals. The bright yellow color indicates CH bond–rich lipids. Images courtesy of S-H. Kim, J.Y. Lee, E.S. Lee, D.W. Moon.