Label‐Free Biomedical Imaging of Lipids by Stimulated Raman Scattering Microscopy

Prasanna V. Ramachandran1, Ayse Sena Mutlu1, Meng C. Wang2

1 These authors contributed equally to this work, 2 Corresponding author (wmeng@bcm.edu)
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 30.3
DOI:  10.1002/0471142727.mb3003s109
Online Posting Date:  January, 2015
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Abstract

Advances in modern optical microscopy have provided unparalleled tools to study intracellular structure and function, yet visualizing lipid molecules within a cell remains challenging. Stimulated Raman Scattering (SRS) microscopy is a recently developed imaging modality that addresses this challenge. By selectively imaging the vibration of chemical moieties enriched in lipids, this technique allows for rapid imaging of lipid molecules in vivo without the need for perturbative extrinsic labels. SRS microscopy has been effectively employed in the study of fat metabolism, helping uncover novel regulators of lipid storage. This unit provides a brief introduction to the principle of SRS microscopy, and describes methods for its use in imaging lipids in cells, tissues, and whole organisms. © 2015 by John Wiley & Sons, Inc.

Keywords: lipid; lipid droplets; metabolism; stimulated Raman scattering; microscopy; C. elegans; cell culture; tissue section

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Instrumental Setup for Stimulated Raman Scattering (SRS) Microscopy
  • Support Protocol 1: Preparation of C. elegans Samples for SRS Imaging
  • Support Protocol 2: Preparation of Cultured Cells for Live‐Cell Imaging by SRS Microscopy
  • Support Protocol 3: Preparation of Frozen Tissue Sections for Imaging by SRS Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Instrumental Setup for Stimulated Raman Scattering (SRS) Microscopy

  Materials
  • Sample for scanning (see Support Protocols protocol 21, protocol 32, and protocol 43)
  • Dodecane: CH 3(CH 2) 10CH 3 (Sigma‐Aldrich, cat. no. 44010)
  • Glass microscope slide
  • Glass coverslips
  • Immersion oil (if oil‐immersion lens is used on microscope)
  • Bright‐field microscope
  • Equipment for SRS microscopy (see Equipment under the introduction to this protocol):
    • Laser system and associated components
    • Laser‐scanning microscope
    • Mirror and filter sets
    • Detector
    • Lock‐in amplifier
    • A/D converter
  • Software for imaging (e.g., Fluoview from Olympus) and quantification (ImageJ from NIH; http://imagej.nih.gov/ij/download.html)

Support Protocol 1: Preparation of C. elegans Samples for SRS Imaging

  Materials
  • Agarose (GeneMate, cat. no. 3119)
  • C. elegans (Caenorhabditis Genetics Center, University of Minnesota)
  • M9 buffer (see recipe)
  • Anesthetizing agent (sodium azide or levamisole)
  • Heat block
  • General‐purpose laboratory labeling tape
  • Glass slides and coverslips
  • Glass Pasteur pipets and pipet bulbs
  • Dissection microscope
  • Worm picker (platinum wire fixed at the tip of a Pasteur pipet; optional)

Support Protocol 2: Preparation of Cultured Cells for Live‐Cell Imaging by SRS Microscopy

  Materials
  • Cells of interest
  • Appropriate growth medium
  • Uncoated glass‐bottom dishes (MatTek, cat. no. P35G‐1.0‐14‐C)
  • Upright or inverted microscope with 60× water objective
  • Water condenser

Support Protocol 3: Preparation of Frozen Tissue Sections for Imaging by SRS Microscopy

  Materials
  • Isopentane (2‐methyl‐butane)
  • Liquid nitrogen
  • Dry ice
  • Tissue sample
  • Tissue freezing medium (Triangle Biomedical Sciences)
  • Gum tragacanth (Sigma‐Aldrich)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Metal beaker
  • Liquid nitrogen canister
  • Petri dish
  • Cryomolds
  • Long metal forceps
  • Cryostat (Leica CM3000)
  • Treated glass slides (e.g., Superfrost Plus Sides from VMR)
  • Coverslips
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Figures

Videos

Literature Cited

Literature Cited
  Evans, C.L. and Xie, X.S. 2008. Coherent anti‐stokes Raman scattering microscopy: Chemical imaging for biology and medicine. Annu. Rev. Anal. Chem. 1:883‐909.
  Folick, A., Min, W., and Wang, M.C. 2011. Label‐free imaging of lipid dynamics using coherent anti‐stokes Raman scattering (CARS) and stimulated Raman scattering (SR). Curr. Opin. Genet. Dev. 21:585‐590.
  Freudiger, C.W., Min, W., Saar, B.G., Lu, S., Holtom, G.R., He, C., Tsai, J.C., Kang, J.X., and Xie, X.S. 2008. Label‐free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 322:1857‐1861.
  Fu, D., Yu, Y., Folick, A., Currie, E., Farese, R.V., Jr., Tsai, T.H., Xie, X.S., and Wang, M.C. 2014. In vivo metabolic fingerprinting of neutral lipids with hyperspectral stimulated Raman scattering microscopy. J. Am. Chem. Soc. 136:8820‐8828.
  Helmchen, F. and Denk, W. 2005. Deep tissue two‐photon microscopy. Nat. Methods 2:932‐940.
  Ji, M., Orringer, D.A., Freudiger, C.W., Ramkissoon, S., Liu, X., Lau, D., Golby, A.J., Norton, I., Hayashi, M., Agar, N.Y., Young, G.S., Spino, C., Santagata, S., Camelo‐Piragua, S., Ligon, K.L., Sagher, O., and Xie, X.S. 2013. Rapid, label‐free detection of brain tumors with stimulated Raman scattering microscopy. Sci. Transl. Med. 5:201ra119.
  Lawrence, V.J. and Kopelman, P.G. 2004. Medical consequences of obesity. Clin. Dermatol. 22:296‐302.
  Min, W., Freudiger, C.W., Lu, S., and Xie, X.S. 2011. Coherent nonlinear optical imaging: Beyond fluorescence microscopy. Annu. Rev. Phys. Chem. 62:507‐530.
  Ogden, C.L., Carroll, M.D., Kit, B.K., and Flegal, K.M. 2014. Prevalence of childhood and adult obesity in the United States, 2011‐2012. JAMA 311:806‐814.
  Raman, C.V. 1928. A new radiation. Ind. J. Phys. 2:387‐398.
  Saar, B.G., Freudiger, C.W., Reichman, J., Stanley, C.M., Holtom, G.R., and Xie, X.S. 2010. Video‐rate molecular imaging in vivo with stimulated Raman scattering. Science 330:1368‐1370.
  Scouten, C.W. and Cunningham, M. 2012. Freezing Biological Samples. Leica Biosystems. Available at http://www.leicabiosystems.com/pathologyleaders/freezing‐biological‐samples/.
  Thomas, G.J. Jr. 1999. Raman spectroscopy of protein and nucleic acid assemblies. Annu. Rev. Biophys. Biomol. Struct. 28:1‐27.
  Wang, M.C., Min, W., Freudiger, C.W., Ruvkun, G., and Xie, X.S. 2011. RNAi screening for fat regulatory genes with SRS microscopy. Nat. Methods 8:135‐138.
  Wei, L., Yu, Y., Shen, Y., Wang, M.C., and Min, W. 2013. Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy. Proc. Natl. Acad. Sci. U.S.A. 110:11226‐11231.
  Wei, L., Hu, F., Shen, Y., Chen, Z., Yu, Y., Lin, C.C., Wang, M.C., and Min, W. 2014. Live‐cell imaging of alkyne‐tagged small biomolecules by stimulated Raman scattering. Nat. Methods 11:410‐412.
  Yen, K., Le, T.T., Bansal, A., Narasimhan, S.D., Cheng, J.X., and Tissenbaum, H.A. 2010. A comparative study of fat storage quantitation in nematode Caenorhabditis elegans using label and label‐free methods. PloS One 5:e12810.
  Yu, Y., Ramachandran, P.V., and Wang, M.C. 2014. Shedding new light on lipid functions with CARS and SRS microscopy. Biochim. Biophys. Acta 1841:1120‐1129.
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