Advertisement

Histochemistry and Cell Biology

, Volume 141, Issue 3, pp 263–273 | Cite as

Chemical imaging of lipid droplets in muscle tissues using hyperspectral coherent Raman microscopy

  • Nils Billecke
  • Gianluca Rago
  • Madeleen Bosma
  • Gert Eijkel
  • Anne Gemmink
  • Philippe Leproux
  • Guillaume Huss
  • Patrick Schrauwen
  • Matthijs K. C. Hesselink
  • Mischa Bonn
  • Sapun H. Parekh
Original Paper

Abstract

The accumulation of lipids in non-adipose tissues is attracting increasing attention due to its correlation with obesity. In muscle tissue, ectopic deposition of specific lipids is further correlated with pathogenic development of insulin resistance and type 2 diabetes. Most intramyocellular lipids are organized into lipid droplets (LDs), which are metabolically active organelles. In order to better understand the putative role of LDs in pathogenesis, insight into both the location of LDs and nearby chemistry of muscle tissue is very useful. Here, we demonstrate the use of label-free coherent anti-Stokes Raman scattering (CARS) microscopy in combination with multivariate, chemometric analysis to visualize intracellular lipid accumulations in ex vivo muscle tissue. Consistent with our previous results, hyperspectral CARS microscopy showed an increase in LDs in tissues where LD proteins were overexpressed, and further chemometric analysis showed additional features morphologically (and chemically) similar to mitochondria that colocalized with LDs. CARS imaging is shown to be a very useful method for label-free stratification of ectopic fat deposition and cellular organelles in fresh tissue sections with virtually no sample preparation.

Keywords

Lipid droplet Microscopy Chemical imaging Raman spectroscopy Multivariate analysis Hyperspectral 

Notes

Acknowledgments

This study was financially supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organization for the Advancement of Research) (M.B., G.R.), the NanoNextNL, a micro and nanotechnology consortium of the Government of the Netherlands and 130 partners (N.B., M.B., and M.K.C.L.), and a Marie Curie Foundation grant #CIG322284 (S.H.P). Ma.B. was financially supported by NUTRIM and the Graduate School VLAG. A Vici (Grant 918.96.618) grant for innovative research from the Netherlands Organization for Scientific Research supports the work of P.S.

G.R. and N.B. performed CARS experiments. P.L. and G.H. helped construct the experimental system. GR and GE did the multivariate analysis using HCA and PCA. A.G., Ma.B., and N.B. performed the fluorescence imaging. N.B., Ma.B., M.K.C.H., and P.S. provided samples. G.R., N.B., M.B., and S.H.P. wrote the paper. M.B. and S.H.P. supervised the research. All authors contributed to discussion of the results and revision of the paper. The authors wish to thank Dr. E. Cánovas and Dr. W. Rock for stimulating discussions and technical support.

Supplementary material

418_2013_1161_MOESM1_ESM.docx (434 kb)
Supplementary material 1 (DOCX 433 kb)

References

  1. Aguer C, Mercier J, Man CYW, Metz L, Bordenave S, Lambert K, Jean E, Lantier L, Bounoua L, Brun JF, de Mauverger ER, Andreelli F, Foretz M, Kitzmann M (2010) Intramyocellular lipid accumulation is associated with permanent relocation ex vivo and in vitro of fatty acid translocase (FAT)/CD36 in obese patients. Diabetologia 53(6):1151–1163. doi: 10.1007/s00125-010-1708-x PubMedCrossRefGoogle Scholar
  2. Bosma M, Minnaard R, Sparks LM, Schaart G, Losen M, de Baets MH, Duimel H, Kersten S, Bickel PE, Schrauwen P, Hesselink MKC (2012) The lipid droplet coat protein perilipin 5 also localizes to muscle mitochondria. Histochem Cell Biol 137(2):205–216. doi: 10.1007/s00418-011-0888-x PubMedCentralPubMedCrossRefGoogle Scholar
  3. Boxer SG, Kraft ML, Weber PK (2009) Advances in imaging secondary ion mass spectrometry for biological samples. Annu Rev Biophys 38(1):53–74. doi: 10.1146/annurev.biophys.050708.133634 PubMedCrossRefGoogle Scholar
  4. Centonze VE, White JG (1998) Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J 75(4):2015–2024PubMedCentralPubMedCrossRefGoogle Scholar
  5. Chavez JA, Knotts TA, Wang LP, Li G, Dobrowsky RT, Florant GL, Summers SA (2003) A role for ceramide, but not diacylglycerol, in the antagonism of insulin signal transduction by saturated fatty acids. J Biol Chem 278(12):10297–10303. doi: 10.1074/jbc.M212307200 PubMedCrossRefGoogle Scholar
  6. Day JPR, Rago G, Domke KF, Velikov KP, Bonn M (2010) Label-free imaging of lipophilic bioactive molecules during lipid digestion by multiplex coherent anti-Stokes Raman scattering microspectroscopy. J Am Chem Soc 132(24):8433–8439. doi: 10.1021/ja102069d PubMedCrossRefGoogle Scholar
  7. Fletcher JS, Vickerman JC (2013) Secondary ion mass spectrometry: characterizing complex samples in two and three dimensions. Anal Chem 85(2):610–639. doi: 10.1021/Ac303088m PubMedCrossRefGoogle Scholar
  8. Gawlik KI, Durbeej M (2011) Skeletal muscle laminin and MDC1A: pathogenesis and treatment strategies. Skelet Muscle 1(1):9. doi: 10.1186/2044-5040-1-9 PubMedCentralPubMedCrossRefGoogle Scholar
  9. Goodpaster BH, Theriault R, Watkins SC, Kelley DE (2000) Intramuscular lipid content is increased in obesity and decreased by weight loss. Metab Clin Exp 49(4):467–472. doi: 10.1016/s0026-0495(00)80010-4 PubMedCrossRefGoogle Scholar
  10. Goodpaster BH, He J, Watkins S, Kelley DE (2001) Skeletal muscle lipid content and insulin resistance: evidence for a paradox in endurance-trained athletes. J Clin Endocrinolo Metab 86(12):5755–5761CrossRefGoogle Scholar
  11. Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2(12):932–940. doi: 10.1038/nmeth818 PubMedCrossRefGoogle Scholar
  12. Lee D-E, Kehlenbrink S, Lee H, Hawkins M, Yudkin JS (2009) Getting the message across: mechanisms of physiological cross talk by adipose tissue. Am J Physiol Endocrinol Metab 296(6):E1210–E1229. doi: 10.1152/ajpendo.00015.2009 PubMedCrossRefGoogle Scholar
  13. Liu Y, Lee YJ, Cicerone MT (2009) Broadband CARS spectral phase retrieval using a time-domain Kramers-Kronig transform. Opt Lett 34(9):1363–1365PubMedCrossRefGoogle Scholar
  14. Matthaus C, Chernenko T, Newmark JA, Warner CM, Diem M (2007) Label-free detection of mitochondrial distribution in cells by nonresonant Raman microspectroscopy. Biophys J 93(2):668–673. doi: 10.1529/biophysj.106.102061 PubMedCentralPubMedCrossRefGoogle Scholar
  15. Matthaus C, Krafft C, Dietzek B, Brehm BR, Lorkowski S, Popp J (2012) Noninvasive imaging of intracellular lipid metabolism in macrophages by Raman Microscopy in combination with stable isotopic labeling. Anal Chem 84(20):8549–8556. doi: 10.1021/ac3012347 PubMedCrossRefGoogle Scholar
  16. Nielsen J, Mogensen M, Vind BF, Sahlin K, Hojlund K, Schroder HD, Ortenblad N (2010) Increased subsarcolemmal lipids in type 2 diabetes: effect of training on localization of lipids, mitochondria, and glycogen in sedentary human skeletal muscle. Am J Physiol Endocrinol Metab 298(3):E706–E713. doi: 10.1152/ajpendo.00692.2009 PubMedCrossRefGoogle Scholar
  17. Pohling C, Buckup T, Motzkus M (2011) Hyperspectral data processing for chemoselective multiplex coherent anti-Stokes Raman scattering microscopy of unknown samples. J Biomed Opt 16(2). doi: 10.1117/1.3533309
  18. Pohling C, Buckup T, Pagenstecher A, Motzkus M (2011b) Chemoselective imaging of mouse brain tissue via multiplex CARS microscopy. Biomed Optics Express 2(8):2110–2116. doi: 10.1364/BOE.2.002110 CrossRefGoogle Scholar
  19. Puppels GJ, Demul FFM, Otto C, Greve J, Robertnicoud M, Arndtjovin DJ, Jovin TM (1990) Studying single living cells and chromosomes by confocal Raman microspectroscopy. Nature 347(6290):301–303. doi: 10.1038/347301a0 PubMedCrossRefGoogle Scholar
  20. Rinia HA, Burger KNJ, Bonn M, Muller M (2008) Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy. Biophys J 95(10):4908–4914. doi: 10.1529/biophysj.108.137737 PubMedCentralPubMedCrossRefGoogle Scholar
  21. Rompp A, Spengler B (2013) Mass spectrometry imaging with high resolution in mass and space. Histochem Cell Biol 139(6):759–783. doi: 10.1007/s00418-013-1097-6 PubMedCentralPubMedCrossRefGoogle Scholar
  22. Seppanen-Laakso T, Laakso I, Hiltunen R (2002) Analysis of fatty acids by gas chromatography, and its relevance to research on health and nutrition. Anal Chim Acta 465(1–2):39–62. doi: 10.1016/s0003-2670(02)00397-5 CrossRefGoogle Scholar
  23. Shaw CS, Jones DA, Wagenmakers AJ (2008) Network distribution of mitochondria and lipid droplets in human muscle fibres. Histochem Cell Biol 129(1):65–72. doi: 10.1007/s00418-007-0349-8 PubMedCrossRefGoogle Scholar
  24. Soboll S SR, Freisl M, Elbers R, Heldt HW (1976). In: JM Tager HS, JR Williamson (ed) Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies, North-Holland, Amsterdam and Oxford, pp 29–40Google Scholar
  25. Sollner TH (2007) Lipid droplets highjack SNAREs. Nat Cell Biol 9(11):1219–1220. doi: 10.1038/ncb1107-1219 PubMedCrossRefGoogle Scholar
  26. Spangenburg EE, Pratt SJP, Wohlers LM, Lovering RM (2011) Use of BODIPY (493/503) to Visualize Intramuscular Lipid Droplets in Skeletal Muscle. J Biomed Biotechnol. doi: 10.1155/2011/598358 PubMedCentralPubMedGoogle Scholar
  27. Srere PA (1980) The infrastructure of the mitochondrial matrix. Trends in biochemical sciences 5(5):120–121. doi:http://dx.doi.org/10.1016/0968-0004(80)90051-1
  28. Stratford S, Hoehn KL, Liu F, Summers SA (2004) Regulation of insulin action by ceramide: dual mechanisms linking ceramide accumulation to the inhibition of Akt/protein kinase B. J Biol Chem 279(35):36608–36615. doi: 10.1074/jbc.M406499200 PubMedCrossRefGoogle Scholar
  29. Tolles WM, Nibler JW, McDonald JR, Harvey AB (1977) Review of theory and application of coherent anti-stokes Raman-spectroscopy (CARS). Appl Spectrosc 31(4):253–271. doi: 10.1366/000370277774463625 CrossRefGoogle Scholar
  30. Vaandrager AB, Testerink N, Ajat M, Houweling M, Brouwers J, Pully VV, van Manen HWJ, Otto C, Helms JB (2009) Raman imaging and lipidomic analysis of lipid droplets in (activated) hepatic stellate cells. Chem Phys Lipids 160:S7–S8. doi: 10.1016/j.chemphyslip.2009.06.109 CrossRefGoogle Scholar
  31. van Manen HJ, Kraan YM, Roos D, Otto C (2005) Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes. Proc Natl Acad Sci USA 102(29):10159–10164. doi: 10.1073/pnas.0502746102 PubMedCrossRefGoogle Scholar
  32. Vartiainen EM, Rinia HA, Muller M, Bonn M (2006) Direct extraction of Raman line-shapes from congested CARS spectra. Opt Express 14(8):3622–3630. doi: 10.1364/oe.14.003622 PubMedCrossRefGoogle Scholar
  33. Wang H, Zhao J, Lee AM, Lui H, Zeng H (2012) Improving skin Raman spectral quality by fluorescence photobleaching. Photodiagn Photodyn Ther 9(4):299–302. doi: 10.1016/j.pdpdt.2012.02.001 Google Scholar
  34. Weigert R, Sramkova M, Parente L, Amornphimoltham P, Masedunskas A (2010) Intravital microscopy: a novel tool to study cell biology in living animals. Histochem Cell Biol 133(5):481–491. doi: 10.1007/s00418-010-0692-z PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Nils Billecke
    • 1
  • Gianluca Rago
    • 1
    • 2
    • 3
  • Madeleen Bosma
    • 4
  • Gert Eijkel
    • 2
  • Anne Gemmink
    • 5
  • Philippe Leproux
    • 6
    • 7
  • Guillaume Huss
    • 6
  • Patrick Schrauwen
    • 4
  • Matthijs K. C. Hesselink
    • 5
  • Mischa Bonn
    • 1
  • Sapun H. Parekh
    • 1
  1. 1.Molecular Spectroscopy DepartmentMax Plank Institute for Polymer ResearchMainzGermany
  2. 2.FOM Institute AMOLFAmsterdamThe Netherlands
  3. 3.Deloitte Consulting B.V.AmstelveenThe Netherlands
  4. 4.Department of Human Biology, School for Nutrition, Toxicology and MetabolismMaastricht University Medical CenterMaastrichtThe Netherlands
  5. 5.Department of Movement Sciences, School for Nutrition, Toxicology and MetabolismMaastricht University Medical CenterMaastrichtThe Netherlands
  6. 6.Leukos Innovative Optical SystemsESTER TechnopoleLimoges CedexFrance
  7. 7.Xlim Research InstituteCNRS-University of LimogesLimoges CedexFrance

Personalised recommendations