Submit Article

Asian Journal of Physics Vol. 33, Nos 1 & 2 (2024) 1-14

Cellular lipid droplets observed in absorption, emission, and scattering – potential and limitations of various spectroscopic methods

Anna M Nowakowska1, Patrycja Dawiec1,2, Karolina Chrabąszcz3,1, Anna Pieczara2,4, Krzysztof Brzozowski1, Mariusz Kępczyński1, and Katarzyna Majzner1
1Department of Chemical Physics, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland
2Doctoral School of Exact and Natural Sciences, Jagiellonian University, Lojasiewicza 11, 30-348 Krakow, Poland
3The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Krakow, Poland
4Jagiellonian Centre for Experimental Therapeutics, Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland

Lipids are a primary source of long-term energy storage and essential in constructing cellular membranes. They also play an important role as mediators in signaling. At the subcellular level, lipids are stored in specialized structures known as lipid droplets (LDs). These droplets serve as dynamic organelles involved in lipid metabolism. LDs can be found in many types of cells, but their number, size, and composition vary greatly and depend on the type, metabolism, and condition of cells.

Tracking LDs in cells provides insights into lipid metabolism, and their presence is frequently linked to the development of diseases. Various imaging techniques, with fluorescence microscopy playing a leading role, are employed for their observation. Although, it allows sensitive detection of LDs in cells, it does not provide insight into their chemical structure. Its limitations include photobleaching and the limited number of dyes that can be detected simultaneously. For this reason, new methods are still being sought to provide better insight into LDs’ molecular structure and function. Among a group of techniques that provide insight into the composition of LDs are spectroscopic methods, such as infrared and Raman imaging.

In our investigation, we performed a multiparameter spectroscopic analysis of LDs in single endothelial and leukemic cells, comparing the results with classical fluorescence staining. Cells were incubated with an exogenous fatty acid, deuterated palmitic acid, to monitor the de novo formation of LDs. We employed three distinct spectroscopic techniques: infrared spectroscopy, spontaneous Raman imaging, and Stimulated Raman Spectroscopy, which were used to verify their efficacy in analyzing LDs in endothelial and leukemic cells. Our findings demonstrate the suitability of these techniques for tracing the chemical composition of LDs, enabling differentiation between newly formed LDs resulting from fatty acid uptake and endogenous lipids within the cell. Additionally, we highlight the advantages and limitations of spectroscopic techniques for studying LDs in individual cells, facilitating the selection of an appropriate tool for investigating lipid metabolism. © Anita Publications. All rights reserved.
Keywords: Lipid droplets, Raman imaging, SRS, Fluorescence, Endothelium, Leukemia, Deuterated palmitic acid.

Peer Review Information
Method: Single- anonymous; Screened for Plagiarism? Yes
Buy this Article in Print © Anita Publications. All rights reserve

References

  1. Maekawa M, Fairn G D, Molecular probes to visualize the location, organization, and dynamics of lipids. J Cell Sci, 127(2014)4801–4812.
  2. Olzmann J A, Carvalho P, Dynamics and functions of lipid droplets. Nat Rev Mol Cell Biol, 20(2019)137–155.
  3. Majzner K, Chlopicki S, Baranska M, Lipid droplets formation in human endothelial cells in response to polyunsaturated fatty acids and 1-methyl-nicotinamide (MNA); confocal Raman imaging and fluorescence microscopy studies, J Biophotonics, 9(2016)396–405.
  4. Murphy D J, Vance J, Mechanisms of lipid-body formation, Trends Biochem Sci, 24(1999)109–115.
  5. Guo Y, Cordes K R, Farese R V, Walther T C, Lipid droplets at a glance, J Cell Sci, 122(2009)749–752.
  6. Milger K, Herrmann T, Becker C, Gotthardt D, Zickwolf J, Ehehalt R, Watkins P A, Stremmel W, Füllekrug J, Cellular uptake of fatty acids driven by the ER-localized acyl-CoA synthetase FATP4, J Cell Sci, 119(2006)4678–4688.
  7. Murphy S, Martin S, Parton R G, Lipid droplet-organelle interactions; sharing the fats, Biochim Biophys Acta, 1791(2009)441–447.
  8. Ohsaki Y, Cheng J, Fujita A, Tokumoto T, Fujimoto T. Cytoplasmic lipid droplets are sites of convergence of proteasomal and autophagic degradation of apolipoprotein B, Mol Biol Cell, 17(2006)2674–2683.
  9. Bagatolli L A, Gratton E, Two Photon Fluorescence Microscopy of Coexisting Lipid Domains in Giant Unilamellar Vesicles of Binary Phospholipid Mixtures, Biophys J, 78(2000)290–305.
  10. Kuerschner L, Moessinger C, Thiele C, Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets, Traffic, 9(2008)338–352.
  11. Vinegoni C, Botnaru I, Aikawa E, Calfon M A, Iwamoto Y, Folco E J, Ntziachristos V, Weissleder R, Libby P, Jaffer F A, Indocyanine Green Enables Near-Infrared Fluorescence Imaging of Lipid-Rich, Inflamed Atherosclerotic Plaques, Sci Transl Med, 3(2011)84ra45, doi:10.1126/scitranslmed.3001577.
  12. Zheng X, Zhu W, Ni F, Ai H, Yang C, A specific bioprobe for super-resolution fluorescence imaging of lipid droplets, Sensors Actuators B Chem, 255(2018)3148–3154.
  13. Listenberger L L, Studer A M, Brown D A, Wolins N E, Fluorescent detection of lipid droplets and associated proteins, Curr Protoc Cell Biol, 2016;71:4.31.1-4.31.14; doi:10.1002/cpcb.7.
  14. Ohsaki Y, Shinohara Y, Suzuki M, Fujimoto T, A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy, Histochem Cell Biol, 133(2010)477–480; doi:10.1007/s00418-010-0678-x.
  15. Greenspan P, Mayer E P, Fowler S D, Nile red: A selective fluorescent stain for intracellular lipid droplets, J Cell Biol, 100(1985)965–973; doi:10.1083/jcb.100.3.965.
  16. DiDonato D, Brasaemle D L, Fixation methods for the study of lipid droplets by immunofluorescence microscopy, J Histochem Cytochem, 51(2003)773–780.
  17. Astanina K, Koch M, Jüngst C, Zumbusch A, Kiemer A K, Lipid droplets as a novel cargo of tunnelling nanotubes in endothelial cells, Sci Rep, 5(2015)1–13.
  18. Toman K, What are the disadventages and disadventages of fluorescence microscopy, In: Frieden T, ed. Toman’s Tuberculosis Case Detection, Treatment, and Monitoring – Questions and Answers, 2nd edn, (World Health Organization), 2004, pp 31–34.
  19. Ami D, Posteri R, Mereghetti P, Porro D, Doglia S M, Branduardi P, Fourier transform infrared spectroscopy as a method to study lipid accumulation in oleaginous yeasts, Biotechnol Biofuels, 7(2014):1-14; doi:10.1186/1754-6834-7-12.
  20. Kodali D R, Small D M, Powell J, Krishnan K, Infrared Micro-Imaging of Atherosclerotic Arteries, Appl Spectrosc, 45(1991)1310–1317.
  21. Czamara K, Majzner K, Pacia M Z, Kochan K, Kaczor A, Baranska M, Raman spectroscopy of lipids: a review. J Raman Spectrosc, 46(2015)4–20.
  22. Freudiger C W, Min W, Saar B G, Lu S, Holtom G R, He C, Tsai J C, Kang J X, Xie X S, Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy, Science, 322(2008)1857–1861.
  23. Kochan K, Maslak E, Krafft C, Kostogrys R, Chlopicki S, Baranska M, Raman spectroscopy analysis of lipid droplets content, distribution and saturation level in Non-Alcoholic Fatty Liver Disease in mice, J Biophotonics, 8(2015)597–609.
  24. Tirinato L, Liberale C, Di Franco S, Candeloro P, Benfante A, Rocca R L, Potze L, Marotta R, Ruffilli R, Rajamanickam V P, Malerba M, Angelis F D, Falqui A, Carbone E, Todaro M, Medema J P, Stassi G, Fabrizio E D, Lipid droplets: A new player in colorectal cancer stem cells unveiled by spectroscopic imaging, Stem Cells, 33(2015)35–44.
  25. Jaeger D, Pilger C, Hachmeister H, Oberländer E, Wördenweber R, Wichmann J, Mussgnug J H, Huser T, Kruse O, Label-free in vivo analysis of intracellular lipid droplets in the oleaginous microalga Monoraphidium neglectum by coherent Raman scattering microscopy, Sci Rep, 6(2016)35340; doi:10.1038/srep35340.
  26. Baena J R, Lendl B, Raman spectroscopy in chemical bioanalysis, Curr Opin Chem Biol, 8(2004)534–539.
  27. He XN, Allen J, Black PN, T Baldacchini, X Huang, H Huang, L Jiang, YF Lu, Coherent anti-Stokes Raman scattering and spontaneous Raman spectroscopy and microscopy of microalgae with nitrogen depletion, Biomed Opt Express, 3(2012)2896; doi:10.1364/boe.3.002896
  28. Ferrara M A, Filograna A, Ranjan R, Corda D, Valente C, Sirleto L, Three-dimensional label-free imaging throughout adipocyte differentiation by stimulated Raman microscopy, PLoS One, 14(2019)e02196811; doi:10.1371/journal.pone.0216811
  29. Zhang C, Li J, Lan L, Cheng J X, Quantification of Lipid Metabolism in Living Cells through the Dynamics of Lipid Droplets Measured by Stimulated Raman Scattering Imaging, Anal Chem, 89(2017)4502–4507.
  30. Huang K C, Li J, Zhang C, Tan Y, Cheng J X, Multiplex Stimulated Raman Scattering Imaging Cytometry Reveals Lipid-Rich Protrusions in Cancer Cells under Stress Condition. iScience, 23(2020)100953; doi:10.1016/j.isci.2020.100953.
  31. Jia H, Yue S, Stimulated Raman Scattering Imaging Sheds New Light on Lipid Droplet Biology, J Phys Chem B, 127(2023)2381–2394.
  32. Tatenaka Y, Kato H, Ishiyama M, Sasamoto K, Shiga M, Nishitoh H, Ueno Y, Monitoring Lipid Droplet Dynamics in Living Cells by Using Fluorescent Probes, Biochemistry, 58(2019)499–503.
  33. Zhao Y, Shi W, Li X, Ma H, Recent advances in fluorescent probes for lipid droplets, Chem Commun, 58(2022)1495–1509.
  34. Neher R, Neher E, Optimizing imaging parameters for the separation of multiple labels in a fluorescence image, J Microsc, 213(2004)46–62.
  35. Jensen E C, Use of Fluorescent Probes: Their Effect on Cell Biology and Limitations, Anat Rec, 295(2012)2031–2036.
  36. Marzec K M, Rygula A, Gasior-Glogowska M, Kochan K, Czamara K, Bulat K, Malek K, Kaczor A, Baranska M, Vascular diseases investigated ex vivo by using Raman, FT-IR and complementary methods, Pharmacol Reports, 67(2015)744–750.
  37. Chrabaszcz K, Kochan K, Fedorowicz A, Jasztal A, Buczek E, Leslie L S, Bhargava R, Malek K, Chlopicki S, Marzec K M, FT-IR- and Raman-based biochemical profiling of the early stage of pulmonary metastasis of breast cancer in mice, Analyst, 143(2018)2042–2050.
  38. Blat A, Dybas J, Chrabaszcz K, Bulat K, Jasztal A, Kaczmarska M, Pulyk R, Popiela T, Slowik A, Malek K, Adamski M G, Katarzyna M, FTIR, Raman and AFM characterization of the clinically valid biochemical parameters of the thrombi in acute ischemic stroke, Sci Rep, 9(2019)15475; doi:10.1038/s41598-019-51932-0.
  39. Chrabaszcz K, Jasztal A, Smęda M, Zieliński B, Blat A, Diem M, Chlopicki S, Malek K, Marzec K M, Label-free FTIR spectroscopy detects and visualizes the early stage of pulmonary micrometastasis seeded from breast carcinoma. Biochim Biophys Acta – Mol Basis Dis, 1864 (2018)3574–3584.
  40. Kochan K, Kus E, Szafraniec E, Wislocka A, Chlopicki S, Baranska M, Changes induced by non-alcoholic fatty liver disease in liver sinusoidal endothelial cells and hepatocytes: Spectroscopic imaging of single live cells at the subcellular level, Analyst, 142(2017)3948–3958.
  41. Wiercigroch E, Staniszewska-Slezak E, Szkaradek K, Wojcik T, Ozaki Y, Baranska M, Malek K, FT-IR Spectroscopic Imaging of Endothelial Cells Response to Tumor Necrosis Factor-α: To Follow Markers of Inflammation Using Standard and High-Magnification Resolution, Anal Chem, 90(2018)3727–3736.
  42. Majzner K, Kochan K, Kachamakova-Trojanowska N, Maslak E, Chlopicki S, Baranska M, Raman imaging providing insights into chemical composition of lipid droplets of different size and origin: In hepatocytes and endothelium, Anal Chem, 86(2014)6666–6674.
  43. Czamara K, Majzner K, Pacia M Z, Kochan K, Kaczor A, Baranska M, Raman spectroscopy of lipids: A review. J Raman Spectrosc, 46(2015)4–20.
  44. Szafraniec E, Kus E, Wislocka A, Kukla B, Sierka E, Untereiner V, Sockalingum GD, Chlopicki S, Baranska M, Raman spectroscopy–based insight into lipid droplets presence and contents in liver sinusoidal endothelial cells and hepatocytes, J Biophotonics, 12(2019); doi:10.1002/jbio.201800290.
  45. Bik E, Ishigaki M, Blat A, Jasztal A, Ozaki Y, Malek K, Baranska M. Lipid droplet composition varies based on medaka fish eggs development as revealed by NIR-, MiR-, and Raman imaging, Molecules, 25(2020)817; doi:10.3390/molecules25040817.
  46. Majzner K, Chlopicki S, Baranska M, Lipid droplets formation in human endothelial cells in response to polyunsaturated fatty acids and 1-methyl-nicotinamide (MNA); confocal Raman imaging and fluorescence microscopy studies, J Biophotonics, 9(2016)396–405.
  47. Czamara K, Majzner K, Selmi A, Baranska M, Ozaki Y, Kaczor A, Unsaturated lipid bodies as a hallmark of inflammation studied by Raman 2D and 3D microscopy, Sci Rep, 7(2017)40889; doi.10.1038/srep40889.
  48. Borek-Dorosz A, Nowakowska A M, Leszczenko P, Adamczyk A, Pieczara A, Jakubowska J, Pastorczak A, Ostrowska K, Ząbczyńska M, Sowinski K, Gruszecki W I, Baranska M, Marzec K M, Majzner K, Raman-based spectrophenotyping of the most important cells of the immune system, J Adv Res, 41(2022)191–203.
  49. Brzozowski K, Matuszyk E, Pieczara A, Firlej J, Nowakowska A M, Baranska M, Stimulated Raman Scattering Microscopy in Chemistry and Life Science – Development, Innovation, Perspectives, Biotechnol Adv, 60(2022) 108003; doi.org/10.1016/j.biotechadv.2022.108003.
Cellular lipid droplets observed in absorption, emissionDownload

Related Posts