Biophysical Reviews

, Volume 4, Issue 4, pp 323–334 | Cite as

Nonlinear optical microscopy in decoding arterial diseases

  • Alex C.-T. Ko
  • Andrew Ridsdale
  • Leila B. Mostaço-Guidolin
  • Arkady Major
  • Albert Stolow
  • Michael G. Sowa


Pathological understanding of arterial diseases is mainly attributable to histological observations based on conventional tissue staining protocols. The emerging development of nonlinear optical microscopy (NLOM), particularly in second-harmonic generation, two-photon excited fluorescence and coherent Raman scattering, provides a new venue to visualize pathological changes in the extracellular matrix caused by atherosclerosis progression. These techniques in general require minimal tissue preparation and offer rapid three-dimensional imaging. The capability of label-free microscopic imaging enables disease impact to be studied directly on the bulk artery tissue, thus minimally perturbing the sample. In this review, we look at recent progress in applications related to arterial disease imaging using various forms of NLOM.


Atherosclerosis Nonlinear optical microscopy Second-harmonic generation Two-photon excited fluorescence Coherent anti-Stokes Raman Artery 


Conflicts of interest



  1. Barad Y, Eisenberg H, Horowitz M, Silberberg Y (1997) Nonlinear scanning laser microscopy by third harmonic generation. Appl Phys Lett 70(8):922–924CrossRefGoogle Scholar
  2. Barlis P, Schmitt JM (2009) Current and future developments in intracoronary optical coherence tomography imaging. EuroIntervention 4(4):529–533PubMedCrossRefGoogle Scholar
  3. Barlis P, Serruys PW, Devries A, Regar E (2008a) Optical coherence tomography assessment of vulnerable plaque rupture: predilection for the plaque ‘shoulder’. Eur Heart J 29(16):2023PubMedCrossRefGoogle Scholar
  4. Barlis P, Ferrante G, Del Furia F, Di Mario C (2008b) In-vivo characterisation of coronary atherosclerosis with optical coherence tomography. Med J Aust 188(12):728PubMedGoogle Scholar
  5. Boulesteix T, Pena AM, Pages N, Godeau G, Sauviat MP, Beaurepaire E, Schanne-Klein MC (2005) Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure. Cytometry A 69A:20–26Google Scholar
  6. Delaney P, Harris M (2006) Fiber-optics in scanning optical microscopy. In: Pawley JB (ed) Handbook of biological confocal microscopy, 3rd edn. Springer, New York, pp 501–515CrossRefGoogle Scholar
  7. Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248(4951):73–76PubMedCrossRefGoogle Scholar
  8. Doras C, Taupier G, Barsella A, Mager L, Boeglin A, Bulou H, Bousquet P, Dorkenoo KD (2011) Polarization state studies in second harmonic generation signals to trace atherosclerosis lesions. Opt Express 19:15062–15068PubMedCrossRefGoogle Scholar
  9. Dudovich N, Oron D, Silberberg Y (2002) Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy. Nature 418:512–514PubMedCrossRefGoogle Scholar
  10. Duncan MD, Reintjes J, Manuccia TJ (1982) Scanning coherent anti-Stokes Raman microscope. Opt Lett 7(8):350–352PubMedCrossRefGoogle Scholar
  11. Freudiger CW, Min W, Saar BG, Lu S, Holtom GR, He C, Tsai JC, Kang JX, Xie XS (2008) Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 322:1857–1861PubMedCrossRefGoogle Scholar
  12. Ganikhanov F, Carrasco S, Xie XS, Katz M, Seitz W, Kopf D (2006) Broadly tunable dual-wavelength light source for coherent anti-Stokes Raman scattering microscopy. Opt Lett 31:1292–1294PubMedCrossRefGoogle Scholar
  13. Hansson GK (2005) Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 352(16):1685–1695PubMedCrossRefGoogle Scholar
  14. Hodgson JM, Reddy KG, Suneja R, Nair RN, Lesnefsky EJ, Sheehan HM (1993) Intracoronary ultrasound imaging: correlation of plaque morphology with angiography, clinical syndrome and procedural results in patients undergoing coronary angioplasty. J Am Coll Cardiol 21(1):35–44PubMedCrossRefGoogle Scholar
  15. Jang IK, Tearney GJ, MacNeill B, Takano M, Moselewski F, Iftima N, Shishkov M, Houser ST, Aretz H, Halpern EF, Bouma BE (2005) In vivo characterization of coronary atherosclerotic plaque by use of optical coherence tomography. Circulation 111(12):1551–1555PubMedCrossRefGoogle Scholar
  16. Jo JA, Fang Q, Papaioannou T, Baker JD, Dorafshar AH, Reil T, Qiao JH, Fishbein MC, Freischlag JA, Marcu L (2006) Laguerre-based method for analysis of time-resolved fluorescence data: Application to in-vivo characterization and diagnosis of atherosclerotic lesions. J Biomed Opt 11(2):021004PubMedCrossRefGoogle Scholar
  17. Kim S-H, Lee E-S, Lee JY, Lee ES, Lee BS, Park JE, Moon DW (2010) Multiplex coherent anti-stokes raman spectroscopy images intact atheromatous lesions and concomitantly identifies distinct chemical profiles of atherosclerotic lipids. Circ Res 106:1332–1341PubMedCrossRefGoogle Scholar
  18. Ko ACT, Ridsdale A, Smith MSD, Mostaço-Guidolin LB, Hewko MD, Pegoraro AF, Kohlenberg EK, Schattka B, Shiomi M, Stolow A, Sowa MG (2010) Multimodal nonlinear optical imaging of atherosclerotic plaque development in myocardial infarction-prone rabbits. J Biomed Opt 15(2):020501PubMedCrossRefGoogle Scholar
  19. Le TT, Langohr IM, Locker MJ, Sturek M, Cheng J-X (2007) Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy. J Biomed Opt 12(5):054007PubMedCrossRefGoogle Scholar
  20. Lee JY, Kim SH, Moon DV, Lee ES (2009) Three-color multiplex CARS for fast imaging and microspectroscopy in the entire CH stretching vibrational region. Opt Express 17(25):22281PubMedCrossRefGoogle Scholar
  21. Libby P (2006) Atherosclerosis: disease biology affecting the coronary vasculature. Am J Cardiol 98(12):S3–S9CrossRefGoogle Scholar
  22. Lilledahl MB, Haugen OA, Davies de Lange C, Svaasand LO (2007) Characterization of vulnerable plaques by multiphoton microscopy. J Biomed Opt 12(4):044005PubMedCrossRefGoogle Scholar
  23. Maffia P, Zinselmeyer BH, Ialenti A, Kennedy S, Baker AH, McInnes IB, Brewer JM, Garside P (2007) Into apolipoprotein-E–deficient mouse carotid artery multiphoton microscopy for 3-dimensional imaging of lymphocyte recruitment. Circulation 115:e326–e328PubMedCrossRefGoogle Scholar
  24. Major A, Sandkuijl D, Barzda V (2009) Efficient frequency doubling of a femtosecond Yb:KGW laser in a BiB3O6 crystal. Opt Express 17:12039–12042PubMedCrossRefGoogle Scholar
  25. Marcu L, Jo JA, Fang Q, Papaioannou T, Reil T, Qiao JH, Baker JD, Freischlag JA, Fishbein MC (2009) Detection of rupture-prone atherosclerotic plaques by time-resolved laser-induced fluorescence spectroscopy. Atherosclerosis 204(1):156–164PubMedCrossRefGoogle Scholar
  26. Megens RTA, Reitsma S, Schiffers PHM, Hilgers RHP, De Mey JGR, Slaaf DW, Oude Egbrink MGA, van Zandvoort MAMJ (2007) Two-photon microscopy of vital murine elastic and muscular arteries. J Vasc A 24(5):1337–1348Google Scholar
  27. Megens RTA, oude Egbrink MGA et al (2008) Two-photon microscopy on vital carotid arteries: imaging the relationship between collagen and inflammatory cells in atherosclerotic plaques. J Biomed Opt 13(04):044022PubMedCrossRefGoogle Scholar
  28. Millard AC, Wiseman PW, Fittinghoff DN, Wilson KR, Squier JA, Müller M (1999) Third-harmonic generation microscopy by use of a compact, femtosecond fiber laser source. Appl Opt 38(36):7393–7397PubMedCrossRefGoogle Scholar
  29. Miyata K, Rotermund F, Petrov V (2009) Efficient frequency doubling of a low-power femtosecond Er-fiber laser in BiB3O6. IEEE Photo Technol Lett 21(19):1417–1419CrossRefGoogle Scholar
  30. Mizutani G, Koyama T, Tomizawa S, Sano H (2005) Distinction between some saccharides in scattered optical sum frequency intensity images. Spectrochim Acta A Mol Biomol Spectrosc 62(4–5):845–849PubMedCrossRefGoogle Scholar
  31. Mostaço-Guidolin LB, Sowa MG, Ridsdale A, Pegoraro AF, Smith MSD, Hewko MD, Kohlenberg EK, Schattka B, Shiomi M, Stolow A, Ko ACT (2010) Differentiating atherosclerotic plaque burden in arterial tissues using femtosecond CARS-based multimodal nonlinear optical imaging. Biomed Opt Express 1:59–73PubMedCrossRefGoogle Scholar
  32. Mostaço-Guidolin LB, Ko AC-T, Popescu DP, Smith MSD, Kohlenberg EK, Shiomi M, Major A, Sowa MG (2011) Evaluation of texture parameters for the quantitative description of multimodal nonlinear optical images from atherosclerotic rabbit arteries. Phys Med Biol 56:5319. doi: 10.1088/0031-9155/56/16/016 PubMedCrossRefGoogle Scholar
  33. Murugkar S, Brideau C, Ridsdale A, Naji M, Stys PK, Anis H (2007) Coherent anti-Stokes Raman scattering microscopy using photonic crystal fiber with two closely lying zero dispersion wavelengths. Opt Express 15:14028–14037PubMedCrossRefGoogle Scholar
  34. Naghavi M, Libby P, Falk E et al (2003) Review: current perspective: from vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I. Circulation 108:1664–1672. doi: 10.1161/01.CIR.0000087480.94275.97 PubMedCrossRefGoogle Scholar
  35. Parasassi T, Yu W, Durbin D, Kuriashkina L, Gratton E, Maeda N, Ursini F (2000) Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides. Free Radic Biol Med 28:1589–1597PubMedCrossRefGoogle Scholar
  36. Paulsen HN, Hilligse KM, Thøgersen J, Keiding SR, Larsen JJ (2003) Coherent anti-Stokes Raman scattering microscopy with a photonic crystal fiber based light source. Opt Lett 28:1123–1125PubMedCrossRefGoogle Scholar
  37. Pegoraro AF, Ridsdale A, Moffatt DJ, Jia Y, Pezacki JP, Stolow A (2009a) Optimally chirped multimodal CARS microscopy based on a single Ti:sapphire oscillator. Opt Express 17:2984–2996PubMedCrossRefGoogle Scholar
  38. Pegoraro AF, Ridsdale A, Moffatt DJ, Pezacki JP, Thomas BK, Fu L, Dong L, Fermann ME, Stolow A (2009b) All-fiber CARS microscopy of live cells. Opt Express 17(23):20700–20706PubMedCrossRefGoogle Scholar
  39. Phipps J, Sun Y, Saroufeem R, Hatami N, Marcu L (2009) Fluorescence lifetime imaging microscopy for the characterization of atherosclerotic plaques. Proc Soc Photo Opt Instrum Eng 7161:71612GGoogle Scholar
  40. Prent N, Green C, Greenhalgh C, Cisek R, Major A, Stewart B, Barzda V (2008) Inter-myofilament dynamics of myocytes revealed by second harmonic generation microscopy. J Biomed Opt 13(4):041318–1–041318–7CrossRefGoogle Scholar
  41. Rinia HA, Burger KNJ, Bonn M, Müller M (2008) Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy. Biophys J 95(10):4908–4914PubMedCrossRefGoogle Scholar
  42. Schenke-Layland K, König K, Riemann I, Stock UA (2005) Imaging of cardiovascular structures using near-infrared femtosecond multiphoton laser scanning microscopy. J Biomed Opt 10:024017. doi: 10.1117/1.1896966 PubMedCrossRefGoogle Scholar
  43. Schenke-Layland K, Madershahian N, Riemann I, Starcher B, Halbhuber KJ, König K, Stock UA (2006) Impact of cryopreservation on extracellular matrix structures of heart valve leaflets. Ann Thorac Surg 81(3):918–926PubMedCrossRefGoogle Scholar
  44. Sheppard CJR, Gannaway JN, Kompfner R, Walsh D (1977) Scanning harmonic optical microscope. IEEE J Q Electron 13(9):100DGoogle Scholar
  45. Smith MSD, Ko ACT, Ridsdale A, Schattka B, Pegoraro A, Hewko MD, Shiomi M, Stolow A, Sowa MG (2009) A single-photon fluorescence and multi-photon spectroscopic study of atherosclerotic lesions. Proc SPIE 7386:73860ICrossRefGoogle Scholar
  46. Spence DE, Kean PN, Sibbett W (1991) 60-fsec pulse generation from a self-mode-locked Ti:sapphire laser. Opt Lett 16(1):42–44PubMedCrossRefGoogle Scholar
  47. Strupler M, Pena AM, Hernest M, Tharaux PL, Martin JL, Beaurepaire E, Schanne-Klein MC (2007) Second harmonic imaging and scoring of collagen in fibrotic tissues. Opt Express 15(7):4054–4065PubMedCrossRefGoogle Scholar
  48. Strupler M, Hernest M, Fligny C, Martin J-L, Tharaux P-L, Schanne-Klein MC (2008) Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling. J Biomed Opt 13(5):054041PubMedCrossRefGoogle Scholar
  49. Sun J, Zhang Z, Lu B, Yu W, Yang Y, Zhou Y, Wang Y, Fan Z (2008) Identification and quantification of coronary atherosclerotic plaques: a comparison of 64-MDCT and intravascular ultrasound. Am J Roentgenol 190(3):748–754CrossRefGoogle Scholar
  50. Suhling K, French PM, Phillips D (2005) Time-resolved fluorescence microscopy. Photochem Photobiol Sci 4(1):13–22PubMedCrossRefGoogle Scholar
  51. Timmins LH, Wu Q, Yeh AT, Moore JE Jr, Greenwald SE (2010) Structural inhomogeneity and fiber orientation in the inner arterial media. Am J Physiol Heart Circ Physiol 298:H1537–H1545PubMedCrossRefGoogle Scholar
  52. Thomas P, Pande P, Clubb F, Adame J, Jo JA (2010) Biochemical imaging of human atherosclerotic plaques with fluorescence lifetime angioscopy. Photochem Photobiol 86:727–731PubMedCrossRefGoogle Scholar
  53. Van Zandvoort M, Engels W, Douma K, Beckers L, Oude Egbrink M, Daemen M, Slaaf DW (2004) Two-photon microscopy for imaging of the (atherosclerotic) vascular wall: a proof of concept study. J Vasc Res 41(1):54–63PubMedCrossRefGoogle Scholar
  54. Yock PG, Fitzgerald PJ (1998) Optimal directional coronary atherectomy final results of the Optimal Atherectomy Restenosis Study (OARS). Am J Cardiol 81:27E–32EPubMedCrossRefGoogle Scholar
  55. Yu W, Braz JC, Dutton AM, Prusakov P, Rekhter M (2007) In vivo imaging of atherosclerotic plaques in apolipoprotein E deficient mice using nonlinear microscopy. J Biomed Opt 12(5):054008PubMedCrossRefGoogle Scholar
  56. Wang H-W, Langohr IM, Sturek M, Cheng J-X (2009) Imaging and quantitative analysis of atherosclerotic lesions by CARS-based multimodal nonlinear optical microscopy. Arterioscler Thromb Vasc Biol 29(9):1342–1348PubMedCrossRefGoogle Scholar
  57. Wang H-W, Simianu V, Locker MJ, Cheng J-X, Sturek M (2011) Stent-induced coronary artery stenosis characterized by multimodal nonlinear optical microscopy. J Biomed Opt 16(2):021110PubMedCrossRefGoogle Scholar
  58. Zoumi A, Lu XA, Kassab GS, Tromberg BJ (2004) Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy. Biophys J 87:2778–2786PubMedCrossRefGoogle Scholar
  59. Zumbusch A, Holtom GR, Xie XS (1999) Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering. Phys Rev Lett 82(20):4142–4145CrossRefGoogle Scholar

Copyright information

© © Her Majesty the Queen in Right of Canada 2012 2012

Authors and Affiliations

  • Alex C.-T. Ko
    • 1
  • Andrew Ridsdale
    • 2
  • Leila B. Mostaço-Guidolin
    • 3
  • Arkady Major
    • 3
  • Albert Stolow
    • 2
  • Michael G. Sowa
    • 1
  1. 1.National Research Council CanadaInstitute for BiodiagnosticsWinnipegCanada
  2. 2.National Research Council CanadaSteacie Institute for Molecular SciencesOttawaCanada
  3. 3.Department of Electrical and Computer EngineeringUniversity of ManitobaWinnipegCanada

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