Molecular Imaging and Biology

, Volume 14, Issue 3, pp 293–300 | Cite as

Spectral Unmixing Imaging of Wavelength-Responsive Fluorescent Probes: An Application for the Real-Time Report of Amyloid Beta Species in Alzheimer’s Disease

  • Chongzhao Ran
  • Anna Moore
Research Article



The goal of the study was to investigate a method for the real-time assessment of a target concentration in vivo using a combination of a spectral unmixing technique and a fluorescent probe specific for amyloid beta (Aβ) species, the biomarkers for Alzheimer’s disease (AD).


The probe CRANAD-3 has a significant emission wavelength shift upon binding to Aβ species. It was used to differentiate a bound probe from an unbound probe in a phantom, ex vivo in brain slices and whole brain, and in vivo in a transgenic mouse model of AD.


The ex vivo unmixing imaging of AD brain clearly showed differential distribution of the bound and unbound probes between the brain tissue and blood vessels. The in vivo unmixed signals of bound CRANAD-3 reached a plateau with increasing dosage, demonstrating that these signals correspond to Aβ content, not probe injected dose.


This study provided evidence that signals processed by the spectral unmixing technique could be used as a real-time reporter of Aβ species loading in vivo and ex vivo.

Key words

Spectral unmixing Alzheimer’s disease Wavelength-responsive fluorescent probe 



This work was supported in part by K25AG036760 award to C.R. We would like to thank Marytheresa Ifediba for proofreading this manuscript.

Conflict of interest

The authors declare they have no conflict of interests pertinent to this study.

Supplementary material

11307_2011_501_MOESM1_ESM.pdf (221 kb)
SI Fig. 1 Raw images of phantom imaging with CRANAD-3 and Aβ 42 species in PBS buffer. The raw images were obtained with 535-nm excitation and with 14 emission filters ranging from 580 to 840 nm. (PDF 220 kb)
11307_2011_501_MOESM2_ESM.pdf (316 kb)
SI Fig. 2 Representative raw image sequence of in vivo imaging of an APP/PS1 mouse with CRANAD-3 (Ex = 570 nm, Em = 620–840 nm with 12 emission filters). (PDF 316 kb)
11307_2011_501_MOESM3_ESM.pdf (1.7 mb)
SI Fig. 3 Fluorescent microscopic images of ex vivo brain slice of APP/PS1 mouse. a Image of Aβ plaque distribution in whole brain slice with ×2 lens. b Image of Aβ plaque in the cortex area (white box in a) with ×10 lens. (PDF 1751 kb)


  1. 1.
    Xu H, Rice BW (2009) In-vivo fluorescence imaging with a multivariate curve resolution spectral unmixing technique. J Biomed Opt 14:064011PubMedCrossRefGoogle Scholar
  2. 2.
    Mayes P, Dicker D, Liu Y, El-Deiry W (2008) Noninvasive vascular imaging in fluorescent tumors using multispectral unmixing. Biotechniques 45:459–464PubMedCrossRefGoogle Scholar
  3. 3.
    Naik S, Piwnica-Worms D (2007) Real-time imaging of beta-catenin dynamics in cells and living mice. Proc Natl Acad Sci USA 104:17465–17470PubMedCrossRefGoogle Scholar
  4. 4.
    Zimmermann T (2005) Spectral imaging and linear unmixing in light microscopy. Adv Biochem Eng Biotechnol 95:245–265PubMedGoogle Scholar
  5. 5.
    Liu J, Lau SK, Varma VA, Kairdolf BA, Nie S (2010) Multiplexed detection and characterization of rare tumor cells in Hodgkin's lymphoma with multicolor quantum dots. Anal Chem 82:6237–6243PubMedCrossRefGoogle Scholar
  6. 6.
    Raymond SB, Skoch J, Hills ID, Nesterov EE, Swager TM, Bacskai BJ (2008) Smart optical probes for near-infrared fluorescence imaging of Alzheimer's disease pathology. Eur J Nucl Med Mol Imaging 35:s93–s98PubMedCrossRefGoogle Scholar
  7. 7.
    Ran C, Xu X, Raymond SB et al (2009) Design, synthesis, and testing of difluoroboron-derivatized curcumins as near-infrared probes for in vivo detection of amyloid-beta deposits. J Am Chem Soc 131:15257–15261PubMedCrossRefGoogle Scholar
  8. 8.
    Nesterov EE, Skoch J, Hyman BT, Klunk WE, Bacskai BJ, Swager TM (2005) In vivo optical imaging of amyloid aggregates in brain: design of fluorescent markers. Angew Chem Int Ed Engl 44:5452–5456PubMedCrossRefGoogle Scholar
  9. 9.
    Mizusawa K, Ishida Y, Takaoka Y, Miyagawa M, Tsukiji S, Hamachi I (2010) Disassembly-driven turn-on fluorescent nanoprobes for selective protein detection. J Am Chem Soc 132:7291–7293PubMedCrossRefGoogle Scholar
  10. 10.
    Dickinson ME, Bearman G, Tille S, Lansford R, Fraser SE (2001) Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. Biotechniques 31:1272, 1274–1276, 1278Google Scholar
  11. 11.
    Ran C, Zhao W, Moir R, Moore A (2011) Non-conjugated small molecule FRET for differentiating monomers from higher molecular weight amyloid beta species. PLoS One 6:e19362PubMedCrossRefGoogle Scholar
  12. 12.
    Selkoe D (2008) Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and behavior. Behav Brain Res 192:106–113PubMedCrossRefGoogle Scholar
  13. 13.
    Haass C, Selkoe DJ (2007) Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol 8:101–112PubMedCrossRefGoogle Scholar
  14. 14.
    Greenspan P, Fowler SD (1985) Spectrofluorometric studies of the lipid probe, Nile red. J Lipid Res 26:781–789PubMedGoogle Scholar
  15. 15.
    Jankowsky JL, Fadale DJ, Anderson J et al (2004) Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. Hum Mol Genet 13:159–170PubMedCrossRefGoogle Scholar
  16. 16.
    Reiserer RS, Harrison FE, Syverud DC, McDonald MP (2007) Impaired spatial learning in the APPSwe + PSEN1DeltaE9 bigenic mouse model of Alzheimer's disease. Genes Brain Behav 6:54–65PubMedCrossRefGoogle Scholar
  17. 17.
    Hintersteiner M, Enz A, Frey P et al (2005) In vivo detection of amyloid-beta deposits by near-infrared imaging using an oxazine-derivative probe. Nat Biotechnol 23:577–583PubMedCrossRefGoogle Scholar
  18. 18.
    Gurskaya NG, Verkhusha VV, Shcheglov AS et al (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol 24:461–465PubMedCrossRefGoogle Scholar
  19. 19.
    Lukyanov KA, Chudakov DM, Lukyanov S, Verkhusha VV (2005) Innovation: photoactivatable fluorescent proteins. Nat Rev Mol Cell Biol 6:885–891PubMedCrossRefGoogle Scholar
  20. 20.
    Johnson I (1998) Fluorescent probes for living cell. Histochem J 30:123–140PubMedCrossRefGoogle Scholar

Copyright information

© Academy of Molecular Imaging and Society for Molecular Imaging 2011

Authors and Affiliations

  1. 1.Molecular Imaging Laboratory, MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Department of RadiologyMassachusetts General Hospital/Harvard Medical SchoolBostonUSA

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