Single-Cell Quantification of Cytosine Modifications by Hyperspectral Dark-Field Imaging

Xiaolei Wang, Yi Cui, Joseph Irudayaraj

Research output: Contribution to journalArticlepeer-review


Epigenetic modifications on DNA, especially on cytosine, play a critical role in regulating gene expression and genome stability. It is known that the levels of different cytosine derivatives are highly dynamic and are regulated by a variety of factors that act on the chromatin. Here we report an optical methodology based on hyperspectral dark-field imaging (HSDFI) using plasmonic nanoprobes to quantify the recently identified cytosine modifications on DNA in single cells. Gold (Au) and silver (Ag) nanoparticles (NPs) functionalized with specific antibodies were used as contrast-generating agents due to their strong local surface plasmon resonance (LSPR) properties. With this powerful platform we have revealed the spatial distribution and quantity of 5-carboxylcytosine (5caC) at the different stages in cell cycle and demonstrated that 5caC was a stably inherited epigenetic mark. We have also shown that the regional density of 5caC on a single chromosome can be mapped due to the spectral sensitivity of the nanoprobes in relation to the interparticle distance. Notably, HSDFI enables an efficient removal of the scattering noises from nonspecifically aggregated nanoprobes, to improve accuracy in the quantification of different cytosine modifications in single cells. Further, by separating the LSPR fingerprints of AuNPs and AgNPs, multiplex detection of two cytosine modifications was also performed. Our results demonstrate HSDFI as a versatile platform for spatial and spectroscopic characterization of plasmonic nanoprobe-labeled nuclear targets at the single-cell level for quantitative epigenetic screening.

Original languageEnglish (US)
Pages (from-to)11924-11932
Number of pages9
JournalACS Nano
Issue number12
StatePublished - Oct 27 2015
Externally publishedYes


  • cytosine modification
  • epigenetics
  • hyperspectral dark-field microscopy
  • plasmonic nanoprobes
  • quantitative imaging

ASJC Scopus subject areas

  • General Materials Science
  • General Engineering
  • General Physics and Astronomy


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