Ex vivo confocal imaging with contrast agents for the detection of oral potentially malignant lesions

Oral Oncol. 2013 Jun;49(6):582-90. doi: 10.1016/j.oraloncology.2013.01.009. Epub 2013 Feb 12.


Objectives: We investigated the potential use of real-time confocal microscopy in the non-invasive detection of occult oral potentially malignant lesions. Our objectives were to select the best fluorescence contrast agent for cellular morphology enhancement, to build an atlas of confocal microscopic images of normal human oral mucosa, and to determine the accuracy of confocal microscopy to recognize oral high-grade dysplasia lesions on live human tissue.

Materials and methods: Five clinically used fluorescent contrast agents were tested in vitro on cultured human cells and validated ex vivo on human oral mucosa. Images acquired ex vivo from normal and diseased human oral biopsies with bench-top fluorescent confocal microscope were compared to conventional histology. Image analyzer software was used as an adjunct tool to objectively compare high-grade dysplasia versus low-grade dysplasia and normal epithelium.

Results: Acriflavine Hydrochloride provided the best cellular contrast by preferentially staining the nuclei of the epithelium. Using topical application of Acriflavine Hydrochloride followed by confocal microscopy, we could define morphological characteristics of each cellular layer of the normal human oral mucosa, building an atlas of histology-like images. Applying this technique to diseased oral tissue specimen, we were also able to accurately diagnose the presence of high-grade dysplasia through the increased cellularity and changes in nuclear morphological features. Objective measurement of cellular density by quantitative image analysis was a strong discriminant to differentiate between high-grade dysplasia and low-grade dysplasia lesions.

Conclusions: Pending clinical investigation, real-time confocal microscopy may become a useful adjunct to detect precancerous lesions that are at high risk of cancer progression, direct biopsy and delineate excision margins.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acriflavine
  • Adult
  • Aged
  • Aged, 80 and over
  • Cell Line
  • Contrast Media*
  • Female
  • Fluorescent Dyes
  • Humans
  • Male
  • Microscopy, Confocal / methods*
  • Middle Aged
  • Mouth Mucosa / pathology
  • Mouth Neoplasms / diagnosis*


  • Contrast Media
  • Fluorescent Dyes
  • Acriflavine