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

Abstract

Objectives We investigated the potential use of real-time confocal microscpy 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 recognise 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.

Simple 3‐criteria‐based ex vivo confocal diagnosis of basal cell carcinoma

“…ex vivo CLSM uses fluorescent contrast agents (eg, acridine orange) and provides sufficient resolution and contrast to produce images on a cellular and even nuclear level in thick tissue sections . Within these thick tissue sections additional optical sectioning of the specimen is possible and allows the examination of the tissue without any damage . ex vivo CLSM examination has been described so far in healthy skin , melanoma , eccrine syringomatous carcinoma , mammary and extramammary Paget's disease ,squamous cell carcinoma , foreign body reactions and in ophthalmology .…”

Fluorescence confocal endomicroscopy of the cervix: pilot study on the potential and limitations for clinical implementation

“…A dichroic mirror allows for efficient separation of excitation and emission light and thus good contrast and comparably high image quality may be achieved. [21][22][23][24] The high imaging contrast of fluorescence endomicroscopy is paired with a limited imaging depth of about 50 μm owing to the finite permeation of the fluorophore dye.…”

Fluorescence confocal microscopy for pathologists

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