Check the orignal paper here: Optical imaging for cervical cancer detection: solutions for a continuing global problem
Change in optical properties in precancerous cells
- Precancerous cells in the cervix undergo changes that can be detected using optical technologies.
- One of these changes is stromal angiogenesis, which is the formation of new blood vessels in the connective tissue of the cervix. This leads to increased absorption of light by the stroma.
- Another change is increased light scattering by the precancerous epithelial cells. This can be further enhanced by the addition of acetic acid.
- In normal cervical tissue, collagen crosslinks within the stroma are strongly fluorescent. This means that they emit light when excited by a specific wavelength of light. However, this fluorescence decreases with the development of precancer.
- In the normal squamous epithelium, different layers of cells show different patterns of fluorescence. Basal epithelial cells show cytoplasmic fluorescence associated with mitochondrial NADH and FAD, which are molecules involved in energy production. Superficial epithelial cells show peripheral fluorescence attributed to keratin, a protein found in skin, hair, and nails.
- With precancer development, the fluorescence patterns change. Keratin fluorescence decreases in the epithelium, and mitochondrial fluorescence is found in cells throughout progressively more superficial regions of the epithelium.
- These changes in fluorescence patterns are biologically predictive of cervical precancer and can be measured using optical technologies at the point of care. This means that doctors can use these technologies to detect precancerous changes in the cervix during a routine exam, without needing to send samples to a lab for analysis.
Light scattering and absorption
- Optical technologies can be used to study changes in tissue architecture, cell morphology, and biochemical composition.
- High-grade precancers often show vascular changes due to the development of new blood vessels, which can be visualized and quantified using image-analysis approaches.
- Haemoglobin, a protein in blood, has a characteristic absorption spectrum with peaks at 420 nm, 542 nm, and 577 nm. By changing the wavelength of illumination, we can enhance vascular contrast and probe vessels at different depths below the visual surface of the cervix.
- Acetic acid, when applied to cervical tissue, increases light scattering from cell nuclei. This means that the light is scattered in different directions, making it easier to see the nuclei.
- Precancerous tissue has a mean scattering coefficient that is approximately three times higher than that of normal epithelium following the application of acetic acid. This difference in scattering is due to increased nuclear size, increased optical density of the nucleus, and changes in chromatin texture that have been documented in cancerous cells.
- Cervical precancer is also associated with decreased stromal scattering, which is attributed to a degradation of collagen fibers. This degradation may be due to proteases secreted by pre-neoplastic epithelial cells.
Autofluorescence
- Autofluorescence is a technique that uses fluorescence interrogation to probe biochemical changes optically.
- In normal cervical tissue, collagen crosslinks cause bright fluorescence in the stroma across a wide range of excitation wavelengths.
- Stromal fluorescence increases with age and menopause in women with normal cervical tissue, but it is greatly reduced in cervical precancers and cancers.
- Ultraviolet (UV) and green excitation wavelengths have been used to monitor autofluorescence in cervical epithelial cells.
- Cytoplasmic autofluorescence seen on exposure to UV excitation wavelengths is due to mitochondrial NADH, while mitochondrial FAD fluoresces on exposure to green excitation wavelengths.
- Cervical epithelial cells also show autofluorescence at the cell periphery, which is often attributed to cytokeratins.
- In normal epithelium, basal epithelial cells show strong cytoplasmic fluorescence, while parabasal, intermediate, and superficial cells show fluorescence only at the periphery of the cell.
- All of this information can be used to determine the presence of precancerous lesions.
- Confocal fluorescence images of organ cultures of normal human cervical tissue and precancerous tissue can be compared to identify differences in cytoplasmic fluorescence.
- In low-grade precancers, cytoplasmic fluorescence is visible in the bottom third of the epithelium, while in high-grade precancers, it is visible throughout the lower two-thirds of the epithelium, with reduced fluorescence attributed to keratin.
- Recent studies have shown that HPV-immortalized keratinocytes exhibit increased NADH and FAD fluorescence relative to normal keratinocytes.
- New imaging technologies need to be developed to take advantage of this knowledge for the benefit of the patient.
High-resolution imaging
- High-resolution imaging techniques can be used to image cervical tissue with sub-cellular resolution.
- Small, flexible confocal microscopes have been developed to image cervical tissue with minimal power requirements.
- Microfabrication techniques can be used to manufacture confocal microscopes.
- High-resolution techniques can probe changes in epithelial cell morphology and epithelial architecture without the need for biopsy, sectioning, and staining.
- Video-rate reflectance confocal microscopy yields images of intact epithelial tissue with 1-2 μm spatial resolution.
- The use of acetic acid can help determine image parameters such as the nuclear to cytoplasmic (N:C) ratio.
- The N:C ratio measured by confocal microscopy can separate high-grade cervical precancers with a sensitivity and specificity greater than 90%.
- Automated image analysis routines can be used to segment nuclei in confocal images of cervical tissue and objectively calculate the N:C ratio.
- Fibre-optic confocal microscopes have become available to acquire confocal images of cervical tissue in vivo at near video rate in both reflectance and fluorescence modes.
- It is difficult to image weak autofluorescence in vivo using confocal fluorescence microscopy owing to photobleaching limits.
- Advances in optically active, targeted contrast agents can be used to tag biomarkers of interest with an optical signal that can be measured and quantified in vivo.
Contrast agents for molecular imaging
- Confocal fluorescence imaging has advanced with the use of new vital stains like intravenously administered fluorescein and topically applied acriflavine.
- Molecular events accompanying carcinogenesis can be understood better with the use of optically active, molecular-targeted contrast agents that can image biomarkers in vivo.
- Targeted optical contrast agents consist of a probe molecule like an antibody or peptide conjugated to an optically interrogatable label like metal nanoparticles, quantum dots, or organic fluorescent dyes.
- Monoclonal antibodies conjugated with fluorescent dyes can target multiple cell surface receptors overexpressed on tumor cells like the epidermal growth factor receptor (EGFR).
- Peptides like EGFR can also be used to bind to specific receptors.
These agents have a smaller molecular weight, making them advantageous for topical use. - Quantum dots have a broad excitation range and narrow emission spectra, allowing for simultaneous imaging of multiple biomarkers, but concerns exist about their cytotoxicity.
- Contrast agents incorporating optically active metal nanoparticles like gold and silver provide a strong source of backscattered light for contrast in wide-field and high-resolution imaging.
- Metal nanoparticles are not susceptible to photobleaching, and gold is non-toxic and biocompatible.
Gold nanoparticles conjugated with anti-EGFR antibodies have been used to image cervical precancer in vitro with high contrast. - EGFR-overexpressing cells induce aggregation of gold nanoparticles, leading to non-linear enhancements in scattering that can magnify signal differences resulting from moderate levels of overexpression.
- In one study, normal cervical tissue and high-grade precancerous tissue labeled with anti-EGFR gold nanoparticles showed a 10-20 fold increase in image-to-contrast ratio due to this aggregation, well beyond values reported for antibody-targeted fluorescent dyes.