26 Jul 2023
Dual-ratio approach enhances fluorescence signal during sensing in biological systems.
Flow cytometry, in which cells tagged with fluorescent markers are illuminated while passing along a narrow channel, is one promising technique, potentially revealing information about cell size and DNA content.
If CTCs are labeled with a fluorescent agent, then a flow cytometry approach with a laser directed directly onto an artery will induce fluorescent emission in any labelled cells present, and the emission can be related directly to the presence and number of those CTCs. This operation is termed diffuse in vivo flow cytometry (DiFC).
However, in practice DiFC performance and the measurement depths achieved can be severely affected by background noise, especially if it originates from the inherent autofluorescence of other cells and tissues in the examined area.
A project at Tufts University and Northeastern University has now developed a new optical measurement method intended to suppress this interference and enhance the signal to noise ratio in deep tissue regions. The study was published in Journal of Biomedical Optics.
The project applied a dual-ratio (DR) approach, originally developed for spectroscopy techniques and now adopted for DiFC, in which two laser sources and two detectors are utilized. This should allow interference from autofluorescence emitted by species in the artery and by the surface skin to be reduced, although how best to apply the principle to DiFC had not been studied until now.
Detection of cancer markers without taking blood samples
To optimize a DR approach, the project first ran Monte-Carlo analysis techniques to simulate various noise and autofluorescence parameters, along with different source/detector configurations. It also conducted DR DiFC experiments using an artificial tissue-mimicking flow phantom, with fluorescent microspheres taking the place of cells.
Lastly, the team used its optimized technique to measure the AF of the skin and the underlying muscle of mice, to assess the variation of noise with tissue type and depth in a real-world scenario. The distribution of autofluorescence sources and the proportion of noise not cancelled by the dual-ratio approach proved to be the key parameters.
"The experiments revealed that dual-ratio DiFC was superior to standard DiFC if the fraction of noise not canceled by DR was under 10 percent, and if the contributions to auto-fluorescence were surface-weighted, being near the surface rather than evenly distributed in the target volume," commented the research team.
"However, as the experiments in mice suggested, autofluorescence is typically much higher in skin than in the underlying muscle, implying that DR DiFC may offer an advantage over standard DiFC in most cases. If autofluorescence was near the surface rather than being homogeneous, the dual-ratio version had a significantly higher penetration range than standard DiFC."
The team anticipates that establishing these principles will now help position DR DiFC as an emerging technique to non-invasively detect fluorescent molecules in the bloodstream, potentially enabling doctors to quickly detect cancer cells in the blood of patients without having to draw samples. Other cell types and molecules of interest may also ultimately be identifiable in the same way.