Diffuse in vivo flow cytometry (DiFC), an optical technique developed at Northeastern University, enables fluorescence detection of tumor cells circulating in the bloodstream. It is a promising technique for detecting cancer that has metastasized. However, due to signal-to-noise ratio (SNR) constraints, which are attributed mainly to background autofluorescence in the tissues, DiFC’s measurement depth is limited. A team from Tufts University and Northeastern University is working to address this issue and take the DiFC method developed by the Northeastern group to a new level. The researchers are investigating whether a new method developed by the Tufts group, called the dual-ratio (DR) approach, can minimize noise and autofluorescence in DiFC. Originally developed for spectroscopy, DR is a diffuse optical measurement technique designed to suppress noise in the optical signal and enhance SNR in deep tissue regions. The researchers focused on whether the combination of DR and near-infrared (NIR) DiFC could improve circulating cells’ maximum detectable depth and SNR, by extending the penetration range of DiFC. The dual-ratio (DR) diffuse in-vivo flow cytometry (DiFC) is a new technique that uses two laser sources and two detectors to mitigate the effects of noise and autofluorescence. DR DiFC may enable doctors to precisely detect fluorescent tags attached to circulating tumor cells in the bloodstream. Courtesy of Blaney et al., 10.1117/1.JBO.28.7.077001. The team’s strategy to improve the accuracy and range of DiFC could further the development of this noninvasive technique and make the diagnosis of metastasis, a hallmark of advanced cancer, faster and simpler. When DiFC is used, circulating tumor cells are labeled with a fluorescent agent. A laser is shined directly onto an artery, and the emitted fluorescent signals are captured using a detector that counts the number of circulating tumor cells. The measurements taken with DiFC can be affected by background noise originating from the inherent fluorescence of the surrounding tissue. DR DiFC uses two laser sources and two detectors to mitigate the effects of noise and autofluorescence. In theory, the noise can be canceled out by combining the signals of the two detectors. The autofluorescence contributions of the surface of the measured medium — for example, skin — can also be minimized using DR. However, the conditions under which DR DiFC truly offers an advantage over standard DiFC required further investigation. To identify the advantages and limitations of the DR DiFC technique, the researchers ran Monte Carlo simulations using various noise and autofluorescence parameters and different source-detector configurations. They conducted phantom experiments using an artificial, tissue-mimicking flow phantom with cell-mimicking fluorescent microspheres to estimate the key parameters in a diffuse fluorescence excitation and emission model. They also measured the autofluorescence of the skin and underlying muscle in mice to gain insight into how noise varied with tissue type and depth. The team found that two key factors must be true to give DR DiFC an advantage over traditional DiFC. First, the fraction of noise not canceled by DR must be under 10%. Second, DR DiFC has an advantage, in terms of SNR, if the distribution of tissue autofluorescence contributors is surface-weighted — that is, higher near the surface instead of being evenly distributed in the target volume. The experiments with mice indicated that autofluorescence is typically higher in the skin than in the underlying muscle. When autofluorescence was higher near the surface, rather than being homogeneous, DR DiFC had a significantly higher penetration range than standard DiFC. The investigation of DR DiFC lays the groundwork for the design of a DR DiFC instrument for diagnosing metastasis rapidly and noninvasively. Although the successful use of DR DiFC depends on the distribution of autofluorescence contributors in vivo and the portion of noncancelable (by DR) noise in the measurement, the results point to DR DiFC having advantages over traditional DiFC. The research was published in Journal of Biomedical Optics (www.doi.org/10.1117/1.JBO.28.7.077001).