Multispectral visualization of surgical safety-margins using fluorescent marker seeds

Abstract

Optical guidance provided by luminescent marker seeds may be suitable for intraoperative determination of appropriate resection margins. In phantom studies we compared the tissue penetration of several organic dyes and inorganic particles (quantum dots; QDs) after incorporation in experimental marker seeds. The tissue penetration of (near infra-) red organic dyes was much better than the penetration of dyes and QDs with an emission in the visible range. By combining 3 dyes in a single marker seed we were able to distinguish four depth ranges. The difference in tissue penetration between the dyes and QDS enabled depth estimation via a 'traffic light' approach.

Keywords: Surgical guidance; fluorescence; marker seeds; phantom studies; tissue penetration.

Figure 1

Experimental setup. A glass seed containing a luminophore (or a combination of several) is placed on a ~4 mm layer of tenderloin/sirloin/lard in the imaging chamber of the IVIS 200 system. After taking a picture of the bare seed, it is then covered with layers of tissue and imaged at every step.

Figure 2

Luminescent characteristics of organic dyes. A) Spectra for FITC, TRITC, Cy5.5 and ICG (peaks normalized to 1) and relevant IVIS 200 emission filter bands (resp. GFP_{515-575 nm}, dsRed_{575-650 nm}, Cy5.5_{695-770 nm} and ICG_{810-885 nm} settings). The excitation filter bands are depicted as bars on the x-axis, with the colors matching those of the dyes in the graph. B) Concentration curve: Comparison of luminescence intensities of all dyes at different concentrations. C) Tissue penetration of 0.5 mg/mL organic dye in a marker seed covered by tenderloin, measured as mentioned in A). Excitation wavelength filters were added to the legend. The lower initial SBR’s found in (C), relative to (B) is a result of the underlying piece of tissue that is only present in (C; see Figure 1)).

Figure 3

Luminescent characteristics of two InP/ZnS QDs. A) Spectra of exciton emissions (peaks normalized to 1) and relevant IVIS 200 emission filter bands (resp. GFP_{515-575 nm} and 660 nm). The GFP excitation filter band is depicted as a gray bar on the horizontal axis. B) Concentration curve: Comparison of luminescence intensities of the two different QDs at different concentrations. C) Tissue penetration of 1 mg/mL and 10 mg/mL green and red QDs in a marker seed covered by tenderloin, measured using the IVIS 200 GFP excitation + GFP_{515-575 nm} emission setting (green QD) or a combination of GFP excitation + 660 nm emission settings (red QD). D) Comparison of tissue penetration of InP/ZnS QD exciton and defect emissions. Figure 4D is adapted from Chin et al. (2009) [13]. The lower initial SBR’s found in (C), relative to (B) is a result of the underlying piece of tissue that is only present in (C; see Figure 1)).

Figure 4

Signal scattering. A) Example of signal scatter of an ICG-containing seed: The uncovered seed (0 mm) gives a signal comparable to the actual size of the seed, while covering the seed with layers of tenderloin tissue increases the diameter of the signal (1-8.5 mm). B) Signal scatter for ICG, Cy5.5 and InP/ZnS QDs covered by tenderloin, determined from the same images as Figures 2C and 3C. C) Signal scatter for ICG and Cy5 in tenderloin, sirloin and lard, determined from the same measurements as used for Figures 2C and 4C-D.

Figure 5

Background luminescence and tissue-dependent penetration. A) Background luminescence of different mouse organs, measured on the IVIS 200 system, using the GFP_{515-575 nm}, dsRed_{575-650 nm}, Cy5.5_{695-770 nm} and ICG_{810-885 nm} settings. B) Background luminescence of tenderloin, sirloin and lard, measured as in A. C) Tissue penetration of ICG in bloody tissue (sirloin), pale tissue (tenderloin) and fat (lard). SBR values were normalized to the value for a bare seed. D) Tissue penetration of Cy5.5 in bloody tissue (sirloin), pale tissue (tenderloin) and fat (lard). Relative fluorescence values were normalized to the value for a bare seed. The blue arrows indicate the tissue depth at which the signal is lost.

Figure 6

Traffic light analogue for depth estimation using a marker seed filled with a mixture of ICG, TRITC and FITC. Depending on how many colors of the traffic light can be detected, an estimation of depth can be made (top illustration). A camera system is required to detect the ICG signal and can aid in visualizing the TRITC and FITC signals; Here the IVIS 200 ICG_{810-885 nm}, dsRed_{575-650 nm} and GFP_{515-575 nm} settings were used, and the signal intensities are shown using the standard (reversed) Rainbow color table. TRITC and FITC can also be seen by eye and the perceived color of the dye mixture provides a direct indication of depth.