1 Quantitative, spectrally- resolved intraoperative fluorescence imaging Pablo A. Valdés 1,2,3, Frederic Leblond 1, Valerie L. Jacobs 2, Brian C. Wilson 5, Keith D. Paulsen 1,2,4, & David W. Roberts 2,3,4 1 Thayer School of Engineering, and 2 Dartmouth Medical School, Dartmouth College, Hanover, NH 03755, USA; 3 Section of Neurosurgery; 4 Norris Cotton Cancer Center, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire 03756, USA; 5 University of Toronto, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada. Correspondence should be addressed to P.A.V. (Pablo.A.Valdes@Dartmouth.edu) or D.W.R. (David.W.Roberts@Dartmouth.edu)
2 SUPPLEMENTARY METHODS AND RESULTS Spatial resolution of optical components. A USAF 1951 (Edmund Optics) contrast resolution target was used to determine the spatial resolution of the qfi imaging system under white light exposure (Suppl. Fig. 3a-c). The target was composed of n = [0, 1, 2, 3] groups, each with i =[1, 2, 3, 4, 5, 6] horizontal and vertical elements consisting of black and white bar patterns (Suppl. Fig. 3c). The crosssectional intensity profiles of each element were extracted to calculate the element-specific contrast transfer function (CTF) from CTF(i) = I max (i) " I min (i) I max (i) + I min (i) *100 where I max and I min are the maximum and minimum intensities of the bar pattern s cross-sectional profile. The calculated element specific resolution was determined from Re solution(i) = 1 2 n + i"1 6 *1000 where n is the group number, i is the element number, and resolution is given in µm. We computed the Rayleigh and Sparrow horizontal (Suppl. Fig. 3a) and vertical (Suppl. Fig. 3b) spatial resolutions by determining the point on the graph were the CTF = 26.4% or 0%, respectively. The optical components of the qfi system demonstrated submillimeter resolutions both in the vertical (214 μm and 125 μm) and horizontal directions (217 μm and 125 μm), which are approximately 5 to 10 times smaller, respectively, than the smallest length scale relevant to the execution of surgical procedures.
3 SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1. Quantitative fluorescence imaging (qfi) system. (A) Schematic of the qfi design integrated with a conventional surgical microscope enabled for fluorescence imaging. The microscope consists of two optical ports (1 and 2) with associated oculars for direct viewing by two surgeons and a third (3) optical port that transmits light onto a RGB CCD camera for video rate and snapshot image acquisition. A fourth (4) free optical port integrates with the portable qfi system. The microscope functions in violet-blue light and white light modes - one for fluorescence imaging and one for white light reflectance imaging, respectively. The qfi system consists of an optical adapter which connects to the free optical port of the surgical microscope, a liquid crystal tunable filter for fast (ms range) wavelength selection and light filtration, and a CCD camera for detection. (B) Illustration of intraoperative use of qfi in a human surgery with fluorescence guidance. Supplementary Figure 2. Scatter plot of qfi-derived C PpIX estimates vs. probe C PpIX values (in triplicate) showing a strong linear relationship with a highly significant Pearson s correlation coefficient, R = 0.79 (p-value<0.0001). Data produced from 41 locations in 4 rats with CNS-1 tumors. Both normal and tumor tissue was sampled. Error bars denote +/- standard deviation. Supplementary Figure 3. Spatial resolution of the optical components comprising the qfi system. Contrast transfer function analysis demonstrates submillimeter vertical spatial resolutions (in A) of 214 μm and 125 μm, and horizontal spatial resolutions (in B) of 217 μm and 125 μm (Rayleigh and Sparrow criteria, respectively) using a standard contrast resolution target (in C).
4 Supplementary Figure 1
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