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1 Supplementary Information for Multi-Functional In Vivo Vascular Imaging Using Near-Infrared II Fluorescence Guosong Hong 1,3, Jerry C. Lee 2,3, Joshua T. Robinson 1, Uwe Raaz 2, Liming Xie 1, Ngan F. Huang 2, John P. Cooke 2 & Hongjie Dai 1 1 Department of Chemistry, Stanford University, Stanford, California 9435, USA 2 Division of Cardiovascular Medicine, School of Medicine, Stanford University, Stanford, California 9435, USA 3 These authors contribute equally to this work. 1
2 Number of molecules per SWNT-PEG-IR conjugate Relative Endothelial Cell Viability Normalized IRDye- fluorescence Abs [OD] Supplementary Figures. Free IRDye- SWNT-PEG-IRDye- (a) 1.1 (b) 5 7 Wavelength [nm] 1 (c) (d) 1 1 maximum concentration circulating in blood IC5: g/l SWNT IR. 1E SWNT concentration (g/l) Figure S1. Chemical composition and cytotoxicity of SWNT-PEG-IRDye- conjugate. (a) Normalized IRDye- fluorescence for free IRDye- and SWNT-PEG-IRDye- at the same concentration of IRDye-, suggesting the photoluminescence of IRDye- was quenched by ~% due to the attachment to SWNTs through the PEG-chains. Inset pictures on top are corresponding photoluminescence images taken in the NIR-I window. (b) UV-Vis-NIR absorption spectrum of SWNT- PEG-IRDye- conjugate taken in a 1 mm cuvette, where the OD values at 65 nm and 785 nm are used for calculating the average number of SWNT and IRDye- molecules in SWNT-PEG-IRDye- conjugate. (c) Calculated number of SWNT and IRDye- molecules in each SWNT-PEG-IRDye- conjugate, indicating on average 167 IRDye- molecules on each SWNT backbone. The large number of IRDye- molecules attached to SWNTs ensured sufficient NIR-I emitters despite fluorescence quenching of IRDye- of ~% in a. (d) Determination of half maximal inhibitory concentration (IC5) of SWNTs for endothelial cells. Original data (black squares) were fitted to sigmoidal function, revealing an IC5 value of.1778±.237 g/l. Errors bars reflect the standard deviation of relative endothelial cell viability values from 3 wells of cells incubated at the same SWNT concentration. 2
3 a t = 4.12 s t = 5.44 s t = s PCA overlay b c t = 4.12 s t = 5.44 s t = s d PCA overlay e t = 4.12 s t = 5.44 s t = s PCA overlay f g t = 4.88 s t = 7.69 s t = s PCA overlay h i t = 4.88 s t = 7.69 s t = s PCA overlay j Figure S2. Differentiation of femoral artery and vein inside healthy, control hindlimbs of Mouse C2 4 (a f) and ischemic hindlimbs of Mouse I2 3 (g j). (a,c,e,g,i) Time course NIR-II fluorescence images showing hindlimb blood flow labeled by SWNT fluorescent tags. (b,d,f,h,j) PCA overlaid images based on the first frames (37.5 s post injection) of each mouse, where arterial vessels are shown in red, while venous vessels are shown in blue. The scale bars all indicate 2 mm. 3
4 t = 12. s t = s t = s t = s (a) (b) (c) (d) t = s t = s t = s t = 249. s PCA overlay (e) (f) (g) (h) (i) Figure S3. Differentiation of femoral artery and vein inside an ischemic hindlimb of Mouse I1 based on a longer post-injection imaging time. (a h) Time course NIR-II fluorescence images showing hindlimb blood flow labeled by SWNT fluorescent tags. Note that femoral vein does not show up until after 2 min due to reduced blood flow of ischemia. (i) PCA overlaid image based on 13 frames (up to s post injection) of the mouse, where arterial vessels are shown in red, while venous vessels are shown in blue. The scale bar indicates 2 mm. 4
5 Distance travelled [cm] Distance travelled [cm] Distance travelled [cm] Distance travelled [cm] t = 12. s t = s t = 14.6 s t = 15. s t = s t = 17.6 s t = 18. s t = s (a) (b) 12 (c) (d) cm/s %/s cm/% t = 7.88 s t = 8.81 s t = 9.75 s t = 19 s t = s t = s t = 13.5 s t = s (f) 18 (g) (h) cm/s %/s cm/% (e) Figure S4. Arterial blood velocity analysis on the hindlimbs of ischemic Mouse I2 (a-d) and I3 (e h). (a,e) NIR-II fluorescence images showing the flow front labeled by SWNT fluorescent tags. (b,f) Distance travelled by the flow front plotted as a function of time. (c,g) Normalized NIR-II signal in the femoral artery plotted as a function of time. (d,h) Linear correlation between the artery blood velocity and NIR-II fluorescence increase in the corresponding artery area. Scale bars in a,e indicate 5 mm and the intensity scale bars range from to 1. 5
6 Coeff. [cm/%] SWNT conc. t = 4.69 s Experimental t = 5.25 s Simulation Flow front front F(t,v) F (v, t) (k) Flow model with axial mixing Inflow of SWNTs (conc., QY and velocity) Flow direction ROI diameter (a) t = 6.56 s (c) (e) Change SWNT conc. Int. Increase=18.95 %/s Coeff.=.739 cm/% (g) Change tube diameter Int. Increase= %/s Coeff.=.766 cm/% (i) Change velocity v=.14 cm/s Int. Increase= %/s Coeff.=.757 cm/% 3 (b) (d) Standard Int. Increase=18.14 %/s Coeff.=.778 cm/% Time [s] Int. Increase=18.55 %/s Coeff.=.756 cm/% 1 3 (f) Change SWNT QY (h) Change ROI length Int. Increase= %/s Coeff.=.789 cm/% (j) Change pre-roi length Int. Increase= %/s Coeff.=.1648 cm/% (l) Standard Int. Increase= %/s Coeff.=.73 cm/% (n) Change SWNT QY Pre-ROI length Int. Increase= %/s Coeff.=.73 cm/% (p) Change ROI length Int. Increase= %/s Coeff.=.7 cm/% (r) Change pre-roi length Int. Increase= %/s Coeff.=.171 cm/% ROI length (m) Change SWNT conc. Int. Increase= %/s Coeff.=.73 cm/% (o) Change tube diameter Int. Increase= %/s Coeff.=.73 cm/% (q) Change velocity v=.14 cm/s Int. Increase= %/s Coeff.=.722 cm/% Experimental (s).14 Simulation.12 Coeff. from mice: cm/% Figure S5. Variable dependency study of the intensity-to-velocity conversion coefficient based on both experimental (a-j) and simulation (l-r) results of the tubing flow setup (k). To find which variable(s) influence(s) the value of the coefficient, six possible parameters are changed one at a time from the Standard change conc change QY change diameter change ROI change velocity change pre-roi 6
7 standard settings while keeping all other parameters unchanged. The standard settings are given by: SWNT concentration =.1 mg/ml, SWNT fluorescence quantum yield (QY) = 2.5%, tubing diameter = 7 m, tubing length within ROI (i.e., ROI length) = 2.5 cm, fluid velocity = 1.4 cm/s and tubing length before ROI (i.e., pre-roi length) = 8.5 cm. (a-c) NIR-II fluorescence images of SWNTs flowing through the tubing at 4.69 s, 5.25 s and 6.56 s post injection. Scale bar indicates 5 mm. (d-j) Experimental normalized NIR-II intensity increase curve as a function of time when: at the standard settings (d), only the SWNT concentration is reduced by 4 times (e), only the QY of SWNT is increased by 2 times (f), only the tubing diameter is reduced by 2 times (g), only the ROI length is reduced by 2 times (h), only the fluid velocity is reduced by 1 times (i), and only the pre-roi is increased by 2 times (j). A linear fit based on the onset increase in each case gives a coefficient of.778 cm/%,.739 cm/%,.756 cm/%,.766 cm/%,.789 cm/%,.757 cm/% and.1648 cm/%, respectively. Note the coefficient remains invariant except in the case of a change in the pre-roi length. (k) A schematic drawing of the tubing flow experiment setup, where all six parameters are labeled. The same setup is used in the numerical simulation, and the flow front is simulated by a sigmoidal function, the shape of which is dependent of both time and fluid velocity, based on a tubing flow model with axial mixing. 1,2 (l-r) Simulational normalized NIR-II intensity increase curve as a function of time when at the standard settings (l), only the SWNT concentration is reduced by 4 times (m), only the QY of SWNT is increased by 2 times (n), only the tubing diameter is reduced by 2 times (o), only the ROI length is reduced by 2 times (p), only the fluid velocity is reduced by 1 times (q), and only the pre-roi is increased by 2 times (r). A linear fit based on the onset increase in each case gives a coefficient of.73 cm/%,.73 cm/%,.73 cm/%,.73 cm/%,.7 cm/%,.722 cm/% and.171 cm/%, respectively. Note the coefficient remains invariant except in the case of a change in the pre-roi length. (s) A bar chart summarizing the conversion coefficients derived from experimental results (red bars) and simulation (blue bars), with comparison to the coefficient derived from the femoral arterial flow in mice (green dashed line), showing the conversion coefficient invariant of SWNT concentration, SWNT quantum yield, tubing diameter, ROI length and fluid velocity, while only influenced by the pre-roi length. 7
8 Velocity [mm/s] Velocity [mm/s] Velocity [mm/s] (a) (b) 5.2±.39 cm/s (d) 5.96±3 cm/s ±6 cm/s (c) ±7 cm/s (f) 5.9±.35 cm/s (e) ±4 cm/s Figure S6. Reproduced femoral artery blood flow quantification in three healthy, control nude mice (Mouse C2 4). (a) Normalized NIR-II signal in the femoral artery plotted as a function of time, revealing a blood velocity of 5.2±.39 cm/s after conversion for Mouse C2. (b) Power Doppler of femoral artery blood flow in Mouse C2 shown in the top graph, revealing an average blood velocity of 5.96±3 cm/s, based on the velocity time integral (VTI) of three cardiac cycles as shown by pulsed wave Doppler in the bottom graph. (c) Normalized NIR-II signal in the femoral artery plotted as a function of time, revealing a blood velocity of 4.58±6 cm/s after conversion for Mouse C3. (d) Power Doppler of femoral artery blood flow in Mouse C3 shown in the top graph, revealing an average blood velocity of 4.89±7 cm/s, based on the VTI of three cardiac cycles as shown by pulsed wave Doppler in the bottom graph. (e) Normalized NIR-II signal in the femoral artery plotted as a function of time, revealing a blood velocity of 5.9±.35 cm/s after conversion for Mouse C4. (f) Power Doppler of femoral artery blood flow in Mouse C4 shown in the top graph, revealing an average blood velocity of 6.49±4 cm/s, based on the VTI of three cardiac cycles as shown by pulsed wave Doppler in the bottom graph. 8
9 Table S1 Establishing the intensity-to-velocity conversion coefficient for translating NIR-II signal increase to blood velocity. Ischemic mouse number Conversion coefficient (cm % 1 ) Average.747 ±.19 Three ischemic mice, mice I1 I3, were analyzed for their blood flow behaviors with SWNTs as NIR-II fluorescence tags. Table S2 Dependency of the intensity-to-velocity conversion coefficient on six different variables based on experimental tubing flow results (left column) and numerical simulation (right column), suggesting the coefficient is independent of SWNT concentration, SWNT fluorescence quantum yield, tubing diameter, ROI length and fluid velocity and influenced only by the pre-roi length. Variable control Experimental results (cm % 1 ) Simulation results (cm % 1 ) Standard settings Change SWNT concentration Change SWNT quantum yield Change tubing diameter Change ROI length Change fluid velocity Change pre-roi length
10 Supplementary Movie Captions Movie S1 Carbon nanotube labeled blood flow in a healthy limb NIR-II video-rate imaging showing blood flow in a healthy hind limb of a control mouse. Total video time: 37.5 s, frame rate: 5.3 frames/s. Movie S2 Carbon nanotube labeled blood flow in an ischemic limb early frames NIR-II video-rate imaging showing significantly reduced blood flow in an ischemic hind limb with occlusion in the femoral artery. The same number of frames as in Movie S1 is shown in this video to highlight the dramatic delay of flow into femoral artery and vein. Total video time: 37.5 s, frame rate: 5.3 frames/s. Movie S3 Carbon nanotube labeled blood flow in an ischemic limb late frames NIR-II video-rate imaging showing significantly reduced blood flow in an ischemic hind limb with occlusion in the femoral artery up to s post injection of carbon nanotubes. Total video time: s, frame rate: 9 frames/s. Movie S4 Differentiation of arterial and venous vessels subserving a larger area NIR-II video-rate imaging showing different blood flow behaviors of arterial and venous vessels subserving a larger area than just the hind limb, which is the basis for differentiation of blood vessel type based on principal component analysis. Total video time: 31.9 s, frame rate: 5.3 frames/s. 9
11 References 1 Zhao, L. L., Derksen, J. & Gupta, R. Simulations of Axial Mixing of Liquids in a Long Horizontal Pipe for Industrial Applications. Energ. Fuel 24, (1). 2 Quinn, J. G. Modeling Taylor dispersion injections: Determination of kinetic/affinity interaction constants and diffusion coefficients in label-free biosensing. Anal. Biochem. 421, 391- (12). 1
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