File Name : figure s1 bca standard_v5.png

Caption : supplementary figure 1. tof-sims analysis of varying concentrations of fibronectin (fn) coated onto plastic slide surfaces, using bca staining as a surrogate marker. (a) representative tof-sims images illustrating copper (cu) ion signals (m/z 62.83–63.03) across fibronectin (fn) coatings ranging from 1 to 100 μg ml-1, with water as a control. (b) cu ion peak intensities at m/z 62.93 for different fn concentrations, displayed using a uniform y-axis scale for comparison. (c) normalized cu ion counts, calculated by dividing cu ion counts by total ion counts for each roi, presented as pink scatter points in a line plot. the water control is indicated by a red dotted line, mean values are shown as blue dots, standard deviations are shown in black, and pink dots represent 4 rois. measurements were acquired in bunched mode.

File Name : figure s2 eosin standard_v4.png

Caption : supplementary figure 2. tof-sims analysis of varying concentrations of fibronectin (fn) coated onto plastic slide surfaces, using eosin staining as a surrogate marker. (a) representative tof-sims images illustrating bromine (br) ion signals (m/z:78.818-79.018) across fn coatings ranging from 1 to 100 μg ml-1, with water as a control. (b) br ion peak at m/z 78.92 for different fn concentrations displayed using a uniform y-axis scale. (c) normalized br ion counts, calculated by dividing br ion counts by total ion counts for each roi, presented as scatter points in a line plot. the water control is indicated by a red dotted line, mean values are shown as blue dots, standard deviations are shown in black, and pink dots represent 4 rois. measurements were acquired in bunched mode.

File Name : figure s3 stats standards_v2.png

Caption : supplementary figure 3. tof-sims analysis of varying concentrations of bodipy–fn (a), fibronectin labeled with surrogate bca (b), fibronectin labeled with eosin (c), and fluorescence measurements of bodipy–fn at corresponding concentrations (d). statistical comparisons were made against the water control (0 µg ml⁻¹) using one-way anova with a post hoc dunnett test. ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

File Name : figure s4 bodipy fn standard fit_v2.png

Caption : supplementary figure 4. power law fits were applied to the bodipy-fn standard concentrations for (a) data in the range of 0.1–500 µg ml-1 and (b) data in the range of 0.1–25 µg ml-1. the fitting equations and r-squared values are provided on the graphs.

File Name : figure s5 bodipy-fn gradient in chip fluorescent_v2 onlyab.png

Caption : supplementary figure 5. bodipy-fn gradient formation in the microfluidic chip visualized by fluorescence imaging. (a) fluorescence images of bodipy-fn gradients formed by 24-hour diffusion from source chambers containing 100 µg ml-1 or 500 µg ml-1 bodipy-fn, compared to the water control. (b) averaged line profiles of fluorescence intensity across chambers a–d, demonstrating gradient formation. 

File Name : figure s6 chip imaging.png

Caption : supplementary figure 6. images of the microfluidic chip used to generate bodipy-fn gradients and prepare samples for tof-sims analysis. (a) overview of the microfluidic chip after removal of the pdms chamber, revealing the underlying plastic surface. (b) sample plate showing the microfluidic slide cut into smaller pieces for tof-sims measurement. (c) close-up image of the microfluidic chambered slide used for tof-sims analysis.