Supplementary Figure 6: Linearity of DSP detection down to a single cell in various model systems.

a, Representative section of FFPE cell pellet (CCRF-CEM cell line) showing of ROIs (UV-cleavage areas) ranging from 331,000 µm² (a circle with a 650 µm diameter) to 1,960 µm2 (a circle with a 50 µm diameter). Concentric areas are displayed for illustration purposes only; in the experiment, independent regions of the cell pellet were illuminated for each ROI. b, Average counts from n=3 replicate measurements for each ROI size. Best fit linear trend line shown. R² calculated from Pearson’s R. Error bars represent one standard deviation. c, Representative CCRF-CEM cell pellet illustrating ROIs illuminated for 1 to 8 cells. For each ROI, the designated number of cells were UV illuminated simultaneously and collected with a single aspiration step. A fluorescently conjugated secondary antibody targeting primary antibodies against CD3 and CD4 was used to visualize cell membranes. This experiment was performed once. d, Counts of replicate ROIs (n=12 for 1 cell, n=9 for 2 cells and 4 cells, and n=6 for 8 cells) for each ROI size plotted versus size of UV-exposed area. Best fit linear trend line shown. R² calculated from Pearson’s R. Limit-of-detection (LOD) value shown as dotted line: the average plus two standard deviations of replicate counts of samples with no illumination, representing assay background. This data shows detectability of CD3 down to a single cell. e, Tonsil stained with CD3 and CD4 rabbit antibodies (T cell markers; green) and CD19 and CD20 mouse antibodies (B cell markers; magenta) and visualized with goat anti-mouse and goat anti-rabbit antibodies labeled with different fluorophores. Average cell diameters for the two cell types differ, and UV-illumination areas, here outlined with circles, were adjusted accordingly. This experiment was performed once. f, CD3 or CD20 counts from single cell illumination of T cells (n=12) and B cells (n=12). Middle black bar represents the average counts for all replicates, and error bars represent one standard deviation. LOD value shown is the average plus two standard deviations of replicate counts of no-UV samples (representing background counts). The expected detectability was seen for single T cells and B cells in this tissue. g, Various 1, 2, or 4 cell ROIs of a PBMC cell pellet were UV illuminated; representative images shown A fluorescently conjugated secondary antibody targeting antibodies against CD3 and CD4 was used to visualize T cells. This experiment was performed once. h, Counts of replicate ROIs for each ROI size plotted versus number of cells illuminated (n=6 for 1 cell, n=6 for 2 cells, and n=5 for 4 cells). Error bars are one standard deviation. Best fit linear trend line shown. R² calculated rom Pearson’s R (three outlier points, gray, outside of one standard deviation were not used for this calculation). Limit-of-detection (LOD) value shown is the average plus two standard deviations of replicate counts of no-UV samples (representing background counts). This data shows detectability of CD3 down to a single cell.