(A) ¹⁵N-thymidine–labeled leukocytes, collected after a 2-day intravenous labeling period or subsequent 4-week label-free chase. Quantitative mass images reveal subcellular details. High phosphate content in nucleic acid accounts for high signal in nuclei of ³¹P images. ³²S images demonstrate intense signal in the granules contained in granulocytes. ¹⁵N/¹⁴N hue saturation intensity image shows label localized to the nuclei of cells born during label administration (arrows). The rainbow scale ranges from blue, set to natural ratio (0.37%, expressed as 0% above natural ratio), to red, where the ratio is above the natural ratio (200% = 3 times natural ratio). Scale bar: 5 μm. (B) Frequency of labeled leukocyte subsets after pulse or chase (n = 6 subjects). Data displayed as Tukey box-and-whisker plots. Mann-Whitney tests were used to assess significance. Note, the y axis ranges from 0%–20%. (C) Labeling schematic for protocol 2, in which subjects were administered intravenous ¹⁵N-thymidine continuously for 3 days concurrently with oral ²H-water. The expected effective labeling period for ²H-water is prolonged relative to ¹⁵N-thymidine due to a total body water half-life of ~1 week. (D) ¹⁵N-thymidine/²H-water doubly labeled cells (arrows). Scale bar: 5 μm. (E) Colocalization of label in D confirmed quantitatively by plotting pixel values, which demonstrate associations among ¹⁵N, ²H, and ³¹P signals, consistent with DNA labeling. Each plotted data point is extracted from a single pixel on the quantitative mass or ratio images. (F) Pixel analysis from ¹⁵N-thymidine–unlabeled cells in D demonstrates no positive correlation between the ²H and ³¹P signals. (G) Data from representative subject undergoing double-labeling protocol (n = 3). After pulse (left), ¹⁵N-thymidine–labeled monocytes demonstrate high ²H-water labeling relative to ¹⁵N-unlabeled cells. After chase (right), high ²H-labeling in ¹⁵N-thymidine–labeled lymphocytes relative to that observed in ¹⁵N-unlabeled cells. Rare ¹⁵N^– outliers that are highly ²H-labeled likely represent cells born after cessation of ¹⁵N-thymidine labeling but within the effective ²H-water–labeling period shown schematically in C. Data displayed as Tukey box-and-whisker plots. Mann-Whitney tests were used to assess significance. b.g., background.