Location and behavior of B-ALL–initiating cells differ in a young vs old BMM. (A) Schematic of the imaging experiments with 2-photon in vivo microscopy. (B) Representative two-photon in vivo microscopy image of the bone marrow (BM) calvarium of nonirradiated BALB/c mice injected with sorted BCR-ABL1⁺ (GFP⁺) BP-1⁺ B-ALL cells from BALB/c mice with established disease labeled with 5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR) (red) in relation to the endosteum. Bones were visualized in blue due to second harmonic generation, and vessels were visualized in green via the injection of dextran-FITC (1 mg per injection). Scale bar, 50 µm. A schematic of how the calculations are performed using the Pythagorean theorem is shown on the right. (C) Distance of sorted BCR-ABL1⁺ BP-1⁺ B-ALL cells (P = .0007; 1-way analysis of variance [ANOVA], Tukey test) or MACS-depleted Lin⁻ cells (n.s., 1-way ANOVA, Tukey test) as random cells to endosteum, as measured by in vivo microscopy of the murine calvarium, in young (red) vs old (blue) mice. Each mouse had been injected with a total of 5 × 10⁵ cells, and in vivo imaging was performed 2 to 3 hours after injection. The distances in micrometers were normalized for each mouse by dividing distances by the surface area of the skull to take into account differences in body size between young and old mice (n = 3). Ten cells per transplanted and imaged mouse were evaluated. Each dot represents an individual cell. (D) Speed (μm/s) of sorted GFP⁺ (BCR-ABL1⁺) BP-1⁺ B-ALL cells from BALB/c mice with established disease labeled with CMTMR and injected into young (red) or old (blue) nonirradiated BALB/c mice (P = .017; Student t test, n = 3). Movies of cells of interest were recorded at 1 frame per second using ImageJ.