Single T-ALL cells can be efficiently transplanted into non-IR CG1 recipient. Transplant recipient fish receiving 1 FACS-sorted dsRED⁺ T-ALL cell at 20 (A,D), 30 (B,E), and 45 days after transplantation (C,F). One representative animal engrafted T-ALL by 20 days (A-C), whereas one never developed leukemia (D-F). Images photographed at 7.0×. All panels are merged images of fluorescent and brightfield photographs.

The LED fluorescence macroscope can image 5 fluorescent fluorophores in normal muscle and T-cell acute lymphoblastic leukemia (T-ALL). Whole animal imaging of wild-type (Negative), mylz2-Amcyan, mylz2-GFP, creatine kinase-zsYellow, mylz2-mCherry transgenic zebrafish (A-D). Amcyan was imaged by the use of a blacklight and 480/30 filter (A,J,N), GFP (blacklight, 520/30 filter; B,K,O), zsYellow (blue light, 575/52 filter; C), dsRED express (green light, 610/40 filter; L,P), and mCherry (green light, 610/40 filter; D). Transgenic zebrafish imaged using an epi-fluorescence stereomicroscope at the lowest magnification (7.0×; E-H). The LED fluorescence macroscope is also capable of imaging engraftment of T-cell acute lymphoblastic leukemia in recipient animals. Whole-animal imaging of recipient fish transplanted with 1.5 × 10⁴ Amcyan-, GFP-, or dsRED express–labeled leukemia cells at 30 days after transplantation (I-L). Animals from panels I through L were mixed to demonstrate that fluorescent-labeled animals can be easily delineated (M-P). Transplant recipients imaged by the use of an epi-fluorescence stereomicroscope at low magnification (7.0×, Q-T). Scale bars are 2 cm in panels A through D and I through L, and 5-mm in panels E through H and Q through T.

T-ALLs from CG1-strain zebrafish engraft into non-IR CG1-strain recipients. GFP-labeled T-ALLs were isolated from primary leukemic fish, and 10³ FACS-sorted GFP-labeled leukemia cells were transplanted into non-IR CG1- and AB-strain animals (A-C and G-I, respectively) or IR AB-strain fish (D-F). Transplant fish were scored for engraftment at 10, 20, and 30 days after transplantation. Panels are merged images of fluorescent and brightfield photographs. The engraftment kinetics differ greatly when T-ALLs are introduced into CG1 (J) or IR AB-strain zebrafish (AB + IR; K). Percent engraftment is the percentage of recipient animals that have visibly engrafted T-ALL at each time point. Panels J and K are combined data from 3 independent experiments (n = 238 transplants total). The raw data for this experiment are available in supplemental Table 2.

T-ALLs from CG1 fish are capable of engraftment into CG1 outcrossed animals. A GFP-labeled T-ALL engrafted into the progeny of CG1 by albino fish (CG1/Alb; A-C). Engraftment of T-ALLs into AB-strain zebrafish required 2 rounds of outcrossing to CG1 fish (D-I). A dsRED-labeled T-ALL engrafted into the progeny of a CG1 by CG1/AB rag2-GFP transgenic animal (D-F) or an Amcyan-labeled T-ALL engrafted into the progeny of a CG1 by CG1/AB mylz2-mCherry (G-I). Images photographed at 7.0×. All panels are merged images of fluorescent and brightfield photographs. GFP-labeled thymus is marked by T.

The LED fluorescence macroscope can detect tumor engraftment of single T-ALL cells and is capable of multispectral, real-time imaging. Animals engrafted with a single dsRED-labeled T-ALL cell can be easily distinguished from nonengrafted animals at 45 days after transplantation (n = 1 of 10 fish, A; n = 2 of 20 fish, B; n = 3 of 29 fish panel C) by LED fluorescence macroscopy. Multispectral imaging with the LED fluorescence macroscope (D-I). Still image capture with dual-spectrum imaging of unanesthetized mylz2-Amcyan and mylz2-mCherry transgenic animals (D, blue and black light with 520/40 filter and 640/35 filter), mylz2-GFP and mylz2-mCherry (E, blue light with 520/40 filter and 640/35 filter), and creatine kinase-zsYellow and mylz2-mCherry (F, blue light with 575/52 filter and 640/35 filter). Animals were imaged from below at 30 frames per second, and a single frame is shown. Multispectral imaging also can be used to visualize fluorescent-labeled T-ALLs engrafted into fluorescent recipient animals. A mylz2-mCherry animal engrafted with an Amcyan-labeled T-ALL at 30 days after transplantation imaged by epi-fluorescence stereomicroscopy (G, 7.0× magnification) or by use of the LED fluorescence macroscope (blue light with 480/30 and 640/35 filters; H-I). Panel H shows a control animal (top) along with a dual transgenic leukemic fish (bottom). Three leukemic fish imaged from below at 30 frames per second and a single frame is shown (I). Scale bars are 1 cm in panels A through F and H through I; 2 mm in panel G.