The Case cryo-imaging system enables high resolution, 3D mapping of fluorescently-labeled stem and cancer cells throughout the mouse. The system consists of a mouse-sized cryomicrotome; microscope; low light camera; three axis robotic positioning system; and automation, visualization, and analysis software. It provides 3D fluoresence/bright-field imaging of the block face following serial sectioning. We made tail vein injections of about 5 million Lewis lung carcinoma (LLC) cells, a cell line used for studies of metastatic cancer and therapies. Seven days post injection, cryo-imaging surveys identified cells in the liver, adrenal gland, and tail near the injection site.
Most cells were in the adrenal gland. In the adrenal gland, we processed images to remove subsurface fluoresence using a light propagation model, segmented fluorescent clusters, and created 3D visualizations that included the vasculature (figure 1). In a representative 2D image of the adrenal gland at 90x, a particle analysis gave 86 cells with an average diameter of 8.2 um, very consistent with 8.4 +/- 1.2 um in culture. Signal was about 6x the autofluorescence noise floor. In one mouse, about 3,850 cell clusters were detected at 50x in the adrenal gland. In a histogram of cluster volumes, discreet peaks corresponding to integer numbers of cells were observed. Most clusters were less than 15 cells, and the largest cluster contained about 310 cells. In the liver, we identified three clusters, and one consisted of about 85 cells. We believe that cryo-imaging can become an important tool to use in concert with in vivo imaging techniques to evaluate important issues in stem and cancer cell biology and therapeutics.
Source: Case Western University