As a prelude to in vivo fluorescence imaging studies, a series of informative cell culture experiments were conducted. First, a set of standard MTT assays, using three different cancer cell lines (HT-29 human colorectal adenocarcinoma, 43 A549 human lung adenocarcinoma, and U87 human glioblastoma ) found that added ICB, like ICG, had no deleterious effect on cell metabolic activity (Figure ). Next, a subsequent series of fluorescence microscopy experiments examined HT-29 cells treated with ICB. Previous work by Onda and coworkers concluded that ICG permeates HT-29 cells by two independent pathways, (a) direct association with the cell plasma membrane followed by clathrin-dependent endocytosis, and (b) facilitated transport through the plasma membrane by endogenous organic anion transporter proteins. We find that analogous cell experiments using ICB produced the same results. That is, cell microscopy experiments that incubated HT-29 cells with 30 µM of ICB for 2 hours observed fluorescent puncta inside the cells with significantly less cell uptake at 4° C compared to 37° C (Figures 4A and S9 This time-dependent pattern of organelle localization is very similar to that reported for ICG. Thus, we conclude that ICB and ICG interacted with the cells in a similar manner. That is, they entered the cells by endocytosis or by facilitated transport through endogenous organic anion transporters and they initially accumulated in mitochondria, but over time they translocated to cell lysosomes. For mouse imaging, we used a commercial in vivo imaging station that was equipped with multiple LEDs as the light sources and a back illuminated CCD for image detection. As noted by others, this camera design detects deep-red light with high sensitivity and we have used it often for imaging the location of deep-red fluorescent probes in living mice. Deep-red light does not penetrate through skin and tissue as far as near-infrared light; nonetheless, we could easily image a phantom containing ICB when it was covered by several millimeters of 1% intralipid solution as a mimic of scattering biological tissue (Figure ). This result provided reassurance that ICB and ICG would be an effective pair of in vivo imaging agents for relatively shallow locations in a living subject.