The IVIS Lumina from Caliper Life Sciences is a highly sensitive imaging system that offers users the flexibility to image fluorescent and/or bioluminescent reporters both in vivo and in vitro. It offers spatial resolution from the scale of one well in a 6-well plate, to a full 6-well plate. The Small Animal Imaging Facility (SAIF) at Penn was very generous and provided the Penn iGEM team with the opportunity to conduct experiments with the IVIS Lumina. Our goal was to characterize our sender and receiver cell separately under the IVIS Lumina Imager, as well as some positive and negative controls. For the sender cell, we have Human Embryonic Kidney (HEK) cells transfected with pRL-CMV-Renilla luciferase + Coelenterazine (CZ). Data is given in radiance: photons/second/cm2/steradian. This information is particularly valuable to us because we can compare the background level of luminescent activity across all images.

Our receiver cell, HEKs with pRRL-CMV-ChR2-eYFP and pRRL-CMV-Aequorin + CaCl2, had no detectable background luminescence (figure 1). This was expected because aequorin needs CZ to luminesce.

Figure 1

Upon the addition of CZ, our luminescence increased (Figure 2). A time course showed that this signal increased over 12 minutes, then began decaying (Figure 3). We then tried to stimulate the ChR2 with 465 nm light, but the signal did not look like it was affected (Figure 4)

Figure 2	Figure 3

Figure 4
We were able to characterize our sender cell, HEKs with pRL-CMV-Renilla + CZ. The radiance of the sender cell was one magnitude more intense than that of the receiver cell. A time course of Renilla showed an increase, then decrease in signal after adding CZ (Figures 5-6).

Figure 5

Figure 6

We then imaged the entire 6-well plate (Figure 7), which showed that Renilla (Well 3) had the highest relative intensity. Our data showed us that Renilla luciferase can cleave CZ substrate almost instantaneously, and the level of luminescent activity (photons generated) would reach a plateau after 10 seconds and remain steady even after 2 hours. This suggests that if we were able to over express Renilla luciferase and saturate the system with CZ, we could potentially generate a strong light signal instantaneously and relay this signal to our receiver cell.

Figure 7

In a separate trial, we imaged an entire 6-well plate (Figure 8). The third well, which was transfected with CatCh and had CZ added, had 3 x 105 average radiance. Nothing else lit up. This shows that either the CatCh is working or we are getting background from CZ.

Figure 8

In a separate 6-well plate, we had in wells 3-6,
3. pcDNA3.1-CMV-ChR2-eYFP + pRRL-CMV-Aequorin
4. pcDNA3.1-CMV-CaTcH-eYFP + pRRL-CMV-Aequorin
5. pRRL-CMV-Aequorin
6. pcDNA3.1-CMV-ChR2-eYFP

We added CaCl2 and CZ to each of these wells, and captured luminescence as well as tried to stimulate with 465 nm light. Wells 3-6 have around the same luminescence, which means that any signal was attributed to the CZ, most likely (Figure 9). Since aequorin requires calcium to cleave coelenterazine, we hypothesized that upon stimulation of the ChR2 gate, our luminescent reporter could capture the influx of calcium ions, begin to fluoresce and cause a corresponding increase in photon counts recorded by the IVIS Lumina system.
Unfortunately, the result was not what we had expected.
When we compared the background luminescent level of aequorin +CZ to excited ChR2+aequorin+CZ, there was no significant difference in number of photons per well between the two samples. After repeated trials and troubleshooting, we believe the problem was that the chromophore of ChR2 was not properly formed. ChR2 requires the supplementation of retinol to properly carry out its light-gated activities. We are currently putting all our efforts into characterizing the receiver cell.

Figure 9