ALZET Technical Sheet 1.1 In vivo Bioluminescence Imaging Using ALZET® Osmotic Pumps eal-time in vivo imaging of small laboratory animals is now feasible through the novel use of luciferase reporters and bioluminescence imaging (BLI) technologies, such as IVIS Imaging Systems.1 This technology allows real-time monitoring of ongoing biological processes in living animals. The technique is highly sensitive, non-invasive, simple to execute, and can reduce the number of animals used for experimentation since data can be acquired from the same animal over time. BLI is based on the detection of visible light produced during luciferase-mediated oxidation of the molecular substrate, luciferin, when the enzyme is expressed in vivo as a molecular reporter. The luciferase reaction is highly dependent on substrate availability. In fact, studies show that if “exogenously administered luciferin is not abundantly present, light emission might not be a true representation of luciferase activity.”2 This constraint can be eliminated with the use of ALZET® Osmotic Pumps, which facilitate steady-state delivery of bioluminescent substrates for extended BLI-Compatible Flow Moderators periods of time. ALZET pumps provide reliable and prolonged substrate delivery, thus eliminating complications of repetitive injections, and ensuring accurate detection of in vivo bioluminescence. When using ALZET pumps with IVIS imaging systems (Caliper Life Sciences, Hopkinton, MA) during BLI studies, researchers should use the BLIcompatible flow moderators in blue or teal color. These were specifically developed by DURECT Corporation to avoid background luminescence caused by the standard (white) flow moderators which may interfere with the real signal from the biological system under study. Improve the quality of your in vivo imaging data. Eliminate the background chemiluminescence observed with the white flow moderators during in vivo BLI with IVIS imaging systems. Caliper Life Sciences Inc., in collaboration with DURECT, performed a series of studies designed to evaluate the in vitro and in vivo chemi-luminescence signal emitted from the new flow moderators and compare it to that of the white flow moderators.3 These studies indicate that the background signal is significantly reduced with the use of blue and teal flow moderators. Therefore, when using ALZET pumps during BLI studies, researchers should replace the white flow moderators, included in each package of ALZET pumps, with BLI-compatible flow moderators (Refer to Table 1 for a list of flow moderators available). ALZET® Osmotic Pumps | DURECT Corporation www.alzet.com │800.692.2990 │ 408.865.14

A Flow Moderator Color White Teal Blue The background chemiluminescence of ALZET pumps containing each of the three different types of flow moderators (white, blue, and teal) was evaluated using the IVIS Imaging System. The open filter was used to measure the maximum chemiluminescence signal. Figure 1 shows the results of this in vitro evaluation. As seen in Figure 1A, the ALZET pump containing the white flow moderator has significant chemi-luminescence, while the teal and blue flow moderator pumps display no visible chemi-luminescence signal. Graphical representation of light emission (in photons/sec)...

Figure 2: In vivo imaging of mice implanted with ALZET Osmotic Pumps containing different color flow moderators (white, teal and blue). Images from the same representative animals subjected to BLI at 0, 15, 30, and 120 minutes after cell injections, are shown. As seen in Figure 2, following in vivo BLI of animals implanted with white flow moderator pumps, a significant background chemiluminescence signal is observed at the location of pump implantation, specifically coinciding with the position of the flow moderator. This signal is absent from the images of animals implanted with the teal and...

Recommendations: These data indicate that the blue and teal flow moderators of ALZET pumps are preferred for in vivo BLI studies using the IVIS imaging systems. These BLI-compatible flow moderators help avoid the background signal associated with the standard (white) flow moderators. Table 1. ALZET BLI-Compatible Flow Moderators* Pump Models ALZET Flow Moderator *Compatibility evaluated using IVIS Imaging Systems ‡ Sold in quantities of 10 per bag Research Applications: A study by Gross et al., published in Nature Methods, explored the benefits of continuous administration of D-luciferin via...

Animals in the injection protocol required D-luciferin injections every 4 hours for 44 hours, and were subjected to prolonged anesthesia during injection and imaging procedures. In contrast, rats implanted with pumps were anesthetized only briefly every 4 hours for imaging. Imaging results demonstrated rhythmic Period1 activity in all olfactory bulbs pump-implanted rats, compared to only 70% in the luciferin-injected rats, indicating that “the pump-implantation procedure yielded a higher success rate.”5 The authors found that a circadian pattern of Period1 driven activity was present in intact...