Annexin vivo 750

Near-infrared fluorescence reflectance imaging (FRI) was performed using the In Vivo FX Pro Imaging System (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with a 400 W halogen illuminator with Cy 7 bandpass excitation (730 nm) and emission filters (790 nm). Fluorescence signals were captured with a cooled charge-coupled device (CCD) camera. Mice received 2 nmol Annexin vivo 750 (Perkin Elmer, Waltham, MA, USA) via the tail vein. After 96 h, the animals were sacrificed and the organs were removed and placed on a Petri dish for fluorescence imaging. Fluorescence images were coregistered with the anatomic white light images and quantified using region of interest (ROI) that includes the whole organ. The mean fluorescence intensity of each sample was reported using Bruker MI 7.1 software. Relative fluorescence signal intensities were visualized in false colors and quantified as AU. To eliminate nonspecific background fluorescence, data from tissue extracts containing no doxorubicin were subtracted.

For studies imaging XenoLight RediJect 2-DG and Annexin Vivo 750, 2 hours after i.v. injection was found to be the optimal time point to image maximal signal and tissue distribution (data not shown), consistent with the manufacturer’s recommendations (PerkinElmer). Total MMP activity was assessed using MMPSense 750 FAST, a probe that becomes fluorescently active upon cleavage by matrix metalloproteinases. Mouse tissues were imaged 6 hours after injection of MMPSense 750 FAST according to the manufacturer’s recommendations (PerkinElmer). All probes were injected in 100 μL via the retro-orbital sinus. At the indicated imaging times, mice were euthanized by isoflurane inhalation and whole organs aseptically removed and placed in a 90-mm-diameter Petri dish. Tissues were immediately imaged with an IVIS Lumina XR imaging system using the ICG filter. Images were analyzed using Living Image 4.0 (PerkinElmer). Regions of interest were drawn around the excised tissue, fluorescent signal intensity measured, and total radiance efficiency calculated. Background autofluorescence was corrected for using tissues from mice in which no fluorescent probe was injected.

Fluorescence molecular tomography (FMT) and microcomputed tomography (µCT) scans were performed in vivo, 2 hours after i.v. injection of 2 nmol of the commercially available Annexin V-based imaging probe Annexin Vivo 750 (PerkinElmer). Mice were scanned and transported between the devices using a multi-modality mouse bed while being anesthetized with isoflurane during the whole procedure [23 (link)]. For better tumor localization, a µCT scan was performed directly before the FMT measurement. Both tubes of the dual energy µCT (Tomoscope Duo, CT Imaging GmbH) were run at energies of 65 kV and currents of 0.38 mA. Each flat panel detector acquired 720 projections at 25 frames per second during a full rotation with a total scan time of 29 seconds. A Feldkamp algorithm was used for image reconstruction with isotropic voxel sizes of 70 µm and a smooth reconstruction kernel. FMT (FMT 2500, Perkin Elmer) was performed using a wave-type specific scanner for transillumination, reflectance, and absorption as described [25 (link)]. Tomographic data sets of µCT and FMT were fused during the post-processing step using fiducial markers in the multi-modality mouse bed [23 (link)]. The tumor was segmented in 3D based on the anatomical µCT-data to quantify the amount of co-localized fluorescence (Imalytics Preclinical, Gremse-IT GmbH) [26 (link)].