In vivo imaging of treatment responses at the molecular level could have a significant impact on the speed of drug discovery and ultimately lead to personalized medicine. Strong interest has been shown in developing quantitative fluorescence-based technologies with good molecular specificity and sensitivity for noninvasive 3D imaging through tissues and whole animals. We show herein that tumor response to chemotherapy can be accurately resolved by fluorescence molecular tomography (FMT) with a phosphatidylserinesensing fluorescent probe based on modified annexins. We observed at least a 10-fold increase of fluorochrome concentration in cyclophosphamide-sensitive tumors and a 7-fold increase of resistant tumors compared with control studies. FMT is an optical imaging technique developed to overcome limitations of commonly used planar illumination methods and demonstrates higher quantification accuracy validated by histology. It is further shown that a 3-fold variation in background absorption heterogeneity may yield 100% errors in planar imaging but only 20% error in FMT, thus confirming tomographic imaging as a preferred tool for quantitative investigations of fluorescent probes in tissues. Tomographic approaches are found essential for small-animal optical imaging and are potentially well suited for clinical drug development and monitoring. drug discovery ͉ quantification ͉ three-dimensional T he ability to noninvasively image molecular processes in vivo is an emerging reality with different reporter and detection approaches (1, 2). Molecular imaging has been heralded to lead to earlier detection than current anatomical imaging approaches, which typically detect late-stage abnormalities. Another important prospect of molecular imaging is the ability to examine and quantify treatment responses in vivo by monitoring specific primary molecules or downstream targets. Therapeutic efficacy could then be probed dynamically on timescales of hours to days. This ability is in contrast to the mainstay of today's healthcare with traditionally late end points of drug efficacy, a practice that often impairs prompt revision and exclusion of ineffective treatment strategies with potentially lethal results.Drug-induced apoptosis is considered a generic biomarker of monitoring the effects of chemotherapeutic drugs (3), antihormonal therapeutics (4), or antiangiogenic therapies (5). Of several different detection methods, optical imaging is emerging as an important alternative to techniques using ionizing radiation and offers the advantages of stable fluorochromes, easier to perform chemistries, and portable and cost-effective imaging practices. Laxman et al. (6), for example, developed a recombinant luciferase reporter molecule that amplifies luminescence in apoptotic cells by specific cleavage of a Asp-Glu-Val-Asp fragment by caspase 3, which can be used in animal studies. Fluorescence detection using an annexin V probe tagged with a cyanine dye has been demonstrated by Schellenberger et al. (7) and Petrovsky et al. (8). Imagin...

The ability to image cardiomyocyte apoptosis in vivo with highresolution MRI could facilitate the development of novel cardioprotective therapies. The sensitivity of the novel nanoparticle AnxCLIO-Cy5.5 for cardiomyocyte apoptosis was thus compared in vitro to that of annexin V-FITC and showed a high degree of colocalization. MRI was then performed, following transient coronary artery (LAD) occlusion, in five mice given AnxCLIO-Cy5.5 and in four mice given an identical dose (2 mg Fe/kg) of CLIO-Cy5.5. MR signal intensity and myocardial T 2 * were evaluated, in vivo, in hypokinetic regions of myocardium in the LAD distribution. Ex vivo fluorescence imaging was performed to confirm the in vivo findings. Myocardial T 2 * was significantly lower in the mice given AnxCLIO-Cy5.5 (8.1 versus 13.2 ms, P < 0.01), and fluorescence target to background ratio was significantly higher (2.1 versus 1.1, P < 0.01). This study thus demonstrates the feasibility of obtaining high-resolution MR images of cardiomyocyte apoptosis in vivo with the novel nanoparticle, AnxCLIO-Cy5. Cardiomyocyte (CM) apoptosis has been implicated in numerous diseases involving the heart including ischemia (1,2), reperfusion injury (3,4), and heart failure (5,6). The myocardium has a limited regenerative capacity, and the prevention of CM loss from apoptosis thus has profound implications and is an area of intense pharmaceutical research. However, despite significant advances in the biochemical characterization of CM apoptosis (7), a gap has developed between the basic molecular understanding of CM apoptosis and the translation of this knowledge into clinical practice. This gap has occurred in large part due to the absence of a reliable biomarker with which to detect and characterize CM apoptosis in the heart in vivo (7,8).Apoptotic cells in all organs express the membrane phospholipid, phosphatidylserine, on the outer surface of their cell membranes early in the apoptotic process (9). Annexin V binds to phosphatidylserine selectively and with a high affinity, and it is thus a highly suitable ligand for targeted apoptosis imaging. Technetium-99m-labeled annexin V (Tcannexin V) has been used to image apoptosis in rat models of cardiac transplant rejection (10) and myocarditis (11) and in patients with cardiac transplant rejection (12) and acute coronary syndromes (13,14). However, the results of these studies have been controversial and adversely affected by the limited spatial resolution of current nuclear imaging technology (15). The utility of Tc-annexin V has also been limited by the need to wait 15-20 h for washout of the probe from the left ventricular blood pool and by the inability to generate tomographic and functional images of myocardial contraction from the Tc-annexin V hot-spot images (13-15). Magnetic resonance imaging does not suffer from any of these limitations and can be used to make high-resolution images of cardiac anatomy and function in both humans and small animals such as mice (16). In addition, delayed enhancement imaging by MRI h...